You are on page 1of 92

B GIO DC V O TO

B Y T

TRNG I HC DC H NI

PHAN TH HUYN

NGHIN CU NNG CAO KH


NNG SINH TNG HP KHNG
SINH T STREPTOMYCES 173.113
LUN VN THC S DC HC

H NI 2014

B GIO DC V O TO

B Y T

TRNG I HC DC H NI

PHAN TH HUYN

NGHIN CU NNG CAO KH NNG


SINH TNG HP KHNG SINH T
STREPTOMYCES 173.113
LUN VN THC S DC HC
CHUYN NGNH: CNG NGH DC PHM BO CH
THUC
M S: 60720402

Ngi hng dn khoa hc: PGS.TS Cao Vn Thu

H NI 2014

Li cm n
Ti xin gi li cm n su sc nht n thy gio PGS.TS Cao Vn
Thu ngi tn tnh hng dn ti t nhng bc u tin cho n khi ti
hon thnh lun vn ny.
Ti xin chn thnh cm n ti thy c gio, cc cn b k thut vin
ang ging dy v lm vic ti b mn Vi sinh - Sinh hc trng i hc
Dc H ni tn tnh gip ti trong sut thi gian ti lm lun vn ti
b mn.
Nhn dp ny ti cng xin gi li cm n ti Ban gim hiu cng ton
th cc thy c gio trng i hc Dc H Ni dy d v to iu kin
thun li cho ti trong sut qu trnh hc tp v rn luyn ti trng.
V cui cng l li cm n ti gi ti gia nh, bn b ng vin,
gip ti trong sut thi gian thc hin lun vn.
Do thi gian thc nghim hn hp, iu kin trang thit b, phng tin
nghin cu cng nh trnh ca bn thn cn hn ch, lun vn ny khng
trnh khi cn nhiu thiu st. Ti rt mong nhn c s gp ca thy c
v bn b lun vn ny c th c hon thin hn.
Ti xin chn thnh cm n!
H Ni, thng 10 nm 2014

Hc vin

Phan Th Huyn

MC LC
T VN

CHNG 1. TNG QUAN

1.1. i cng v Streptomyces

1.1.1. c im ca x khun chi Streptomyces

1.1.2. Kh nng sinh tng hp khng sinh ca Streptomyces

1.1.3. S tng qut sinh tng hp khng sinh t x khun..

1.1.4. Nhng im lu khi nghin cu cc cht khng sinh t x khun

1.2. Bc u nghin cu ln men sinh tng hp khng sinh nh Streptomyces 173.113

1.3. Nghin cu tng hp Actinomycin X2 t Streptomyces 15.29 Streptomyces


microflavus..

1.4. Cng ngh ln men Daunorubicin v Adriamycin.

1.5. Tng hp cirramycin B t Streptomyces cyaneus.

1.6. Khng sinh chng nm non-polyen t Streptomyces albidoflavus.

1.7. Tng hp khng sinh antimycin A9 t Streptomyces Sp. K01-0031

10

1.8. Sparsomycin khng sinh c tng hp t Streptomyces Sp. AZ-NIOFD1.

12

CHNG 2: I TNG V PHNG PHP NGHIN CU.

14

2.1. Nguyn vt liu v thit b

14

2.1.1. Nguyn vt liu

14

2.1.2. My mc thit b.

16

2.2. Phng php thc nghim

16

2.2.1. Nui cy v gi ging x khun.

16

2.2.2. nh gi hot tnh khng sinh bng phng php khuch tn...

17

2.2.3. Sng lc ngu nhin

18

2.2.4. t bin bng nh sng UV

18

2.2.5. Ln men chm tng hp khng sinh ..

20

2.2.6. Chit khng sinh t dch ln men bng dung mi hu c ..

20

2.2.7. Tch cc thnh phn trong khng sinh bng sc k lp mng.

21

2.2.8. Thu khng sinh th bng phng php ct quay chn khng

22

2.2.9. Tinh ch khng sinh th bng sc k ct

22

2.2.10. Xc nh cu trc ha hc thnh khng sinh chnh tinh khit thu c

23

CHNG 3: KT QU THC NGHIM V NHN XT

24

3.1. Chn lc v ci to ging x khun

24

3.1.1. Kt qu sng lc ngu nhin ..

24

3.1.2. Kt qu t bin ln th nht ..

25

3.1.3. Kt qu t bin ln th hai ..

27

3.2. Xc nh iu kin ln men, chit tch khng sinh tt nht...

29

3.2.1. Kt qu chn chng ln men ..

29

3.2.2. Kho st chn mi trng ln men chm.

31

3.2.3. Kt qu ln men, chit v ct quay thu khng sinh th.

32

3.2.4. Kt qu tinh ch khng sinh ln th nht

32

3.2.5. Kho st h dung mi tinh ch khng sinh ln th hai

35

3.2.6. Kt qu tinh ch khng sinh ln th hai.

37

3.2.7. Kho st h dung mi tinh ch khng sinh ln th ba.

39

3.2.8. Kt qu tinh ch khng sinh ln th ba..

40

3.3. Nghin cu tnh cht, cu trc khng sinh.

43

3.3.1. Kt qu o nng chy.

43

3.3.2. Kt qu ph t ngoi .

43

3.3.3. Kt qu ph hng ngoi

..

44

46

3.3.4. Kt qu o ph khi

3.3.5. Kt qu o ph cng hng t ht nhn

46

CHNG 4. BN LUN

49

4.1. Chn lc v ci to ging x khun

49

4.2. Xc nh iu kin ln men, chit tch khng sinh ti thch

50

52

4.3. Nghin cu cu trc khng sinh

KT LUN V KIN NGH..


TI LIU THAM KHO
PH LC

54

DANH MC CC BNG
Bng 1.1: Gi tr MIC v MFC ca khng sinh t Streptomyces. albidoflavus ....... ...
10
Bng 1.2: Gi tr MIC ca cc antimycin trn Caenorhabditis elegans v
Artemia salina .................................................................................................... .
11
Bng 2.1: Cc vi sinh vt kim nh ....................................................................
14
Bng 2.2.: Cc MT nui cy VSV kim nh...........

15

Bng 2.3.: Cc dung mi s dng

15

Bng 3.1: Kt qu th HTKS sng lc ngu nhin...............................................


24
Bng 3.2.: Tnh ton t l sng st sau t bin ln th nht

25

Bng 3.3: Kt qu th HTKS t bin ln 1 ........................................................


26
Bng 3.4.: Tnh ton t l sng st sau t bin ln th hai

28

Bng 3.5: Kt qu th HTKS t bin ln 2 ........................................................


28
Bng 3.6: Kt qu chn chng ln men ..............................................................
30
Bng 3.7.: Kt qu chn mi trng ln men...........
Bng 3.8: Kt qu sc k ct ln th nht .......................................... ..

31

33..

Bng 3.9: Kt qu SKLM cc phn on chy ct ln 1 ..................................... .


34
Bng 3.10: Kt qu SKLM cc phn on kho st dung mi tinh ch KS ln 2

36

Bng 3.11: T l cc thnh phn h dung mi Ethylacetat: methanol: n-hexan ....... ..


36
Bng 3.12: Kt qu SKLM vi cc h dung mi Ethylacetat: methanol: n-hexan

37

Bng 3.13: Kt qu th hot tnh KS ca cc phn on sau tinh ch ln 2 ..........


38
Bng 3.14: Kt qu SKLM cc phn on sau tinh ch ln th hai ......................
38
Bng 3.15: Thnh phn cc h dung mi tinh ch khng sinh ln 3 .................... .
40
Bng 3.16.: Kt qu SKLM vi cc h dung mi tinh ch khng sinh ln th 3.

40

Bng 3.17.: Kt qu th HTKS cc phn on sau tinh ch ln 3

41

Bng 3.18.: Kt qu SKLM cc phn on sau tinh ch ln th 3

42

Bng 3.19.: Bin gii cc nhm chc ca KS1 t ph IR

44

Bng 3.20.: Bin gii cc nhm chc ca KS2 t ph IR

45

Bng 3.21.: So snh ph NMR ca KS2 vi khung phenoxazone ca actinomycin D

48

DANH MC CC HNH
Hnh 1.1 : Mt s khng sinh c tng hp t Streptomyces ............... 3
Hnh 1.2 : S ln men tng hp khng sinh ...................................... 4
Hnh P1 : Ph UV ca cht khng sinh KS1
Hnh P2 : Ph UV ca cht khng sinh KS2
Hnh P3 : Ph IR khng sinh KS1
Hnh P4 : Ph IR khng sinh KS2
Hnh P5 : Ph khi MS khng sinh KS1
Hnh P6 : Ph khi MS khng sinh KS2
Hnh P7 : Ph 1H NMR ca khng sinh KS2
Hnh P7a, b, c: Ph 1H NMR gin ca khng sinh KS2
Hnh P8 : Ph 13C NMR ca khng sinh KS2
Hnh P8a, b, c : Ph 13C NMR gin ca khng sinh KS2
Hnh P9 : Ph COSY ca khng sinh KS2
Hnh P9a, b, c : Ph COSY gin ca khng sinh KS2
Hnh P10 : Ph DEPT ca khng sinh KS2
Hnh P10a, b : Ph DEPT gin ca khng sinh KS2
Hnh P11 : Ph HSQC ca khng sinh KS2
Hnh P11a, b : Ph HSQC gin ca khng sinh KS2
Hnh P12 : Ph HMBC ca khng sinh KS2
Hnh P12a, b, c, d : Ph HMBC gin ca khng sinh KS2

T VN
Vi tnh cht l mt nc c kh hu nhit i m gi ma, Vit Nam nm trong
s cc nc c nhu cu s dng khng sinh rt cao. Tuy nhin, trn th trng dc
phm nc ta hin nay, hu ht cc thuc khng sinh u c nhp khu dng thnh
phm hoc bn thnh phm, ngnh cng nghip sn xut khng sinh cha thc s hnh
thnh. V vy, n Quy hoch chi tit pht trin cng nghip Dc Vit Nam giai
on n nm 2020 v tm nhn n nm 2030 xc nh y mnh vic sn xut
nguyn liu khng sinh th h mi p ng nhu cu v nguyn liu sn xut khng
sinh trong nc [4].
Tng hp ho hc, bn tng hp v sinh tng hp l ba hng pht hin v
tng hp khng sinh. Trong sinh tng hp khng sinh vi nhiu u im c s
dng ch yu tm ra khng sinh mi.
Trong tt c khng sinh c bit n hin nay, c ti 55% khng sinh c
phn lp t x khun, 75% trong s t chi Streptomyces [8, 23]. B mn Vi sinh
Sinh hc trng i hc Dc H Ni u t nghin cu v pht hin nhiu chng
x khun phn lp t t, bn, c kh nng sinh tng hp khng sinh, trong c
chng x khun Streptomyces 173.113. Bc u nghin cu cho thy Streptomyces
173.113 c kh nng sinh tng hp khng sinh ph rng, tuy nhin hiu sut tng hp
cng nh hiu sut tinh ch cha cao, cha bin gii c cu trc ha hc ca khng
sinh thu c [7]. Bi vy, chng ti la chn ti Nghin cu nng cao kh nng
sinh tng hp khng sinh t Streptomyces 173.113 lm lun vn thc s vi cc mc
tiu c th nh sau:
1. Chn lc, ci to ging nng cao hiu sut ln men sinh tng hp khng
sinh.
2. Nng cao kh nng ln men, tch chit, tinh ch khng sinh.
3. Bc u nghin cu cu trc khng sinh chnh thu c.

CHNG 1. TNG QUAN


1.1. i cng v Streptomyces
1.1.1. c im ca x khun chi Streptomyces
Cc loi x khun thuc Streptomyces c kh nng to ra nhiu khng sinh
c cu trc phc tp. Trong tng s cc khng sinh c tm thy do x khun
tng hp th c ti 75% l t Streptomyces [8, 23].
- c im hnh thi: h si ca x khun gm c khun ty c cht v
khun ty kh sinh:
+ Khun ty c cht: mc su vo mi trng nui cy, khng phn ct
trong sut qu trnh pht trin, b mt nhn hoc sn si, c th tit ra mi
trng mt s loi sc t, c sc t tan trong nc, c sc t ch tan trong dung
mi hu c.
+ Khun ty kh sinh: do khun ty c cht pht trin di ra trong khng kh.
Sau mt thi gian pht trin trn nh khun ty kh sinh s xut hin cc chui
bo t.
+ Chui bo t (si bo t) c rt nhiu hnh dng khc nhau: thng, sng,
mc cu, xon Chui bo t phn chia to thnh cc bo t trn, l c quan
sinh sn ch yu ca x khun. B mt bo t c th c dng trn nhn (sm), x
x da cc (wa), c gai (sp) hoc c tc (sp).
- c im sinh l: Streptomyces l sinh vt d dng, c tnh oxi ha cao.
pht trin, chng phn gii cc hydratcarbon lm ngun cung cp vt cht v
nng lng, ng thi thy phn cc hp cht nh gelatin, casein, kh nitrat
thnh nitrit. Streptomyces l loi x khun h hp hiu kh. Nhit ti thch ca
chng l 25-30C, pH ti thch thng l 6,8-7,5.

- Kh nng to sc t: Sc t to thnh t Streptomyces c chia lm 4


loi: sc t ha tan, sc t ca khun ty c cht, sc t ca khun ty kh sinh, sc
t melanoid [1, 3, 5, 27].
1.1.2. Kh nng sinh tng hp khng sinh ca Streptomyces
Trong s cc khng sinh do x khun tng hp c bit n hin nay th
phn ln c tng hp t Streptomyces. Cc cht khng sinh c tng hp t
x khun bao gm cc cht chng nm, cc cht khng sinh, v ha cht tr liu
ha hc trong ung th. Hnh 1 di y gii thiu mt s khng sinh, trong
phn ln l do Streptomyces tng hp [2, 8, 23].

Hnh 1.1: Mt s khng sinh c tng hp t Streptomyces


3

1.1.3. S tng qut sinh tng hp khng sinh t x khun


Cc bc c bn trong qu trnh ln men sn xut khng sinh t x khun c
th hin trong hnh 1.2 di y.
Ging truyn trong phng th nghim
Bnh nhn ging trong phng th nghim
Bnh nhn ging trn thit b nhn ging
Bnh ln men to khng sinh
Dch ln men
Sinh khi

Dch lc

Sinh khi th

Dch chit khng sinh

Dch chit sinh khi

Dch chit m c

Sn phm tinh ch

Sn phm tinh ch

Sn phm kim nghim


Sn phm c ng gi
Hnh 1.2: S tng qut ln men sn xut khng sinh

1.1.4. Nhng im lu khi nghin cu cc cht khng sinh t x khun


X khun l vi khun tht thuc nhm c th nhn s, nhng pht trin
thnh h si v thng sinh sn bng bo t. Ging nh nm si, x khun c
kh nng pht trin tt trn mi trng xp. Hin nay, do iu kin k thut m
bo mi trng nui v trng, ngi ta rt quan tm n vic nghin cu pht
trin phng php ln men trn mi trng xp sinh tng hp enzyme, cc
cht khng sinh v cc cht c hot tnh sinh hc khc.
X khun c cu to ging vi khun, nhng thi gian sinh trng pht
trin chm hn vi khun. Do , thi gian sinh trng, pht trin v sinh tng
hp cc cht khng sinh thng ko di n 96 -120 gi ln men.
Hu ht cc chng x khun pht trin tt trong mi trng trung tnh v
nhit ti u l 28-300C. Hn na, sau giai on sinh trng, pht trin, x
khun thng chuyn sang giai on t phn, do trong qu trnh sn xut phi
lu n thi gian thu nhn sn phm. [8]
1.2. Bc u nghin cu ln men sinh tng hp khng sinh t
Streptomyces 173.113
Streptomyces 173.113 c b mn Vi sinh Sinh hc trng H Dc
H Ni phn lp t t Ty Nguyn. y l mt chng x khun n nh, c kh
nng tng hp khng sinh ph rng trn c vi khun Gram dng v Gram m,
c tim nng trong vic ng dng trong lnh vc phng v cha bnh. Tuy nhin,
hot tnh khng sinh cha cao, kh nng sinh tng hp khng sinh cn thp, do
cn phi tin hnh ci to ging la chn chng c kh nng sinh tng hp
khng sinh cao v nghin cu phng php tinh ch khng sinh thu c hiu
sut cao nht.
Qua sng lc ngu nhin, v t bin bng UV v tc nhn ha hc thu
c bin chng B3.04 l bin chng c hot tnh khng sinh cao nht. T l
5

sng st sau 3 ln t bin ca VSV l 0,15%. Sau ln men chm bin chng
B3.04 to khng sinh. Mi trng ln men thch hp nht l MT1dt. Lng
khng sinh th thu c t dch ln men l 0,1033 g/l.
Tch chit khng sinh:
Sau khi ln men, dch lc ln men c chit bng ethylacetat pH 5. Dch
chit dung mi hu c c em ct quay chn khng thu khng sinh th.
Qu trnh tch cc thnh phn trong hn hp khng sinh th cho thy hn
hp khng sinh c t nht 3 thnh phn. Cc thnh phn chnh c tinh ch
bng sc k ct Silicagel, vi hiu sut tinh ch cui cng khong 33,48%
Mt s tnh cht l ha khng sinh chnh thu c:
Nhit nng chy : 217,40C.
Ph t ngoi: cho cc nh hp th : 204 nm, 333 nm v 443 nm. T ,
d on cu trc KS c nhn thm, ni i lin hp, d t, hoc kt hp cc c
im trn.
Ph khi lng phn t: d on phn t lng l 1268, 49 vC [7].
Nh vy, nghin cu bc u tin hnh sng lc, t bin thu c
bin chng kh nng sinh tng hp khng sinh cao hn, v tch chit c
thnh phn khng sinh chnh trong qu trnh ln men. Tuy nhin, hiu sut sinh
tng hp khng sinh cha cao, qu trnh tinh ch khng sinh cha xc nh c
khng sinh tinh khit hay cha, do cn tip tc nghin cu nng cao kh
nng sinh tng hp, hiu sut qu trnh tinh ch, v tinh khit ca khng sinh
chnh thu c.
1.3. Nghin cu tng hp Actinomycin X2 t Streptomyces 15.29
Streptomyces microflavus
Streptomyces 15.29 l chng x khun c phn lp t t Vit Nam,
ti phng th nghim vi sinh, b mn Vi sinh sinh hc trng H Dc H Ni.
6

Tin hnh phn loi Streptomyces 15.29 theo ISP th thy 92,68% cc c im
trng khp vi Streptomyces sinensis. Tuy nhin, khi gii trnh t gen 16s rADN
bng k thut khuch i gen PCR thy h gen ca Streptomyces 15.29 hon
ton trng vi gen ca Streptomyces microflavus. Do kt lun tn khoa hc
ca Streptomyces 15.29 l Streptomyces microflavus.
Tin hnh ln men Streptomyces microflavus thu dch ln men sau chit
bng ethylacetat. Dch chit ethylacetat c ct chn khng thu c cn (5g),
sau tin hnh cc phng php tinh ch bng sc k lp mng v sc k ct
silica gel thu c cht khng sinh chnh 1(1g).
Khng sinh chnh 1 c xc nh cc thng s v c tnh l ha v cng
thc phn t, cng thc cu to.
Khng sinh chnh 1 : Cht rn c mu hng sm, Ph khi lng ESI-MS
m/z 1292 vC [M+Na]+ C62H84N12O17.
Nhit nng chy: 249 - 250oC.
quay cc: []22D -360o (c, 0,5 trong MeOH).
Tin hnh o ph cng hng t ht nhn v bin gii ph, tc gi xc
nh c hp cht 1 l actinomycin X2, mt hp cht cng c phn lp t
chng Streptomyces spp. v c hot tnh chng ung th cng nh hot tnh khng
sinh rt mnh [16, 17, 20].
1.4. Cng ngh ln men Daunorubicin v Adriamycin.
Daunorubicin v Adriamycin l cc khng sinh c hot tnh chng ung th
t cc x khun chi Streptomyces. Daunorubicin c sinh tng hp t chng S.
peucetius v S.coeruleorubidus c c cao nn ngy nay khng c s dng
iu tr ung th, thay vo ngi ta nghin cu ra Adriamycin l cht khng
sinh tng hp t bin chng ca S. peucetius c hot tnh cao hn v ph iu tr
ung th rng hn, t c tnh hn. Ngoi ra, mt nhm nghin cu Lin X
7

cng thu c mt dn xut tng t khc l carmynomycin sinh tng hp t


x khun Actinomadura carminata.
Qu trnh ln men sn xut Adriamycin nh sau: Chng S. peucetius c
t bin bng ha cht thu bin chng. Bin chng c nui cy trong ng
thch nghing 26-270C, trong khong 10 ngy, sau loi b h si nm c
mu m cy chuyn sang mi trng bnh tam gic 2 lt v nui trn my lc
tc 120 v/pht, 280C trong 2 ngy. Sau cy sang thit b nhn ging th
tch 170 lt ( cha 120 lt mi trng), nhn ging 26-270C trong 27 gi, ri
cy truyn tip sang thng ln men 800 lt ( cha 500 lt MT). Qu trnh ln men
ko di khong 145 gi.
Sn phm adriamycin tn ti trong c mi trng ln h si, v vy ngi
ta phi x l c phn sinh khi v phn dch trong thu sn phm. u tin,
dch ln men c lc tch ring dch trong v b lc. Phn b lc c ngm
x l trong hn hp dung mi aceton: acid sulfuric 0,1N t l 4:1, sau lc
tch b. Phn dch c trung ha v pH 7, v c c chn khng xung nng
Adriamycin khong 250mg/l. Dch c c c loi lipid bng trch ly vi dung
mi chloroform pH 3. Pha nc c kim ha bng NaOH v pH 8,6 ri trch
ly bng h dung mi Chloroform Methanol. Dung mi trch ly loi nc c
lm kh bng natri sulfate khan, lc, c c, cui cng b sung ether vo kt
ta thu adriamycin th. Phn dch trong thu c sau khi lc dch ln men cng
c x l qua cc cng on tng t thu bt adriamycin th. Bt th c ha
tan li trong nc tch qua ct cellulose, ra ct bng m phosphate pH 4,5
v n-butanol. Dch thu gom cc phn on sn phm c c c chn khng ri
trch ly sang chloroform ( t l dung mi 4:1). Bc tip theo l phn tly thu pha
dung mi, lm kh bng Natri sulphat khan, c c chn khng ri b sung dung
dch HCl trong methanol v lm lnh kt tinh thu kt ta. Cui cng, ha tan
8

v kt tinh li kt ta trong ethanol thu sn phm Adriamycin hydrochlorid


tinh sch.[2, 8].
1.5. Tng hp cirramycin B t Streptomyces cyaneus
Atta v cng s tin hnh nghin cu ln men sinh tng hp v tinh ch
cirramycin B t S. Cyaneus
Sau khi ln men, dch lc ln men c chit xut bng Ethylacetat t l
1:1. Loi dung mi bng ct chn khng di p sut gim thu c cn, cn
c ha tan trong lng ti thiu DMSO, sau c lc. Cc thnh phn
khng sinh c tch bng sc k lp mng vi h dung mi chy sc k l
Chloroform: Methanol ( 24:1). C t nht 3 thnh phn trong hn hp khng
sinh, trong thnh phn c Rf = 0,4 l thnh phn chnh.
Tinh ch khng sinh chnh bng sc k ct silicagel vi dung mi chy ct
l Chloroform: methanol (9:1).
Khng sinh chnh thu c c nhit nng chy l 228 0C, ha tan khng
gii hn trong CHCl3, ethylacetat, n butanol, aceton, ethanol, methanol, isopropanol, khng tan trong du m, hexan, benzene. o ph UV, bc sng hp
thu cc i l 240nm, ph IR c 11 nh hp th. o ph khi xc nh khi
lng phn t l 679,4 vC, cng thc phn t l C36H59NO12.
Tin hnh phn tch ph cng hng t ht nhn v bin gii ph xc
nh c khng sinh chnh l cirramycin mt khng sinh th h II nhm
macrolide, c kh nng c ch c vi khun Gr + v Gr-[20].
1.6. Khng sinh chng nm non-polyen t Streptomyces albidoflavus
Khng sinh chng nm ch chim mt t l rt nh trong cc nhm thuc
hin nay , tuy nhin li ng vai tr rt quan trng trong vic kim sot cc bnh
v nm.

S K. Augustine v cng s tin hnh nghin cu tng hp mt khng


sinh chng nm non polyen t Streptomyces albidoflavus PU23.
Lng khng sinh thu c t Streptomyces albidoflavus PU23 cao nht
l vo ngy th 8 ca qu trnh nui cy. Dch ln men c chit bng n-hexan,
sau bc hi thu hi dung mi thu c khng sinh th. Cc thnh phn trong
khng sinh th ny c phn tch bng sc k lp mng vi h dung mi l
ethanol: nc: chloroform (2:2:1). Dng sc k ct vi h dung mi l ethanol:
nc ( 50:50) tinh ch thu khng sinh tinh khit chnh.
Da vo phn tch ph FTIR, ph cng hng t ht nhn d on cng
thc phn t ca khng sinh chnh l C16H32O19, c mt nhm hydroxymethyl,
mt ni i, cn li l cc lin kt CHOH-. Nh vy khng sinh chnh c cng
thc cu to kiu chui polyhydroxy polyether mch thng, c mt lin kt i,
do thuc nhm chng nm non-polyen [18].
V hot tnh sinh hc ca khng sinh chnh i vi cc chng nm c
th hin bng di y:
Bng 1.1 : Gi tr MIC v MFC ca khng sinh t S.albidoflavus.

1.7. Tng hp khng sinh antimycin A9 t Streptomyces Sp. K01-0031


10

Chng K01-0031 c phn lp t mu t trn ni ti Chuxiong,


Yunnan, Trung Quc. Qua phn loi ISP, xc nh c l thuc nhm x khun
Streptomyces.
Tin hnh ln men chng x khun trn, dch ln men c chit bng
ethylacetat, sau ct thu hi dung mi thu c cn. Tin hnh sc k ct hn
hp khng sinh th, thu c hn hp khng sinh nhm antimycin. Hn hp ny
c chy qua sc k lng hiu nng cao HPLC, dung mi ra gii l CH 3CN
70% thu c khng sinh tinh khit chnh l antimycin A9 (1). Bn cnh cn
thu c antimycin A3a (2), A3b (3), A4 (4), A7 (5).
Antimycin A9 l khng sinh chnh, n tan c trong MeOH, aceton,
acetonitril, ethyl acetat v chloroform, khng tan trong nc v n-hexan. Cng
thc ca antimycin A9 c xc nh l C29H34N2O9 . Ph IR ca antimycin A9
tng t nh ph ca mt s antimycin bit. Phn tch ph

H,

13

C NMR

thy cng thc cu to ca (1) c mt vng dilactone, mt nhm bn 3formamidosalicyl, mt chui n-butyl. Nhng c im ny ging vi cng thc
cu to ca cc antimycin khc. Tuy nhin, (1) l cht duy nht c nhm
phenylacetyl gn vi C8 [25].
V kh nng khng khun, nghin cu nh gi trn hai chng vi khun
Caenorhabditis elegans, v Artemia salina. Kt qu c th hin trn bng sau:
Bng 1.2: Gi tr MIC ca cc antimycin trn Caenorhabditis elegans, v
Artemia salina
Hp cht

Caenorhabditis elegans

Artemia salina

Antimycin A9

0.2

0.05

Antimycin A3a

0.2

Antimycin A3b

0.2

11

Antimycin A4

0.2

Antimycin A7

>1

0.2

1.8. Sparsomycin khng sinh c tng hp t Streptomyces Sp. AZNIOFD1


Atta v cng s tin hnh phn lp mt chng x khun Streptomyces
sp. AZ-NIOFD1 t nc sng Nin-Ai Cp, sau tin hnh ln men, chit xut
v tinh ch thu c mt hp cht c hot tnh khng sinh.
Tin hnh gii trnh t gen 16s rADN ng dng k thut khuch i gen
PCR nhn thy Streptomyces sp. AZ-NIOFD1 tng t Streptomyces
violaceusniger , AZ-NIOFD1.
Dch ln men c chit bng n-butanol pH 7, ct chn khng loi dung
mi. S dng sc k lp mng tch cc thnh phn trong hn hp khng sinh
th, dung mi l chloroform : methanol (24:1), ch cht c Rf = 0.75 l c hot
tnh khng sinh. Tin hnh tinh ch bng sc k ct silica gel vi h dung mi
chy ct l chloroform : methanol (8:2), phn on t 23 n 30 l c hot tnh
khng sinh mnh nht.
Khng sinh chnh tinh khit c xc nh cc thng s ha l v cu
trc. Khng sinh chnh tan c trong chloroform, n-buthanol, acetone,CCl4,
methanol, ethanol, DMSO, khng tan trong du ha, hexan, v benzen. Nhit
nng chy l 2050C. Cng thc phn t l C13H21N3O6S2, phn t lng l 361.3
vC, ph IR c 23 nh hp th. Ph UV hp th cc i 270 v 302 nm. Phn
tch ph cng hng t kt hp vi cc thng s trn c th kt lun khng sinh
chnh sinh tng hp t Streptomyces Sp. AZ-NIOFD1 l sparsomycin.

12

Nghin cu hot tnh sinh hc ca khng sinh ny thy n c kh nng c


ch Gram dng ( MIC dao ng t 11,7 n 31,25 g/ml), Gram m ( MIC dao
ng t 11,7 n 15,6 g/ml) [19].

13

CHNG 2. I TNG V PHNG PHP NGHIN CU


2.1. Nguyn vt liu v thit b
2.1.1. Nguyn vt liu
Chng x khun: chng Streptomyces 173.113 do B mn Vi sinh Sinh
hc trng i hc Dc H Ni phn lp t mu t ti Ty Nguyn.
Chng vi sinh vt kim nh: do B mn Vi sinh Sinh hc cung cp, c
trnh by bng 2.1.
Bng 2.1: Cc vi khun kim nh
Vi khun Gram(+)
Vi khun Gram(-)
Staphylococcus aureus ATCC 1228

Proteus mirabilis BV 108

Mi trng
Cc mi trng nui cy x khun: Theo nghin cu trc [7], chng ti
la chn MT1 lm mi trng nui cy x khun vi thnh phn trong 100 ml
mi trng nh sau: Tinh bt 2g, KNO3 0,1 g, K2HPO4 0,05g, MgSO4.7H2O 0,5
g, NaCl 0,5 g, thch 1,8 g, pH 7.
Cc MT ln men (MTdt): C thnh phn tng ng MT nui cy x khun
nhng loi b thch.
Cc MT nui cy VSV kim nh: thnh phn cc MT c trnh by
bng 2.2.
Ch : Cc MT nui cy x khun, MT ln men v MT nui cy VSV kim nh
u c tit trng 118-120C trong 30 pht

14

Bng 2.2: Cc MT nui cy VSV kim nh


NaCl

Cao tht

Pepton

Thch

MT canh thang

0,5

0,3

0,5

MT thch thng

0,5

0,3

0,5

1,8

Thnh phn

pH

7,0-7,4

Dung mi
Bng 2.4 gii thiu cc dung mi c s dng trong nghin cu cng cc
c trng c bn ca chng.
Bng 2.3: Cc dung mi s dng
Dung mi

Khi lng ring (g/ml)

Nhit si (C)

Aceton

0,79

56

Ethanol

0,789-0,791

78,3

Ethylacetat

0,889-0,901

70,4

Methanol

0,791-0,793

88,6-88,8

Triethylamin

0,726-0,728

90

n-hexan

0,655

68,7

n-propanol

0,803

97-98

Vt liu chy sc k
- Bn mng sc k: Silicagel 60 F254, Merck.
- Dung mi chy sc k: Cc dung mi trong bng 5 trn
- Bnh chy sc k, buret, Silicagel 60 F254 ht c ng knh 0,04-0,063
mm

15

2.1.2. My mc thit b
- Tc nhn gy t bin: nh sng UV (=254nm), ngun pht Toshiba
60W, in th 220V.
- My lc Taitec Bio Shaker BR 300 Lf
- T m Memmert, Binder
- T cy v trng Aura VF 48
- T sy SL shellab
- T lnh Sanyo
- Ni hp v trng Hirayama Model HL3030
- Cn k thut Sartorius TE 210
- Cn phn tch Sartorius BP 121 S
- My o nhit nng chy E2-Melt
- My ct quay Buchi Waterbath B480
- Thc kp Palmer chnh xc 0,02mm
- Hp Petri ng knh 9cm
- Buret, bnh chit, patuyn, pipet cc loi, ng nghim, bnh nn, ng
ong, cc c m cc loi, nt thy tinh, bng gc, giy lc, que cy, giy th pH,
n cn, a thy tinh, kim mi mc,
2.2. Phng php thc nghim
2.2.1. Nui cy v gi ging x khun
- Cy zigzac x khun trn ng thch nghing c cha MT nui cy thch
hp, cho pht trin 28-29C.
- Sau 6-10 ngy, x khun pht trin tt, cho cc ng ging vo t lnh
bo qun 2-3C, nh k 3-6 thng cy chuyn.

16

2.2.2. nh gi hot tnh khng sinh bng phng php khuch tn


- Nguyn tc: Mu th c t trn lp thch dinh dng cy VSV
kim nh, khng sinh t mu th khuch tn vo MT thch s c ch s pht
trin ca VSV kim nh to thnh vng v khun.
- Tin hnh:
+ To hn dch VSV: Ly mt vng que cy VSV kim nh cy vo 2.5
ml MT canh thang, 37C trong 18-24h (i vi vi khun) to thnh hn
dch VSV c nng 106-108 t bo/ml.
+ Cy hn dch VSV vo mi trng thch thng tit trng v
ngui n 45-50C vi t l 2,5:100 (105 t bo/1ml), lc u, vo cc a
Petri v trng vi th tch 20ml/a.
+ a mu th vo mi trng kim nh theo 1 trong 3 cch sau:
Phng php khoanh giy lc: Khoanh giy lc (=6,0mm)
c tm 3 ln dch chit dung mi hu c ca mu th, sy kh nhit di
500C, sau t ln b mt mi trng cy VSV kim nh.
Phng php khi thch: t khi thch (=6,0mm) c cha VSV
sinh khng sinh ln b mt MT cy VSV kim nh.
Phng php ging thch: c cc ging thch (=6,0mm) trn
MT cy VSV kim nh. Nh 0,05ml dch th vo mi ging.
+ Cc a Petri c mu th c trong t m 37C trong 18-24h (i
vi vi khun). Mi mu c th song song trn 3 a vi cng 1 loi VSV kim
nh.
+ nh gi kt qu: o ng knh vng v khun bng thc kp Palmer
c chnh xc 0,02mm. Kt qu c nh gi da trn ng knh vng v
khun x l theo cng thc:

17

Di

i 1

( D D)

i 1

n 1

Trong :
D (mm): ng knh trung bnh vng v khun

Di (mm): ng knh vng v khun th i

s: lch thc nghim chun c hiu chnh


n: S thc nghim tin hnh song song (Thng thng n=3)
2.2.3. Sng lc ngu nhin
- Pha hn dch bo t: Ly mt vng que cy bo t Streptomyces
173.113, cho vo ng nghim cha 10ml nc ct v trng, lc cho bo t phn
tn u, thu c hn dch bo t pha long 10 -1 (nh c). Sau , pha
long tng t n nng 10-6 bng nc v trng.
- Cy bo t: Dng pipet v trng ht chnh xc 0,1ml hn dch bo t
cc pha long 10-4, 10-5, 10-6, nh ln b mt thch ca MT2 trong a Petri.
Dng que trang v trng dn u hn dch bo t ln b mt thch. Tin hnh
trn 3 a Petri cho mi nng pha long. Cho cc a vo t m 28C trong 6
ngy cho xut hin cc khun lc mc ring r.
- Php chn lc ngu nhin: Chn ngu nhin cc khun lc pht trin c
th, mc ring r sau 6 ngy ri ly ra cy th HTKS. Cc khun lc ny, sau khi
nui cy 6 ngy c th hot tnh khng sinh bng phng php khi thch.
2.2.4. t bin bng nh sng UV
- Pha hn dch bo t: Ly mt vng que cy bo t Streptomyces
173.113, cho vo ng nghim cha 10ml nc ct v trng, lc cho bo t phn
tn u, thu c hn dch bo t pha long 10 -1 (nh c). Sau dung
18

1ml hn dch bo t ny pha long n nng 10 -6 lm mu chng, 9ml cn li


c a vo a Petri v trng.
- t bin di tc dng ca nh sng UV: 9ml hn dch bo t nng
10-1 ng trong a Petri c chiu nh sng UV (=254nm) vi khong cch
v thi gian thch hp (khong cch 60cm, thi gian 5 pht), sau ly ra,
ch ti 2h, ri dng pipet v trng pha long n nng 10 -5 bng nc v
trng.
- Cy cc bo t sau t bin: Dng pipet v trng ht chnh xc 0,1ml
mu chng 10-6 v 0,1ml hn dch t bin cc nng 10 -4 v 10-5 vo cc
a Petri c cha MT1, sau dng que trang v trng dn u git hn dch bo
t trn b mt thch, mi pha long lm 3 a song song. Cc a Petri sau khi
cy c nhit 28C trong 6 ngy n khi xut hin khun lc. m cc
khun lc, xc nh % sng st theo cng thc:
% sng st =
Trong :

Nm
x 10-6+k x 100%
No

Nm: S khun lc sau t bin nng 10-k


No: S khun lc trong mu chng nng 10-6.

- Tin hnh sng lc vi cc khun lc sau khi t bin tng t nh


phng php sng lc ngu nhin. Lm song song mu chng. Phn trm bin
i hot tnh c xc nh theo cng thc:
% bin i hot tnh =

Di
100%
Do

Trong :
D i : ng knh trung bnh vng v khun ca bin chng th i sau t

bin
D o : ng knh trung bnh vng v khun ca mu chng.

19

- Sau khi tin hnh t bin, da vo kt qu thu c la chn ra cc


bin chng c % bin i hot tnh (+) cao nht dng cho cc nghin cu tip
theo.
2.2.5. Ln men chm tng hp khng sinh
- Nguyn tc: Sau khi c kt qu xc nh c cc mi trng nui cy
b mt tt nht cho vic sn xut khng sinh, s dng ging cp 1 ln men
gin on trong cc mi trng lng tng ng.
- Tin hnh
+ To ging cp 1: Chng ging Streptomyces 173.113 sau khi nui cy
trn thch nghing (MT1) 6 ngy tui c cy v trng sang bnh nn 500ml
cha 100ml MT1dt bng 10ml nc v trng, lc trn my lc trn nhit
28C 0,1C, tc quay 140 vng/pht trong 48h.
+ Ln men: Sau 48h nhn ging, tin hnh ln men trn mt s MT khc
nhau chn MT ln men tt nht. Ging cp 1 c cy v trng sang bnh nn
500ml cha cc MT dinh dng vi t l Vging:Vmi trng = 1:10. Cc bnh ln
men c lc nhit 28C 0,1C, tc quay 140 vng/pht trong 120h.
chn chng c kh nng ln men sinh tng hp khng sinh tt nht:
ging cp 1 trn MT1dt c ln men trn mi trng ti thch cho vic sinh
tng hp khng sinh, sau la chn chng cho dch ln men c HTKS tt nht.
2.2.6. Chit khng sinh t dch ln men bng dung mi hu c
- Mc ch: Xc nh dung mi v pH chit ti u cho dch lc.
- Tin hnh: Dng phng php chit 1 ln.
+ Dch lc c chnh v cc pH 3, 5, 7, 9, 11 bng dung dch NaOH 1M
v HCl 1M.

20

+ Chit dch lc chnh pH vi nhiu dung mi khc nhau: Dch lc v


dung mi cho vo bnh gn theo t l Vdung mi:Vdch lc = 1:5. Sau lc k trong
1 pht. yn 1h cho phn lp. Tch ring ly lp dung mi v dch lc.
+ Sau mi ln chit, ly lp dung mi v lp dch lc th hot tnh khng
sinh theo phng php khoanh giy.
2.2.7. Tch cc thnh phn trong khng sinh bng sc k lp mng
- Mc ch: Xc nh cc thnh phn khng sinh trong dch chit, dch lc
hoc xc nh khng sinh tinh khit hay cha.
- Tin hnh:
+ Ct bn mng c kch thc thch hp, hot ha 110C/30 pht.
+ Pha hn hp dung mi theo ng t l, cho vo bnh sc k (lp dung
mi khng qu 1cm), yn bnh khong 1h bo ha dung mi.
+ Dng ng mao qun ng knh 0,5mm chm dung dch cn sc k ln
bn mng. Chm nhiu ln, kh t nhin hoc sy 50C.
+ t bn mng vo bnh sc k. Cho dung mi chy ln khong chiu
di bn mng th ly ra, nh du ng dung mi chy.
+ Xc nh vt theo cc phng php sau:
Pht hin bng mt thng.
Soi n t ngoi: t bn mng sc k vo n t ngoi, vt cht
th s hin mu.
Phng php hin hnh VSV: t bn mng vo a Petri tit
trng, mt lp va phi MT thch thng cy VSV kim nh sao cho bn
mng chm trong thch. vo t 37C, trong 18-24h, vng v khun xut
hin nu c khng sinh.

21

Phng php hin mu ha hc: Bn mng c phun thuc th


hin mu, sy 70C trong 10 pht hin vt. Dng thuc th ninhydrin 1%
trong cn pht hin cc khng sinh c cha nhm amin.
+ Tnh h s Rf (Retardation factor)
Rf

Trong :

a
b

a: Khong cch t vch xut pht n tm ca vt hat cht


b: Khong cch t vch xut pht n vch dung mi.

2.2.8. Thu khng sinh th bng phng php ct quay chn khng
Pha dung mi hu c thu c sau khi chit c ct quay bng my ct
chn khng p sut -0,09 atm. Cn thu c c ha tan bng mt lng nh
methanol hoc aceton, vo bnh nn sch, bc hi 50C n kh. Co, thu
ly bt khng sinh th.
2.2.9. Tinh ch khng sinh th bng sc k ct
- Pha h dung mi chy theo ng t l, 30 pht cho bo ha.
- Chun b ct sc k: Ct c ng knh 1cm, di 50cm c ra sch,
trng cn, sy kh. Cn khong 8g ht Silicagel nhi ct, hot ha 110C trong
30 pht, em ha thnh hn dch vi dung mi chy v a ln ct.
- a mu th ln ct: Bt khng sinh th ha tan trong mt lng ti
thiu methanol ri trn vi khong 2g Silicagel hot ha. Sy 50C cho bay
ht methanol ri cho ln ct, n nh ct trong 30 pht.
- Cho dung mi chy qua ct vi tc 0,5ml/pht. Ly cc phn on
vo ng nghim sch, khong 30 phn on, mi phn on 5ml.
- Pht hin v xc nh cc phn on cha khng sinh cn tch: Th
HTKS ca cc phn on bng phng php khoanh giy lc. Cc phn on c
22

HTKS c tin hnh sc k lp mng vi h dung mi tch tt nht. Cc phn


on chnh l cc phn on t khi xut hin vt n khi khng cn vt c R f
tng ng v vn c hot tnh khng sinh mnh.
- Cc phn on chnh sau khi chy ct c thu vo bnh cu 250 ml, ct
quay chn khng loi dung mi thu c cn. Co thu bt tinh th khng sinh
vo ng nghim sch. Sau , bt tinh th khng sinh c kt tinh li bng hn
hp dung mi, lc, sy kh 50C thu khng sinh tinh khit.
2.2.10. Xc nh tnh cht, cu trc ha hc thnh khng sinh chnh tinh
khit thu c
- o nhit nng chy.
- Ph hng ngoi.
- Ph t ngoi.
- Ph khi MS.
- Ph cng hng t ht nhn: 1H NMR, 13C NMR, DEPT, COSY, HSQC,
HMBC.

23

CHNG 3: KT QU THC NGHIM V NHN XT


3.1. Chn lc v ci to ging x khun
3.1.1. Kt qu sng lc ngu nhin
K tha kt qu ca nghin cu trc [7] chng ti la chn vi khun
Staphylococcus aureus v Proteus mirabilis l 2 chng VSV kim nh i din
thc hin cc nghin cu v sau. Tin hnh sng lc ngu nhin trn chng
Streptomyces 173.113. B3.04 l chng tt nht t nghin cu trc ang c
lu gi ti b mn Vi sinh sinh hc, trng H Dc H Ni.
* Mc ch:
Chn dng chng c HTKS cao trong qun th.
* Tin hnh v kt qu:
Chng Streptomyces 173.113.B3.04 c SLNN, gi li 32 dng chng,
th HTKS bng phng php khi thch. Kt qu c th hin trn bng 3.1.
Bng 3.1: Kt qu th HTKS sng lc ngu nhin
Dng
chng
SL.01
SL.02
SL.03
SL.04
SL.05
SL.06
SL.07
SL.08
SL.09
SL.11
SL.12
SL.13

Hot tnh khng sinh


P. mirabilis
S. aureus
D

(mm)
17,97
19,23
16,02
16,64
18,02
17,04
18,63
18,76
19,35
16,11
18,66
19,39

Dng
chng

s
1,23
0,51
0,97
1,17
0,53
0,82
0,44
0,52
0,55
2,24
0,63
0,55

(mm)
20,05
21,45
20,46
20,73
19,97
20,27
21,29
21,01
21,37
20,50
21,18
21,51

S
0,39
0,64
1,41
0,88
1,10
1,63
0,77
0,40
0,23
0,40
0,45
0,26
24

SL.17
SL.18
SL.19
SL.20
SL.21
SL.22
SL.23
SL.24
SL.26
SL.27
SL.28
SL.29

Hot tnh khng sinh


P. mirabilis
S. aureus
D

(mm)
14,59
17,42
16,87
16,07
16,20
14,45
16,61
15,27
17,95
18,29
18,95
18,65

S
1,19
0,41
1,98
1,16
0,59
0,97
1,02
0,94
0,36
0,64
0,99
0,47

(mm)
19,31
19,50
20,65
19,31
18,99
19,47
19,55
19,23
19,05
19,69
20,93
20,89

s
0,33
0,37
1,15
0,90
0,80
0,37
1,10
0,50
0,37
1,46
0,93
0,25

SL.14
SL.15
SL.16

14,89 1,60
17,01 0,84
16,33 0,71

19,71 0,39
19,63 0,73
19,13 0,20

SL.30
SL.31
SL.32

17,27 0,49
18,47 0,54
18,29 1,51

19,49 0,73
20,58 0,35
20,45 0,49

* Nhn xt:
Theo bng 3.1 c th thy 3 dng chng SL.02, SL.09 v SL.13 l nhng
dng chng c hot tnh khng sinh cao nht. Do , gi 3 dng chng ny li
cho nghin cu tip theo.
3.1.2. Kt qu t bin ln th nht
* Mc ch:
S dng nh sng UV gy t bin nhm tng hot tnh khng sinh ca
chng x khun thu c sau SLNN.
* Tin hnh v kt qu:
Chn dng chng c HTKS cao nht sau SLNN l SL.13, mang t bin
bng nh sng UV (=254nm), khong cch chiu 60 cm, thi gian 5 pht.
T l sng st l 0,73%, cch tnh c th hin trong bng 3.2.
Bng 3.2. Tnh ton t l sng st sau t bin ln th nht
a

x i (S khun lc trung

bnh)
Sau t bin nng 10-4
Sau t bin nng 10
Chng nng 10

22,67

5,13

6,33

1,53

116,33

5,51

-5

-6

T l sng st

0,73%

Chn v nui cy 32 bin chng, th HTKS bng phng php khi


thch. Kt qu c th hin bng 3.3.

25

Bng 3.3. Kt qu th HTKS sau t bin ln th nht


Hot tnh khng sinh
K hiu
bin
chng

P. mirabilis
D

(mm)

S. aureus

% hot
tnh so vi
MC

(mm)

% hot
tnh so vi
MC

MC

21,02

0,09

100,00

23,08

2,31

100,00

B1.01

20,87

1,11

99,30

22,79

0,96

103,20

B1.02

22,45

0,77

106,82

23,28

0,87

105,43

B1.03

21,21

0,66

100,92

23,21

1,11

105,13

B1.04

23,09

0,05

109,86

24,01

0,93

108,76

B1.05

22,27

0,93

105,96

22,86

0,71

103,53

B1.06

22,25

1,17

105,87

19,52

2,17

88,41

B1.07

19,46

0,75

92,58

22,36

2,13

101,27

B1.08

20,51

1,89

97,56

22,13

0,55

100,24

B1.09

22,10

1,21

105,14

23,12

0,87

104,71

B1.10

19,71

1,67

93,78

20,61

0,68

93,33

B1.11

21,45

0,88

102,06

23,01

0,37

104,23

B1.12

22,39

2,92

106,50

23,75

0,99

107,55

B1.13

23,81

0,37

113,26

24,15

0,26

109,39

B1.14

21,41

0,28

101,84

23,11

0,80

104,68

B1.15

21,87

0,88

104,03

23,75

0,10

107,58

B1.16

22,59

0,12

107,48

21,98

0,64

99,55

B1.17

20,25

0,41

96,35

21,32

0,57

96,56

B1.18

21,87

0,29

104,06

22,88

0,53

103,62

B1.19

22,46

0,61

106,85

20,89

0,85

94,63

B1.20

22,56

1,00

107,33

23,89

0,18

108,18

B1.21

20,45

3,86

97,30

23,29

0,44

105,50

26

B1.22

19,60

1,28

93,24

22,31

1,50

101,03

B1.23

21,16

1,53

100,67

22,61

1,06

102,42

B1.24

18,44

0,96

87,73

20,71

0,73

93,78

B1.25

18,69

0,55

88,90

21,39

0,59

96,86

B1.26

18,93

0,53

90,07

21,21

0,40

96,04

B1.27

20,11

0,85

95,65

20,86

1,07

94,47

B1.28

21,17

0,69

100,73

20,57

1,44

93,18

B1.29

22,10

0,78

105,14

22,87

0,81

103,56

B1.30

23,57

0,29

112,15

24,28

0,18

109,98

B1.31

19,29

0,87

91,79

22,29

0,50

100,94

0,97

104,12

23,91

1,00

108,30

B1.32
21,89
* Nhn xt:

Sau khi t bin bng nh sng UV vi t l sng st l 0,73% th hot


tnh khng sinh ca chng x khun tng ln ng k trn c P. mirabilis v S.
aureus, nht l cc bin chng B1.04, B1.13 v B1.30. Do , tin hnh
nui cy v gi ging 3 bin chng ny. La chn bin chng B1.13 tip
tc t bin ln 2.
3.1.3. Kt qu t bin ln th hai
* Mc ch:
Tip tc s dng nh sng UV gy t bin nhm tng hot tnh khng
sinh ca chng x khun thu c sau t bin ln th nht.
* Tin hnh v kt qu:
Chn dng chng c HTKS cao nht sau t bin ln th nht l B1.13,
mang t bin bng nh sng UV (=254nm), khong cch chiu 60 cm, thi
gian 4 pht.
T l sng st l 0,58%, cch tnh c th hin trong bng 3.4.
27

Chn v nui cy 32 bin chng, th HTKS bng phng php khi


thch. Kt qu c th hin bng 3.4.
Bng 3.4. Tnh ton t l sng st sau t bin ln th hai
a

x i (S khun lc trung bnh)

20,67

9,87

5,33

3,21

127

42,53

Sau t bin nng 10-4


Sau t bin nng 10
Chng nng 10

-5

-6

T l sng st

0,58%

Bng 3.5. Kt qu th HTKS sau t bin ln th hai


Hot tnh khng sinh
K hiu
bin
chng
MC
B2.01
B2.02
B2.03
B2.04
B2.05
B2.06
B2.07
B2.08
B2.09
B2.10
B2.11
B2.12
B2.13
B2.14

P. mirabilis
D

(mm)
22,62
23,01
24,94
23,05
23,09
23,27
22,25
21,13
22,90
23,33
24,56
23,05
22,39
23,81
22,39

s
0,56
0,74
0,77
1,14
0,05
0,60
1,17
2,65
1,54
0,60
0,43
1,50
0,97
0,37
1,11

S. aureus
% hot
tnh so vi
MC
100,00
101,74
110,26
101,92
102,09
102,89
98,38
93,43
101,24
103,15
108,58
101,92
98,97
105,25
98,97
28

(mm)
23,38
23,85
26,03
24,21
24,01
23,95
23,08
22,38
23,42
24,11
25,01
23,59
24,65
24,15
25,16

s
0,58
0,63
0,07
0,96
0,93
0,45
1,92
2,57
0,75
0,79
0,02
0,52
0,34
0,26
0,82

% hot
tnh so vi
MC
100,00
102,02
111,32
103,54
102,71
102,45
98,72
95,72
100,17
103,14
106,96
100,88
105,45
103,31
107,61

B2.15
B2.16
B2.17
B2.18
B2.19
B2.20
B2.21
B2.22
B2.23
B2.24
B2.25
B2.26
B2.27
B2.28
B2.29
B2.30
B2.31
B2.32

23,01
22,59
24,35
22,21
22,95
23,39
22,50
23,34
22,99
21,84
24,60
20,67
21,91
24,37
22,10
23,77
20,97
22,79

0,87
0,12
0,91
0,49
0,95
0,87
0,97
1,80
1,13
2,16
0,63
1,92
1,86
0,46
0,78
0,45
2,98
1,28

101,71
99,88
107,65
98,17
101,44
103,42
99,47
103,18
101,65
96,55
108,75
91,36
96,88
107,75
97,70
105,10
92,72
100,74

23,75
24,48
21,47
22,88
20,93
24,19
23,58
22,59
22,26
20,71
25,35
21,21
20,86
20,57
22,87
24,28
22,29
23,91

0,10
1,56
0,69
0,53
0,90
0,59
0,51
1,86
1,03
0,73
0,37
0,40
1,07
1,44
0,81
0,18
0,50
1,00

101,60
104,70
91,84
97,86
89,51
103,45
100,86
96,64
95,21
88,57
108,41
90,70
89,22
88,00
97,80
103,86
95,32
102,28

* Nhn xt:
Sau khi t bin bng nh sng UV vi t l sng st l 0,58% th hot
tnh khng sinh ca chng x khun tng ln ng k trn c P. mirabilis v S.
aureus, nht l cc bin chng B2.02, B2.10 v B2.25. Do , tin hnh
nui cy v gi ging 3 bin chng ny.
3.2. Xc nh iu kin ln men, chit tch khng sinh tt nht
3.2.1. Kt qu chn chng ln men
K tha kt qu ca nghin cu trc [7], chng ti bit MT1dt l mi
trng ln men chm tt hn cc mi trng cn li i vi chng Streptomyces

29

173.113. Do , tip tc tin hnh chn chng ln men chm tt nht trn
MT1dt.
* Mc ch:
Chn c bin chng (hoc dng chng) c kh nng ln men chm tt
nht t cc bin chng, dng chng c lu gi qua cc th nghim sng lc
ngu nhin, t bin ln 1, ln 2.
* Tin hnh v kt qu:
Thc hin ln men chm ring r cc bin chng ny trong MT1dt. Dch
ln men c lc qua giy lc loi sinh khi ri th HTKS dch lc bng
phng php ging thch. Kt qu c trnh by bng 3.6.
Bng 3.6. Kt qu chn chng ln men
Hot tnh khng sinh
Dng chng, bin chng
P. mirabilis
S. aureus
s
s
D (mm)
D (mm)
SL.02
19,06
0,70
20,83
0,93
SL.09
19,23
0,51
21,63
0,42
SL.13
20,07
0,08
21,31
0,56
B1.04
23,06
0,65
24,49
0,58
B1.13
23,85
0,79
24,81
0,38
B1.30
23,52
0,46
23,53
0,53
B2.02
25,06
0,20
26,36
0,48
B2.10
24,71
0,38
24,37
0,61
B2.25
24,50
0,62
25,61
0,52
* Nhn xt:
Trong 9 dng chng (bin chng) em ln men chm th dch lc ca dch
ln men chng B2.02 c HTKS cao nht. Do , chn chng B2.02 ln
men to khng sinh.

30

3.2.2. Kho st chn mi trng ln men chm


T kt qu ca nghin cu trc [7], thy MT1dt c kh nng ln men
chm tt hn so vi cc mi trng ln men khc. Nay, chng ti tin hnh kho
st nng cao kh nng ln men ca MT1dt bng cch cho thm cao nm men
vi cc t l 0,1%, 0,5%, 1%, 2% c k hiu ln lt l MT1dt.1; MT1dt.2;
MT1dt.3; MT1dt.4 t tm ra t l tt nht lm mi trng ln men chm
cho cc nghin cu v sau.
Kt qu kho st c trnh by bng 3.7.
Bng 3.7. Kt qu chn mi trng ln men
Hot tnh khng sinh
K hiu
mi
trng

P. mirabilis
D

(mm)

S. aureus

% hot
tnh so vi
MTdt1

(mm)

% hot
tnh so vi
MTdt1

MT1dt

25,50

0,45

100,00

26,65

0,43

100,00

MT1dt.1

25,53

0,57

100,13

26,84

0,85

100,71

MT1dt.2

27,33

0,42

107,16

28,43

0,64

106,69

MT1dt.3

26,95

0,17

105,67

27,77

0,89

104,22

MT1dt.4

25,31

1,71

99,27

26,32

0,52

98,76

Nhn xt: Khi thm 0,1% cao nm men vo MT1dt hot tnh khng sinh
khng thay i, khi thm 0.5% cao nm men vo th hot tnh khng sinh tng r
rt. Vi t l cao nm men 1% hot tnh khng sinh cng tng nhng khng bng
mi trng 0,5%, cn mi trng 2% cao nm men th li lm gim hot tnh
khng sinh ca chng ln men. Do , MT1dt.2 vi t l cao nm men c
thm vo l 0,5% c la chn lm mi trng ln men cho cc m ln men
chm sau.
31

3.2.3. Kt qu ln men, chit v ct quay thu khng sinh th


Tin hnh ln men chm bin chng B2.02 trn MTdt1.2, lc qua giy
lc loi sinh khi, thu c 7,5 lt dch lc.
Chit 7,5 lt dch lc ny bng 1,5 lt Ethylacetat th thu c 1,1 lt dch
chit khng sinh trong Ethylacetat.
Tin hnh ct quay 1,3 lt dch chit ny thu c 0,9134 g khng sinh th
c mu nu.
Hm lng khng sinh th trong dch ln men chm = 0,1218 g/l.
Lng khng sinh th ny c gi li nghin cu v tin hnh tinh
ch.
3.2.4. Kt qu tinh ch khng sinh ln th nht
* Mc ch:
Loi tp, bc u tch cc thnh phn khng sinh khc nhau t bt khng
sinh th.
* Tin hnh v kt qu:
Theo kt qu ca nghin cu trc [7], chn h dung mi chy ct sc k
l butylacetat: aceton: triethylamin (1:2:1) nhm loi tp v bc u tch cc
thnh phn khng sinh t bt khng sinh th.
Bt khng sinh th chy qua ct sc k silicagel, h dung mi khai trin
l h dung mi : Butylacetat: Aceton: Triethylamin (1:2:1).
Thu mi phn on 5ml vo mt ng nghim sch. Nhn thy khi thu
c 15 phn on th dch chy ra t ct ch cn mu vng rt nht nn kt thc
chy ct, lc ny trn ct silicagel cn lu li mt hp cht mu nu.
Th hot tnh khng sinh tng phn on bng phng php khoanh giy
lc vi chng vi sinh vt kim nh l P. mirabilis.
32

Kt qu chy sc k lp mng v kt qu th HTKS mi phn on c


th hin trong bng 3.8 v bng 3.9.
Bng 3.8. Kt qu sc k ct ln th nht
Phn on
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

D (mm)

21,91

0,12

22,51

0,69

21,87

0,49

21,97

0,29

17,87

0,46

17,45

1,23

16,45

1,02

10,06

0,81

9,24

0,72

10,25

0,96

10,13

0,28

8,19

0,89

7,93

1,46

9,06

0,51

0,00

0,00

Thc hin chy sc k lp mng mi phn on thu c vi h dung mi


Butylacetat: Aceton: Triethylamin (1:2:1). o Rf tng ng vi tng phn on.

33

Bng 3.9. Kt qu sc k lp mng cc phn on


Phn on

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

Vt 1

Vt 2

Vt 3

Rf

Rf

0,53

0,45

0,54

0,44

0,51

0,44

0,54

0,45

0,54

0,46

0,54

0,45

0,54

0,46

0,54

0,44

0,55

0,47

0,53

0,44

0,53

0,46

0,46

0,36

0,47

0,36

0,44

0,35
-

Rf
-

* nh gi cm quan: C mt cht mu nu c lu gi trn ct ( cht ny c


th l tp trong dch ln men b gi li ct). Cc phn on t 1-7 c mu cam
m, t phn on 8 -11 c mu vng cam nht hn, v phn on 12-15 c mu
vng chanh nht.
* Nhn xt:
Nhn vo bng kt qu 3.8, c th thy phn on 15 khng c hot tnh
khng sinh. Cc phn on cn li u c hot tnh khng sinh trong phn
34

on t 1 n 7 c hot tnh khng sinh mnh nht (cng l cc phn on c


mu cam m nht khi quan st bng mt thng).
Cn t bng kt qu 3.9 thy h dung mi chy ct c kh nng tch cc
khng sinh trn bn mng, nhng cha tch ring cc thnh phn trn ct
silicagel (do cc phn on thu c u cho 2 vt khng sinh trn sc k lp
mng). Do , cn phi kho st tm h dung mi c kh nng tch ring cc
thnh phn khng sinh.
Cng t bng 3.9 thy c t nht 3 khng sinh khc nhau trong hn hp
khng sinh th thu c. Chia cc phn on thu c thnh 2 phn: Phn I t
cc phn on 1-11 gm 2 khng sinh, y l phn chnh, c dng cho cc
nghin cu v sau, phn II t phn on 12-14 cng gm 2 khng sinh nhng
lng nh l cc phn on ph (mu rt nht).
Cc phn on phn I c mang gp chung, em ct quay chn khng
500C n khi bc hi ht dung mi, thu c 0,4859g khng sinh tinh ch ln
1 (t y gi l khng sinh P1).
Nh vy, hiu sut tinh ch khng sinh ln th nht l:
H1% =

x 100% = 53,20 %

3.2.5. Kho st h dung mi tinh ch khng sinh ln th hai


T kt qu nghin cu trc [7], tc gi la chn h dung mi
ethylacetat: methanol (10:1) lm h dung mi chy ct tinh ch khng sinh.
Tuy nhin, khi kho st li chng ti thy kh nng tch ca h ny trn SKLM
cha tt, do chng ti tin hnh kho st tm ra h dung mi chy ct tt
hn cho qu trnh tinh ch tip theo.
u tin, kho st mt s h dung mi bng cch thay i t l
ethylacetat. Cc h dung mi c kho st bao gm ethylacetat: methanol vi
cc t l l 20:1; 25:1; 30:1; 35:1. Kt qu SKLM cho thy h dung mi
35

ethylacetat: methanol vi t l 20:1 cho kh nng tch cc khng sinh trn bn


mng tt nht, vt sc k trn, gn nht so vi cc h dung mi khc.
a h dung mi ethylacetat: methanol (20:1) ln ct Silicagel tch
hn hp khng sinh P1, thu 30 phn on, mi phn on 3ml, tin hnh SKLM
tng phn on thu c kt qu nh sau:
Bng 3.10: Kt qu SKLM cc phn on
Phn on

S vt

Rf vt 1

Rf vt 2

0,76

2-16

0,75

0,68

17-30

0,68

T bng 3.10 cho thy khi s dng h ethylacetat: methanol (20:1), cc vt


sc k tch tt trn bn mng nhng trn ct cc khng sinh tch vn cha tt.
Do cn ci tin h ethylacetat: methanol (20:1) tch cc khng sinh trn ct
tt hn.
Kho st cc nghin cu trc, thm mt dung mi na l n-hexan vo h
ethylacetat: methanol (20:1), tin hnh kho st tm t l n-hexan thch hp.
Bng 3.11. T l cc thnh phn h dung mi ethylacetat: methanol: nhexan
H dung mi

T l cc thnh phn
Etylacetat

Methanol

n-hexan

1.1

20

1.2

20

1.3

20

36

Tin hnh sc k lp mng khng sinh P1 vi cc h dung mi 1.1, 1.2, 1.3 thu
c kt qu trong bng 3.12.
Bng 3.12. Kt qu SKLM vi cc h dung mi Ethylacetat: methanol: nhexan
H dung mi

Rf vt 1

Rf vt 2

1.1

0,68

0,52

1.2

0,66

0,53

1.3

0,33

0,38

Kt qu bng 3.12 cho thy, h 1.1 so vi h ethylacetat: methanol


(20:1) ch c thm 1 lng rt nh n-hexan nhng kt qu tch ci thin nhiu,
cn h 1.2 c tc dng ko di khong cch gia 2 vt khng sinh nhng khng
khc h 1.1 nhiu thm ch cn km hn h 1.1. H 1.3 cha nhiu n-hexan nht
th dung mi khng ko c vt khng sinh P1 ln , cc vt khng sinh gn nh
khng tch nhau. Do , chng ti la chn h 1.1 tin hnh tinh ch khng
sinh P1.
3.2.6. Kt qu tinh ch khng sinh ln th hai
* Mc ch:
Tip tc tinh ch khng sinh P1 nhm thu c thnh phn khng sinh
tinh khit hn.
* Tin hnh v kt qu:
Khng sinh P1 c chy ct Silicagel bng h dung mi ethylacetat:
methanol: n-hexan vi t l 20:1:1 n khi dung mi chy ra ch cn mu vng
nht th thu c 30 phn on mi phn on 3ml.
Tin hnh th hot tnh khng sinh tng phn on bng phng php
khoanh giy lc vi VSV kim nh l vi khun P. mirabilis. Kt qu ca th
nghim ny c th hin trn bng 3.13.
37

Bng 3.13. Kt qu th HTKS cc phn on sau tinh ch ln 2.


Phn on
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

D (mm)

0,00

0,00

11,13

0,82

11,95

0,74

16,37

0,48

18,06

0,82

18,83

0,56

19,31

0,56

20,53

0,52

21,64

0,43

22,53

0,38

22,84

0,17

23,25

0,89

23,46

0,70

22,24

0,89

21,78

0,78

Phn on

D (mm)

21,76

0,44

20,57

0,40

18,86

0,55

17,88

0,65

16,77

0,30

15,52

0,53

12,91

0,39

10,02

0,19

7,39

0,50

5,97

1,53

5,87

0,68

0,00

0,00

0,00

0,00

0,00

0,00

0,00

0,00

16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Cc phn on c hot tnh khng sinh c mang chy sc k lp mng vi h


dung mi 1.1. Kt qu ny c th hin trong bng 3.14.
Bng 3.14. Kt qu SKLM cc phn on sau tinh ch ln th hai
Phn on

S vt

Rf vt 1

Rf vt 2

2-7

1 vt

0.68

8-16

2 vt

0,68

0,53

17-26

1 vt

0,53

38

* Nhn xt
Bng 3.13 cho thy phn on u tin v bn phn on cui cng
khng c HTKS. Cc phn on cn li c HTKS kh cao, c bit l cc phn
on t 4 n 20.
Bng 3.14. cho thy cc phn on t 2 n 7 l cng 1 khng sinh c
Rf=0,68. Cc phn on t 17 n 26 cho cng 1 cht khng sinh c Rf=0,53.
Trong cc phn on t 8 n 16 cha 2 khng sinh nhng cha tch ring c.
Do , tin hnh gp cc phn on t 2 n 7, ct quay chn khng
nhit 500C n ht dung mi, kt tinh thu c 0,0423g khng sinh c
Rf=0,68 (t y gi l khng sinh X1).
Gp cc phn on t 17 n 26, ct quay chn khng nhit 500C
n ht dung mi, kt tinh thu c 0,0532g khng sinh c Rf=0,53 (t y gi
l khng sinh X2).
Th SKLM khng sinh X1 v X2 vi 4 h dung mi khc nhau th u
thy 1 vt trn, gn, khng ko ui. Nh vy, X1 v X2 l cht tinh khit cao.
Hiu sut tinh ch khng sinh X1 t khng sinh P1 l:
HX1=

x 100% = 8,71 %

Hiu sut tinh ch khng sinh X2 t khng sinh P1 l:


HX2=

x 100% = 10,95 %

Gp phn on t 8 n 16, ct quay chn khng nhit 500C n ht


dung mi, kt tinh thu c 0,2068g hn hp khng sinh X1 v X2 ( gi l P2).
Nh vy, lng khng sinh trong cc phn on t 8 n 16 l rt ln, cn tm
c dung mi tt hn tch khng sinh X1 v X2 t hn hp khng sinh P2
ny.
3.2.7. Kho st h dung mi tinh ch khng sinh ln th ba
39

Tin hnh kho st cc h dung mi khc bng cch thay i thnh phn
methanol trong h 1.1 bng cc ancol mch di hn c th l ethanol v npropanol
Bng 3.15: Thnh phn cc h dung mi tinh ch khng sinh ln 3
H dung mi

Thnh phn

T l

1.1

Ethylacetat: Methanol: n-hexan

20:1:1

2.1

Ethylacetat: Ethanol: n-hexan

20:1:1

2.2

Ethylacetat: n-Propanol: n-hexan

20:1:1

Tin hnh sc k lp mng khng sinh P2 vi cc h dung mi 1.1, 2.1, 2.2 thu
c kt qu trong bng 3.12.
Bng 3.16. Kt qu SKLM vi cc h dung mi tinh ch khng sinh ln 3
H dung mi

Rf vt 1

Rf vt 2

1.1

0,68

0,55

2.1

0,58

0,40

2.2

0,35

0,31

* Nhn xt: Kt qu bng 3.16 cho thy, so vi h 1.1, th h 2.1 tch tt hn,
vt sc k trn, gn hn. Cn h 2.2 th cc cht gn nh l khng tch. Do ,
chng ti la chn h 2.1 tin hnh tinh ch khng sinh P2.
3.2.8. Kt qu tinh ch khng sinh ln th ba
* Mc ch:
Tip tc tinh ch khng sinh P2 nhm thu c thnh phn khng sinh
tinh khit hn.
* Tin hnh v kt qu:
40

Khng sinh P2 c chy ct Silicagel bng h dung mi ethylacetat:


ethanol : n-hexan vi t l 20:1:1 n khi dung mi chy ra ch cn mu vng
nht th thu 30 phn on mi phn on 3ml.
Tin hnh th hot tnh khng sinh tng phn on bng phng php
khoanh giy lc vi VSV kim nh l vi khun P. mirabilis. Kt qu ca th
nghim ny c th hin trn bng 3.17.
Bng 3.17. Kt qu th HTKS cc phn on sau tinh ch ln 3
Phn on
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

D (mm)

0,00

0,00

0,00

0,00

0,00

0,00

9,24

0,30

11,25

0,67

11,07

0,67

16,03

0,80

17,33

0,52

18,83

0,56

19,31

0,56

20,85

0,44

21,00

0,83

21,88

0,58

21,99

1,33

22,87

0,58

Phn on
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

D (mm)

20,33

0,58

20,01

1,31

19,93

0,51

18,86

0,55

17,88

0,65

15,84

0,55

15,52

0,53

13,15

0,09

10,02

0,19

7,67

0,57

6,31

1,53

0,00

0,00

0,00

0,00

0,00

0,00

0,00

0,00

Cc phn on c hot tnh khng sinh c mang chy sc k lp mng


vi h dung mi 2.1. Kt qu ny c th hin trong bng 3.18.
41

Bng 3.18. Kt qu SKLM cc phn on sau tinh ch ln th ba


Phn on

S vt

Rf vt 1

Rf vt 2

4-10

1 vt

0,58

11-15

2 vt

0,58

0,40

16-26

1 vt

0,40

* Nhn xt
Bng 3.17 cho thy 3 phn on u tin v 4 phn on cui cng
khng c HTKS. Cc phn on cn li c HTKS kh cao, c bit l cc phn
on t 4 n 18.
Bng 3.18. cho thy cc phn on t 4 n 10 l cng 1 khng sinh c
Rf=0,58. Cc phn on t 16 n 26 cho cng 1 cht khng sinh c Rf=0,40.
Trong cc phn on t 11 n 15 cha 2 khng sinh.
Tin hnh nh ln tinh ch khng sinh th 2, gp cc phn on t 4 n
10, ct quay chn khng nhit 500C n ht dung mi, kt tinh thu c
0,0321g khng sinh c Rf=0,58 (k hiu N1). Gp cc phn on t 16 n 26,
ct quay chn khng nhit 500C n ht dung mi, kt tinh thu c 0,0424
g khng sinh c Rf=0,4 ( k hiu N2):
Hiu sut tinh ch khng sinh N1 t hn hp khng sinh P2 l:
HN1=

x 100% = 15,52 %

Hiu sut tinh ch khng sinh N2 t hn hp khng sinh P2 l:


HN2=

x 100% = 20,52 %

Th SKLM khng sinh N1, N2 ny vi cc khng sinh X1, X2 thu c


trn vi cc h dung mi khc nhau thy cho Rf tng t nhau, cc vt sc k
u trn, gn, do khng sinh N1 thu c chnh l khng sinh X1 ca ln tinh
ch th 2. V khng sinh N2 chnh l khng sinh X2 ca ln tinh ch 2. Gp
42

khng sinh N1 vi khng sinh X1 bn trn thu c khng sinh tinh khit 1 ( k
hiu l KS1).
Tng t nh vy, gp khng sinh N2 vi khng sinh X2 thu c khng
sinh tinh khit 2 ( k hiu KS2).
Khi lng ca khng sinh KS1 sau ba ln tinh ch l:
mKS1= 0,0423 + 0,0321 = 0,0744 g
Hiu sut tinh ch khng sinh KS1 t khng sinh th l:
HKS1=

x 100% = 8,15 %

Khi lng ca khng sinh KS2 sau ba ln tinh ch l:


mKS2= 0,0532 + 0,0424 = 0,0956 g
Hiu sut tinh ch khng sinh 2 t khng sinh th l:
HKS2=

x 100% = 10,47 %

3.3. Nghin cu tnh cht, cu trc khng sinh


3.3.1. Kt qu o nng chy
Th nghim o nng chy ca khng sinh KS1 v KS2 c thc hin
ti phng K thut tng hp Ha dc- b mn Cng nghip Dc, trng i
hc Dc H Ni. Kt qu nh sau:
Khng sinh KS1 nng chy 143-145 oC.
Khng sinh KS2 nng chy 231-236 oC.
3.3.2. Kt qu ph t ngoi
Tin hnh o ph t ngoi ca khng sinh KS1 v KS2 ti khoa Ha li hc Dc H Ni. Kt qu ph UV cho thy khng sinh KS1 c 3 nh hp
th bc sng 1 = 444 nm, 2 = 239,5 nm v 3= 203 nm.

43

Khng sinh KS2 c 2 nh hp th bc sng 1 = 443,5 nm v bc


sng 2 = 204 nm.
Hnh nh ph UV ca KS1 v KS2 c th hin trong ph lc (Hnh P1, P2).
3.3.3. Kt qu ph hng ngoi
Ph IR ca 2 khng sinh KS1 v KS2 c o ti vin Ha hc bng
phng php vin nn KBr vng bc sng t 4000 500 cm-1.
Kt qu cho thy:
Ph IR ca cht KS1 c 12 bc sng hp th cc i l 3366,32 cm-1,
2931,99 cm-1, 1731,56 cm-1, 1655,90 cm-1, 1577,44 cm-1, 1462,55 cm-1, 1266,39
cm-1, 1101,06 cm-1, 980,57 cm-1, 733,98 cm-1, 588,26 cm-1 v 479,13 cm-1.
Ph IR ca cht KS2 c 14 bc sng hp th cc i l 3393,74 cm-1,
3266,85 cm-1, 2963,72 cm-1, 1744,99 cm-1, 1644,30 cm-1, 1575,31 cm-1, 1478,20
cm-1, 1301,87 cm-1, 1197,10 cm-1, 1120,93 cm-1, 813,78 cm-1, 627,23 cm-1,
545,45 cm-1, 479,01 cm-1 .
Bng 3.19: Bin gii cc nhm chc ca KS1 t ph IR
Bc sng Nhm chc c trng

Bc sng Nhm chc c trng

(cm-1)

(cm-1)

3366,32

OH trong phenol, hoc 1266,39

-C-O trong alcol, ether,

carboxylic acid.

ester, carboxylic, anhydrid.

-N-H trong amin hoc amid.

-C-F.
S=O trong sulfon, sulfunyl
chlorid, sulfat, sufonamid.

2931,99

- C-H ca alkan.

1101,06

-C-O trong alcol, ether,


ester, carboxylic, anhydrid.
-C-F.

44

S=O trong sulfoe, sulfunyl


chlorid, sulfate, sufonamid.
C-N ca amin
1731,56

-CO ca aldehyd, hoc ester. 980,57

-CH trong alken.

1655,90

-CO ca amid hoc C=N ca 733,98

-C-Cl.

imin hoc oxim.

-C-H trong alken, hoc nhn

C=C ca alken.

thm.

1577,44

- N-H trong amin hoc amid. 588,26

-C-Br hoc C-I.

1462,55

- CH3 trong alkan.

-C-Br hoc C-I.

479,13

Bng 3.20: Bin gii cc nhm chc ca KS2 t ph IR


Bc sng Nhm chc c trng

Bc sng Nhm chc c trng

(cm-1)

(cm-1)

3393,74

OH trong phenol, hoc 1301,87

-CN trong amin.

3266,85

carboxylic acid.

S=O trong sulfon, sulfunyl

-N-H trong amin hoc amid .

chlorid, sulfat, sufonamid.


C-F.

2963,72

-C-H ca alkan.

1197,10

-C-O trong alcol, ether, ester,

1120,93

carboxylic, anhydrid.
-C-N ca amin.
S=O trong sulfon, sulfunyl
chlorid, sulfat, sufonamid

1744,99

-CO ca ester

813,78

-CH trong alken hoc nhn


thm.

45

1644,30

-CO ca amide hoc C=N 627,23

C-Cl

ca imine hoc oxime


C=C ca alken
1575,31

-N-H trong amin hoc amid

545,45

C-X (X= Br, I)

1478,20

C=C trong nhn thm

479,01

C-X (X= Br, I)

* Nhn xt: Nh vy c th s b d on khng sinh KS1, KS2 c th c cc


nhm chc amin, amid, acid carboxylic, aldehyd, este, c nhn thm, cc mui
ca amin bc 2, bc 3....Hnh nh ph IR ca KS1 v KS2 c th hin trong
ph lc ( Hnh P3, P4).
3.3.4. Kt qu o ph khi
Tin hnh o ph khi ca KS1 v KS2 ti Vin Ha hc. Kt qu cho
thy khi lng phn t d kin ca KS1 v KS2 ln lt l: 1269,45 vC v
1255,44 vC. Hnh nh ph MS ca hai cht ny c th hin trong ph lc
(Hnh P5, P6).
3.3.5. Kt qu o ph cng hng t ht nhn
Tin hnhh o ph cng hng t ht nhn i vi mu KS2, gm c: 1H
NMR, 13C NMR, DEPT, COSY, HSQC, HMBC.
Ph 13C-NMR xut hin 13 tn hiu ti vng trng thp t 160-180 ppm
ti 166,32; 166,485; 166,522; 166,601; 167,572; 167,671; 168,624; 169,046;
173,256; 173,279; 173,317; 173,652; 179,063. Trong c 12 tn hiu u in
hnh cho s c mt ca 12 nhm C=O trong lin kt peptid N-C=O, v mt
nhm C=O trong vng ti 179,063

46

Xut hin 12 tn hiu ca 12 nhm methyl ti 39,216; 39,144; 34,956;


34,889; 19,093; 19,070; 19,029; 18,985; 17,716; 17,313; 15,025; 7,767. Trong
, 4 tn hiu ti 39,216; 39,144; 34,956; 34,889 dch chuyn v trng thp
hn so vi tn hiu nhm methyl thng thng=> in hnh cho 4 nhm methyl
gn trc tip vi nguyn t Nit.
Cng t ph 13C-NMR , ph DEPT 135 xut hin tn hiu ca 7 nhm
CH2 ti 22,838; 23,005; 30,981; 31,226; 47,350; 47,590; v 51,374. Trong
ngoi cc tn hiu 22,838; 23,005, cn li 5 tn hiu u dch chuyn mnh v
pha trng thp hn cc nhm methylene thng thng. iu ny chng t 5
nhm methylene ny u c lin kt vi cc nhm ht in t mnh nh Nit v
C=O.
Ph 13C-NMR , ph DEPT 90 xut hin tn hiu ca 16 nhm CH- ti
26,945; 27,008; 31,562; 31,804; 54,818; 55,171; 56,315; 56,554; 58,758; 58,918;
71,184; 71,384; 74,985; 75,053; 125,663; 130,276. Trong , ngoi 4 tn hiu ti
26,945; 27,008; 31,562; 31,804 th cc tn hiu khc u b dch chuyn mnh
v trng thp hn cc nhm CH- thng thng, chng t cc nhm ny nm
trong vng thm.
c bit ph

13

C-NMR v

H cho thy cc tn hiu ca khung

phenoxazone ti C=O : 179,063; CH3 : 15,025 v 7,767; CH : 130,276, 125,663;


C : 147,496; 145,845; 145,067; 140,482; 132,606, 129,122; 127,660; 113,527;
101,800. Ph 1H cho hai tn hiu H: 7,607; 7,353, da vo tng tc trn ph
HSQC c th kt lun hai tn hiu l tn hiu ca 2 proton olefin ca khung
phenoxazone ti v tr octo ca khung (C7 v C8).
Di y l bng so snh d kin ph NMR ca KS2 vi khung phenoxazone
ca phn t actinomycin D [16, 17, 20].

47

Bng 3.21. So snh ph NMR ca KS2 vi khung phenoxazone ca


actinomycin D
V tr

Actinomycin D

KS2

101,6

101,800

146,0

145,845

179,1

179,063

113,5

113,527

4a

145,1

145,047

5a

140,5

140,482

127,5

127,660

130,2

130,276

125,9

125,663

132,6

132,606

9a

129,1

129,122

-CH3 v tr 4

7,7

7,767

-CH3 v tr 6

15,0

15,025

C=O v tr 3

179,1

179,063

H+ v tr 7

7,6

7,607

H+ v tr 8

7,3

7,353

Nh vy, c th kt lun, khng sinh KS2 phn lp t x khun


Streptomyces 173.113 l khng sinh thuc nhm actinomycin.
Kt qu o ph NMR c th hin trong phn ph lc ( T hnh P7 n
hnh P12).

48

CHNG 4. BN LUN
4.1. Chn lc v ci to ging x khun
T nghin cu trc [7], sau khi tri qua bc sng lc ngu nhin v 3
ln t bin bng UV v ha cht th hot tnh khng sinh ca chng
Streptomyces 173.113 tng r rt. La chn chng c hot tnh khng sinh cao
nht t nghin cu trc, tip tc tin hnh sng lc ngu nhin v t bin bng
UV tng hot tnh khng sinh. Mc d nghin cu trc, chng c hot tnh
khng sinh cao nht c ng knh vng v khun ln ti 28,19 mm (VSV kim
nh l S. aureus), v 26,12 mm (VSV kim nh l P. mirabilis), cn nghin
cu ny ng knh vng v khun ca chng c hot tnh khng sinh mnh
nht sau sng lc ngu nhin v 2 ln t bin ch t c 26,03 mm (VSV kim
nh l S. aureus), v 24,94 mm (VSV kim nh l P. mirabilis) nhng so vi
mu chng th vn cao hn ( iu ny c th do iu kin nhit , m lc
lc tin hnh nui cy khc nhau, nh hng ti ng knh vng v khun).
Qua ln t bin bng nh sng UV ln th nht, vi sng st l 0,73%
th a phn cc bin chng thu c c hot tnh cao hn so vi mu chng (t
bin dng). Trong , cao nht l bin chng B1.13 vi hot tnh khng sinh
bng 113,26% so vi mu chng.
Tip tc t bin bin chng B1.13 bng nh sng UV ln th hai,
sng st l 0,58% th vn thu c t bin dng. Cao nht l bin chng
B2.02 c hot tnh khng sinh bng 110,26% so vi mu chng.
Nh vy, kt qu 2 ln t bin bng nh sng UV lm tng hot tnh
khng sinh ca chng x khun gc (hot tnh khng sinh sau 2 ln t bin bng
124,88% so vi mu ban u).
Do gii hn v thi gian, nn chng ti cha th tin hnh t bin bng
tc nhn khc nh gi s thay i hot tnh khng sinh ca x khun. V vy,
49

chng ti s xut thay i tc nhn t bin cho nghin cu sau ny (c th


dng tc nhn ha hc).
4.2. Xc nh iu kin ln men, chit tch khng sinh ti thch
Tin hnh ci to ging qua sng lc ngu nhin v t bin UV, chng ti
la chn c 9 dng chng, bin chng tt la chn chng ln men tt
nht, l: SL.02, SL.09, SL.14, B1.04, B1.13, B1.30, B2.02, B2.10,
B2.25. Tin hnh ln men ring bit v so snh hot tnh khng sinh ca cc
dch lc dch ln men th thy bin chng B2.02 ln men chm tt nht. Kt qu
ny l hp l bi v B2.02 l bin chng c HTKS cao nht thu c sau t
bin ln hai.
Nng cao kh nng ln men ca chng / bin chng bng cch thm vo
mi trng MT1dt mt lng cao nm men. Kt qu thu c mi trng ln
men ti thch l MT1dt.2 (thm 0,5% cao nm men vo MT1dt), dch ln men
c hot tnh khng sinh cao hn dch ln men ca MT1dt ( hot tnh khng sinh
dch ln men MT1dt.2 bng 107,16% MT1dt).
Lng khng sinh trong dch ln men thu c l 0.1218 g/l, so snh vi
nghin cu trc lng khng sinh trong dch ln men ch t 0,1033 g/l [7].
Nh vy, qu trnh chn lc, ci to ging, thay i mi trng nui cy lm
tng hiu qu sinh tng hp khng sinh ca Streptomyces 173.113 so vi nghin
cu trc.
Khng sinh th thu c sau ct quay l mt hn hp nhiu cht khng
sinh v cc tp cht khc (c th l cht mu,). Do , vic tch v tinh ch
khng sinh c ngha rt quan trng trong qu trnh nghin cu khng sinh.
Trong ti ny, khng sinh c tinh ch ba ln. Ln th nht, s dng
h dung mi butylacetat: aceton: triethylamin (1:2:1) tin hnh sc k ct.
H ny c tc dng gi li mt s tp cht (c th l cht mu trong khng sinh
50

th, do trc chy ct khng sinh th c mu nu, sau chy ct c mu


ti). Tuy nhin, cc phn on khng sinh P1 sau khi sc k ct, mc d cc
khng sinh c tch nhau trn SKLM nhng li khng tch nhau trn sc k ct (
mi phn on u c t nht 2 cht khng sinh). Nh vy, cn phi tm h dung
mi khc c th tch ring c cc thnh phn khng sinh trn sc k ct.
Ln tinh ch th hai s dng h dung mi ethylacetat: methanol: nhexan ( 20:1:1) sc k ct tch cc thnh phn khc nhau ca hn hp P1.
Nhn vo bng kt qu 3.13 v 3.14 c th thy cc phn on t 2 n 7,
t 17 n 26 cho ta c 2 khng sinh tinh khit l X1 v X2 c hot tnh khng
sinh kh cao. Tuy nhin, cc phn on xen ph gia cht X1 v X2 l cc phn
on 8 n 16 cng c hot tnh khng sinh rt cao (khi quan st bng mt
thng th cc phn on 2 cht ny c mu cam rt m), ct loi dung mi
thu c 0.2068g. Nh vy, phn on hn hp t 8 n 16 cn cha mt lng
lng khng sinh tinh khit rt ln m h ethylacetat: methanol: n-hexan (
20:1:1) cha tch c. Do , chng ti kho st tm ra h dung mi c
kh nng tch ring c khng sinh X1 v X2 tt hn.
Thay i methanol trong h dung mi trn bng cc alcol c mch di hn
nhm tng thi gian lu trn ct ca cc cht, do d tch ring cc cht ra
hn. Kho st cc h, thu c h dung mi ethylacetat: ethanol: n-hexan (
20:1:1) c kh nng tch cc cht tt hn h s dng methanol. C th, hiu sut
ln chy ct th 3 ca cht khng sinh tinh khit 1, v 2 ln lt l 15,52% v
20,52%, tng cao hn so vi hiu sut chy ct ln 2 ca cht 1, v 2 l 8,71%
v 10,95%. Cc khng sinh tch ra t ln tinh ch 3 cng chnh l cc khng sinh
tch ra t ln tinh ch 2. Gp ln lt cc khng sinh ca hai ln tinh ch thu
c hai khng sinh tinh khit KS1, v KS2

51

Hiu sut tinh ch sau cng ca cc khng sinh KS1 v KS2 ln lt l


8,15% v 10,47%. So snh vi nghin cu trc, hiu sut tinh ch sau cng
ca khng sinh chnh l 33,48% tuy nhin, tinh khit ca khng sinh thu c
cha cao. Trong nghin cu ny, mc d hiu sut tinh ch cha bng nghin
cu trc, tuy nhin tinh khit cao hn do cc nghin cu v sau c
chnh xc cao hn, nht l trong vic xc nh cu trc khng sinh thu c.
Nh vy, sau 3 ln chy ct tinh ch thu c hai cht tinh khit l KS1 v
KS2 v qu trnh nghin cu cng cho thy, chng c vai tr tng ng nhau
trong vic to thnh hot tnh khng sinh ca chng x khun ny, khng th
phn r u l cht chnh, u l cht ph.
4.3. Nghin cu cu trc khng sinh
* Kt qu o ph UV: cho thy KS1 c 3 nh hp th chnh tng ng
vi 3 bc sng 1 = 444 nm, 2 = 239,5 nm v 3= 203 nm. KS2 c 2 nh hp
th chnh tng ng vi 2 bc sng max l 1 = 443,5 nm v 2 = 204 nm.
Cc nh hp th ny tng ng vi s chuyn dch in t t n ln *.
Trong n l cp in t t do (khng lin kt) cc d nguyn t O, N, S,
halogen. Nh vy khng sinh nghin cu l hp cht c cha in t n v lin
kt . Hay ni cch khc trong cu trc khng sinh c th c nhn thm, ni i
lin hp, c cha d t, hoc kt hp cc yu t trn.
* Kt qu o ph IR: Ph IR ca cht KS1 v KS2 cho php d on cc
nhm chc c trng trong cu trc ha hc ca khng sinh KS1 v KS2.
Nh vy, t bng 3.19 v bng 3.20 c th s b d on khng sinh KS1
v KS2 c th c cc nhm chc amin, amid, acid carboxylic, aldehyd, este, c
nhn thm, cc mui ca amin bc 2, bc 3....
Tuy nhin, nhn vo kt qu ph IR c th thy cu trc ha hc ca KS1
v KS2 ht sc phc tp vi nhiu nhm chc khc nhau, do cn tin hnh o
52

thm cc ph khi, ph cng hng t ht nhn xc nh c chnh xc cu


trc ca KS1 v KS2
* Kt qu o ph NMR v MS:
T hnh nh cc ph NMR ca cht KS2 cho thy cu trc ca khng sinh
ny rt phc tp. Do hn ch v trnh v thi gian, trong nghin cu ny
chng ti ch bin gii c mt s nhm chc c trng ca KS2 m cha bin
gii c cu trc y ca KS2.
T qu trnh bin gii cho thy cu trc KS2 c mt ca khung
phenoxazone, mt khung c trng cho cu trc cc khng sinh thuc nhm
actinomycin.
Nh vy, c th kt lun, khng sinh KS2 phn lp t x khun
Streptomyces 173.113 l khng sinh thuc nhm actinomycin.

53

KT LUN V KIN NGH


Kt lun
Sau mt thi gian nghin cu, chng ti c bn hon thnh c mc
tiu ban u ca lun vn. Cc kt lun c th nh sau:
- Qua 2 ln t bin bng UV th hot tnh khng sinh ca Streptomyces
173.113 tng ln nhiu ( hot tnh khng sinh sau t bin bng 124,88% trc
t bin).
- Tng kh nng ln men bng cch cho thm cao nm men vo mi
trng ln men ban u vi t l 0,5%: kh nng ln men tng 107,16% so vi
MT1dt.
- Thay i h ethylacetat: methanol: n-hexan ( 20:1:1) ln tinh ch 2
bng h ethylacetat: ethanol: n-hexan ( 20:1:1) ln tinh ch 3 lm tng hiu
sut tinh ch khng sinh KS1 ( tng t 8,71% ln 15,52%) v KS2 (tng t
10,95% ln 20,52%).
- Tinh ch c 2 cht khng sinh c tinh khit cao l KS1 v KS2 vi
hiu sut ln lt l: 8,15% v 10,47%.
- Khng sinh KS1 c cc c im:
+ Tinh th hnh kim, mu cam.
+ Nng chy 143-145C.
+ Ph UV c 3 nh hp th: 1 = 444 nm, 2 = 239,5 nm v 3=
203 nm.
+ Ph IR cho thy cu trc ha hc rt phc tp vi rt nhiu nhm
chc
+ Khi lng phn t: 1269,45 vC.
- Khng sinh KS2 c cc c im:
54

+ Tinh th hnh kim, mu gch.


+ Nng chy 231-236C.
+ Ph UV c 2 nh hp th: 1 = 443,5 nm v 2 = 204 nm.
+ Ph IR cho thy cu trc ha hc rt phc tp vi rt nhiu nhm
chc.
+ Khi lng phn t: 1255,44 vC.
+ tin hnh o ph NMR, kt lun thuc nhm khng sinh
actinomycin, tuy nhin cng thc cu to c th vn cha bin lun c
Kin ngh
T nhng kt qu thu c, chng ti xut tip tc pht trin ti
nghin cu su hn theo cc hng sau:
- Tip tc bin gii cu trc ca KS1 v KS2.
- Tin hnh t bin bng ha cht nng cao hn na hiu sut sinh
tng hp khng sinh.
- Nghin cu tm phng php tinh ch khng sinh cho hiu sut cao hn.

55

TI LIU THAM KHO


Ting Vit
1. B Y T (2008), Vi sinh vt hc, Nh xut bn Gio Dc, H Ni, tr. 2287.
2. Nguyn Vn Cch (2004), Cng ngh ln men khng sinh, NXB Khoa hc
v k thut, tr 130-136.
3. L Huy Chnh (2007), Vi sinh vt y hc, NXB Y hc, tr. 25-57
4. Cc Qun l Dc (2010), Quy hoch chi tit pht trin cng nghip
Dc Vit
Nam giai on n nm 2020 v tm nhn n 2030, H Ni.
5. Nguyn Ln Dng (2001), Vi sinh vt hc, NXB Gio dc, tr. 39-67.
6. L Huy Dng (2010), Nghin cu ln men sinh tng hp khng sinh t
Streptomyces 168.27, Kha lun tt nghip dc s, Trng i hc Dc
H Ni, H Ni.
7. L Th Thu Hin ( 2013), Gp phn nghin cu ln men sinh tng hp
khng
sinh nh Streptomyces 173.113. Kha lun tt nghip Dc s kha 20082013, Trng i hc Dc H Ni.
8. L Gia Hy (2013), Cng ngh sn xut khng sinh, NXB Khoa hc v k
thut, tr 161-193.
9. T Minh Kong (2006), K thut sn xut dc phm II, NXB Y hc, tp
2
10. on Th Nguyn (2009), Vi sinh vt, NXB Gio dc Vit Nam, tr. 7-37.
11. Lng c Phm (1999), Cng ngh vi sinh vt, NXB Nng nghip.
12. Nguyn Hunh Minh Quyn (2011), iu tra, nghin cu mt s hot
cht c

kh nng khng vi sinh vt v khng dng t bo ung th t x khun, i


hc Quc gia H Ni, tr. 16-46.
13. Nguyn Vn Thch (2009), Cng ngh sinh hc dc, NXB Gio dc
Vit Nam, tr. 35-57.
14. Trn Th Thanh (2001), Cng ngh vi sinh, NXB Gio dc, tr. 40-52.
15. Cao Vn Thu (2008), Vi sinh vt hc, NXB Gio dc.
16. Cao Vn Thu, Phan Vn Kim, Bi Vit H (2010), Actinomycin X2,
cht
khng sinh c ln men t qu trnh sinh tng hp, Tp ch Ha hc, T.48
(4), tr 469-474.
17. Cao Vn Thu, Bi Vit H, Quch Th L H, Nguyn Hng Hnh,
Nguyn Th
Thy Hin, Phan Vn Kim, V Th Thy Linh, V Nguyn Thnh, V Th
Thu Thy (2010), Nghin cu ln men sinh tng hp khng sinh t
Streptomyces 15.29 Streptomyces microflavus, Tp ch khoa hc v cng
ngh, T.48 (5), tr 105-210.

Ting Anh
18. Augustine S.K, Bhavsar S.P, Kapadnis B.P. (2005), A non-polyene
antifungal
antibiotic from Streptomyces albidoflavus PU23, The Journal of
bioscience, No. 30 (2), p. 201-211.
19. Atta H.M. (2009), Sparsomycin antibiotic production by Streptomyces
sp.
AZ-NIOFD1: Taxonomy, fermentation, purification and biological
activities, Americam-Eurasian journal of Agricultural and environmental
science, No. 5 (3), p. 368-377.
20. Atta H.M. (2011), Cirramycin-B antibiotic production by Streptomyces
Cyaneus-AZ-13Zc: fermentation, purification and biological activities,
NewYork Science journal, No. 4 (2), p. 35-42.
21. Chater, Brios K.F. (2010), The complex extracellular biology of
Streptomyces, FEMS microbiology review, No. 34(2), p. 171-198.
22. Chojnacka K. Fermentation products, Chemical engineering and
chemical
process technology, Vol. V.
23. Emerson R. (2012), Antibiotics produced by Streptomyces, The
Brazilian
journal of infectious diseases, No. 16 (5), p. 466-471.
24. Hollstein U. (1974), Actinomycin Chemistry and metabolism of
action,
Chemical review, No 74(6), p. 625-652.
25. Kazuro Shiomi (2005), A new antibiotic, Antimycin A9, produced by
Streptomyces sp. K01-0031, The journal of antibiotics, No 58(1), p. 7478.
26. Keeler J. (2007), Understanding NMR Spectroscopy, University of
California

Publisher.
27. Paul Dyson (2011), Streptomyces: Molecular biology and biotechnology,
Horizon Scientific Press.
28. Pavia D.L, Lampman G.M, Kriz G.S, Vyvyan J.R. (2008), Introduction to
spectroscopy, Brooks/Cole, Cengage Learning, Fourth edition.
29. Shirling E.B. (1996), Methods of characterization of Streptomyces sp.,
Int J.
Bacterial, vol 16 (3), p. 313-340.
30. Vladislav B. (2000), Bioactive products from Streptomyces,

Adv.

Appl.
Microbiol, No. 47, p. 113-156.
31. Waksman S.A., Woodruff H.B. (1940), How many antibiotics are
produced by
the genus Streptomyces?, Arch. Microbiology, No. 176 (5), p. 386-390.
32. Wiliams S.T. (1983), Numerical classification of Streptomyces and
related
genera, Journal of microbiology, No. 129, p. 1743- 1813.

PH LC

Hnh P1: Ph UV ca cht khng sinh KS1

Hnh P2: Ph UV ca cht khng sinh KS2

Hnh P3: Ph IR khng sinh KS1

Hnh P4: Ph IR khng sinh KS2

Hnh P5: Ph khi MS khng sinh KS1

Hnh P6: Ph khi MS ca khng sinh KS2

Hnh P7: Ph 1H NMR ca khng sinh KS2

Hnh P7a: Ph 1H NMR gin ca khng sinh KS2

Hnh P7b: Ph 1H NMR gin ca khng sinh KS2

Hnh P7c: Ph 1H NMR gin ca khng sinh KS2

Hnh P8: Ph 13C NMR ca khng sinh KS2

Hnh P8a: Ph 13C NMR gin ca khng sinh KS2

Hnh P8b: Ph 13C NMR gin ca khng sinh KS2

Hnh P9: Ph COSY ca khng sinh KS2

Hnh P9a: Ph COSY gin ca khng sinh KS2

Hnh P9b: Ph COSY gin ca khng sinh KS2

Hnh P9c: Ph COSY gin ca khng sinh KS2

Hnh P10: Ph DEPT ca khng sinh KS2

Hnh P10a: Ph DEPT gin ca khng sinh KS2

Hnh P10b: Ph DEPT gin ca khng sinh KS2

Hnh P11: Ph HSQC ca khng sinh KS2

Hnh P11a: Ph HSQC gin ca khng sinh KS2

Hnh P11b: Ph HSQC gin ca khng sinh KS2

Hnh P12: Ph HMBC ca khng sinh KS2

Hnh P12a: Ph HMBC gin ca khng sinh KS2

Hnh P12b: Ph HMBC gin ca khng sinh KS2

Hnh P12c: Ph HMBC gin ca khng sinh KS2

Hnh P12d: Ph HMBC gin ca khng sinh KS2