PHAN LAP TH RITNG NGAP MAN CAN Gid

T?p chl KHOA HQC DHSP TPHCM Tran Thj Minh Binh va tgk

NGHIEN ClTU DIEU K I ( : N NUOI CAY THICH HglP

DE CHUNG ASPERGILLUS TERSEUS OI

PHAN LAP TH RITNG NGAP MAN CAN Gid

SINH TONG HOP CHAT KHANG SINH

T R A N THI MINH B|NH', T R A N THANH THCIY"

T 6 M TAT Tir 58 chung ndm sgi phdn lap dugc td ritng ngdp man Cdn Gia. da tuyin chgn dugc

chiing ndm sgi Bl co hogt tinh khdng sinh mgnh. Kit qud gidi trinh tu gen 2SS rRNA cho thdy chung Bl thugc lodi Aspergillus terreus. Cdc dieu kien nudi cdy thich hgp cho chung tidy sinh tong hap chdt khdng sinh da dugc khdo sdt vd xdc dinh.

Tir khda: Aspergillus, chat khdng sinh, tdi uu dieu kien nudi cay. ABSTRACT

Optimization of culture conditions for antibiotic production of Aspergillus terreus Dl Isolated from Can Gio mangrove

From 58 filamentous fungi strains isolated from Can Gio mangrove soil, a strain which has the best antimicrobial activity was obtained. Based on 28S rRNA sequence, this strain was classified to Aspergillus terreus species. The culture conditions for antibiotic production of this strain were optimized.

Keywords: Aspergillus, antibiotic, culture conditions optimization.

1. Md diu

Trong nhimg nam gan day, cdc nhd khoa hpc dde biet quan tdm nghien cuu cac ddi tupng vi sinh vft (VSV) tfl cac hf sinh thdi dac bift, trong dd cd rung ngap man (RNM). Bdi theo edc nha khoa hpc, chiing Id ngudn sinh cdc ehat cd boat tinh sinh hpe ddi ddo, ttong dd ed ehat khdng sinh (CKS). Vd thye tl, hp da phat hien nhilu hpp ehat khang khuin, khdng nira, khdng virus, khdng ung thu,... tfl VSV rirag ngfp rajn vdi hoat tinh mjnh hem rdt nhieu so vdi VSV tu dat liln.

Trong sd cae VSV sinh khang sinh, nam spi (NS) la ddi tupng cd y nghta ed ve I mat ljch sfl lin y hpc. NS Id ddi tupng sinh ra mpt lupng ldn CKS khdng vi khuin ; (VK), khdng virus, khdng khdi u va khdng nim. Trong sl 23.000 chit tfl VSV ed hoat tinh sinh hpc nhu khang ndm, khdng khuan, khang virus,... ed den 42% ttong sd dd ed nguon gde tfl cac lodi thude gidi nim. [2]

Cdc nghien cflu eho thdy ndra bien cd ho^t tinh khang sinh (KS) khang dupc phd rfng cdc VSV [5]. Cuomo va cdng sy (1995) so sanh boat tinh KS cua 1500 chung ndra

i j i iS, Trudng 0?i hpc Su pham TPHCM TS, Tn/dng Bai hoc Su pham TPHCM

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Tap chi KHOA HOC DHSP TPHCM SB 4/ nam 2013

phan lap tu biln vdi 1450 miu phan lap tfl dit liln, kit lufn rdng cd nhieu miu nim phan ldp tfl biln cd hoat tinh KS hon tfl dit liln. Carsten Christophersen va cf ng sy (1999) nghien cflu tten 227 chung NS phdn lap tfl biln (trong do cd RNM), ket qua co 7 chimg ed kha ndng khdng Vibrio parahaemoliticus, 55 chung khang S. aureus. [3]

Cho din nay, cac nghien cuu vl CKS eua ndra biln, trong dd cd ndm RNM, da phat hien dupc nhilu chit, bao gdm:

Chit khang khuin: nigrospoxydon A tfl ndm Nigrospora sp. khang lai 5. aureus (Trisuwan va cpng su, 2008) va khdng MRSA. [5]

Chit khang nim: isoeulmorin (Alam vd cpng sy, 1996), culmorin (Strongman va cpng sy 1987), ierosphaeropsin tfl Microsphaeropsis sp. (Holler vd cdng sy, 2000), mactanamide (Lorenz va edng sy, 1998), 2 chit thudc nhdra macrodiolide tii Cladosporium herbarum (Jadulco va cpng su, 2001), 3 chit thudc nhdm lipodepsipeptide tac ddng len qua trinh tdng hpp thdnh tl bao cua nim vd ed tiem nang trong viec trj benh ndm ngodi da (Schilingham va cdng su, 1998). [5]

Chit chdng ung thu: penochalasins A-C chdng lai ddng tl bao P388 leukemia (Numata vd cpng sy, 1996; Iwamoto vd cdng su, 1999), trichdenones A-C (Araagata va cpng sy, 1998), communesins A va B tfl Penicillium sp. (Numata vd cpng sy, 1993, 1996), paeciloxoein A chdng lai ddng tl bao HepG2 (Wen va cdng sy, 2010), pentostatin A-D, nigrosporanene A chdng lai ddng te bdo MCF-7 va Vero (IC50: 9.37, 5.42 pg/ml) (Rukachaisirikul va cpng sy, 2010); Xylaria psidii cd hoat tinh manh is IC50 4 pg/ml va 1,5 pg/ml (Tarman vd cdng sy, 2011). [5]

(3 Viet Nam, cd mpt sd cdng trinh nghien cflu ve nam spi RNM:

'Tdng kit kit qua nghien cflu ve tinh da dang va vai ttd cua NS phdn Ifp tfl mpt sd RNM 6 hai tinh Nam Djnh vd Thai Binh" cua Mai Thj Hdng (2002).

"Khao sat hoat tinh ddi khang va tilm nang flng dung cua cdc chung NS phan Ifp tfl mdt sl khu RNM Nam Djnh va Thdi Binh" cua Mai Thi Hdng, Le Thanh Huyen (2002).

"Khao sat kha nang ki sinh gay benh cdn trflng va tiem nang kiem soat sinh hgc cua nam RNM Nam Djnh" ciia Mai Thi Hang, Nguyen Vinh Ha (2002).

Phan Thanh Phuang (2007), Bui Thi Nha (2008), Pham Thj Thiiy Trinh (2008), Pham Gia Cin (2009), Phung Van Phiie (2009), Khdng Thj Thu Hd (2010), Trucmg Mai Thanh Thanh (2010) nghien cflu ve khd nang sinh KS ciia mdt sd ehung NS du?c phan lap tfl RNM Cin Gid. Cac nghien cflu nay da phat hifn dupc nhilu loai NS cd kha nang sinh KS flc ehe VK, NS, nam men.

Nhin chung, nhung nghien ciru ve nam spi RNM sinh KS tten thi gidi va hong nude cho thdy, cae CKS tfl nim spi RNM cd boat tinh manh hon nhilu so vdi CKS til cdc VSV cd ngudn gdc tfl dit liln. Dilu nay chiing td vifc tim kilm cdc CKS mdi ti khu hf VSV RNM la rapt hudmg di dung ddn.

T^p chi KHOA HOC OHSP TPHCM TrSn Thj Minh Binh vi tgk

1. Vat lien va phuong phdp nghidn ciru

2.L Vftlifu

Cic giSng VSV dd sir dung

Cdc chflng NS phan lap tfl dit RNM Cdn Gid.

Vi khuan Bacillus subtilis, Escherichia coli nhdn t bd suu tap gidng eua Phdng Thi nghifm Vi sinh - Sinh hoa, Tr ng DHSP TPHCM.

Cdc MT nghiin cdru da su dung

MTl. Glucose 20 g, cao ndm men 2 g, peptone 5 g, MgS04.7H20 0,5 g, KH2PO4 lg, nude cat I lit. [10]

MT2. Cao malt 2,1 g, glicerol 4 g, peptone lg, nude cit 1 lit. [9]

MT3. Cao nira men 9g, glucose 20 g, KH2PO4 1 g, MgS04.7H20 0,5 g, peptone l,8g, nude'cit 1 lit. [9]

MT4. NaNOj 3 g, K2HPO4 1 g, KCl 0,5 g, MgS04.7H20 0,5 g, FeS04.7H20, cao nim men 5 g, sucrose 30 g, nude cit 1 lit. [7]

MT5. Glucose 30 g, bdt ddu tuong 2,5 g, cao nim men 0,5 g, KH2PO4 1 g, MgS04.7H20 1 g, NaCl 0,5 g, CaCl2.2H20 0,5 g, FeCl3.2H20 2 mg, ZnS04.7H20 2 mg, nude cit 1 lit.

MT6. Cao nim men 4 g, glucose 20 g, agar 20 g, nuoc biln 1 lit. [6]

MT7. Cao malt 20 g, peptone 1 g, glucose 20 g, agar, nude biln 1 lit.

MTS. NaNO, 3,5 g, K2HPO4 1,5 g, MgS04.7H20 0,5 g, KCl 0,5 g, FeS04.7H20 0,01 g, glucose 20 g, agar 20 g, nude biln 1 lit.

MT9. (MTnudi vi khuin kiim djnh) Cao thjt 5 g, peptone 5 g, NaCl 5 g, agar 20 g, nude cit 1 lit.

2.2. Phuang phip nghiin ciru

Cdc chiing NS dupe phan Ifp tfl dit RNM theo phuang phap eua Agate A.D. (1988). Sau do, dl tuyln chpn chiing cd hoat tinh khang sinh manh, cae chung NS nay dupc thfl ho^t tinh ddi khang vdi vi khudn B. subtilis va E. coli theo phuong phdp cua EgorovN.X(1985). [4]

Chung Dl dupc phdn loai den chi theo md td dac diem hinh thai cua Bui Xudn Ding (2004) [1], A. Samson (1996) [8]. Gidi ttinh ty gen 28S rRNA cua ehung nay vd so sdnh vdi ngdn hang gen NCBI dl phan loai din lodi.

Khdo sdt anh hudng eua ede dieu kien nudi edy (MT, ngudn carbon, ngudn nittogen, nong df NaCl, pH, nhiet dp, thdi gian) din boat tinh khdng sinh eua chiing Dl bdng edeh thay ddi cdc dilu kifn nudi cay ndi tten vd thfl boat tinh khdng sinh theo phuong phdp eua Kirby - Bauer (1966).

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Tap chi KHOA HOC DHSP TPHCM S8 47 ndm 2013

3. Kit qud vi bifn lufn 3.L Tuyin chgn chung ndm sfi cd hogt tinh khing khuin

Tfl 58 chung NS phdn Ifp dupc tfl RNM Cin Gid (ki hifu tfl DI - D58), chiing tdi tiln hanh hiyln chpn chung NS ed boat tinh doi khdng mynh bdng cdch thu hojt tinh khdng khuin ciia edc chung NS ndy vdi VK kilm djnh Id B. subtilis vd E. coU. Kit qud dupc trinh bdy ttong bdng 1.

Bing 1. Hogt linh dSi khdng cua cdc chung NSphdn lgp tie RNM Cdn Gid

STT

1 2 3 4 5 6 7 8 9 10

11

12

13 14

15 16

17

18 19

20

21

22

,23 24

25

26

27

28

29

Kihifu chiing

S l D2

D3

D4

D5

D6

D7

D8

D9

DIO

Di l

D12

D13

D14

DIS

D16

D17

D18 D19

D20

D21

D22

D23

D24

D25

D26

D27

D28

D29

Hoft tinh dii khang. D-d (cm)

B. subtilis

2,74

. 1,40

. 0,75

1,45

1,05

1,00

1,60

. 0,60

0,60

.

. 1,10

--

0,60

1,60

0,60

0,90

-1,70

0,70

1,00

£. coli

1,10

-

-. . -

--. . . . -. . . . . --------

STT

30 31 32

33 34

35

36

37

38

39

40

41

42

43

44

45 46

47

48

49

50 51 52

53 54

55

56

57

58

Ki hieu chiing

D30

D31

D32

D33

D34

D35

B36

D37

D38

D39

D40

D41

D42

D43

D44

D45

D46

D47

D48

D49

D50

DSI

D52

D53

D54

D55

D56

DS7

D58

Hoat tinh dii Idiang, D-d (cm)

B. subtilis

0,50

0,75

0,60

0,50

1,45

0,45

0,60

0,60

1,10

1,20

0,95

. 0,65

1,50

1,20

-1,60

1,50

1,70

1,85

-1,80

1,10

E. coli

---

-1,00

1,75

-0,90

---.

.

1

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T?p chi KHOA HOC BHSP TPHCM Trdn Thf Minh Bjnh va tgk

Kit qud dupc trinh bdy trong bdng 3.1 cho thiy trong sd 58 chflng NS cd:

1/58 chung ddi khdng vdi cd B. subtilis vd E. coli. 19/58 chung khdng cd hoyt tinh ddi khdng vdi cd B. subtilis vd E. coli.

3/58 chung cd hoyt tinh dii khdng vdi E. coli, ttong do 1/58 ehung dii khdng d mflc ttung binh, 2/58 chiing doi khdng d mflc ylu.

37/58 chung cd hoyt tinh doi khdng vdi B. subtilis. 1/58 chiing dii khdng vdi B. subtilis a mflc rit manh. 9/58 chiing dii khdng vdi B. subtilis a raiic ttung binh. 26/58 chflng khdng B. subtilis a raflc ylu.

Chiing Dl ed boat tinh khdng vdi B. subtilis a mflc rit manh (D-d = 2,74 ± 0,01 cm), khdng E. coli a mflc ylu (D-d = 1,10 ± 0,00 cm). CKS cfla chflng Dl khdng VKG(-H) d mflc rat mynh ndn cd gif trj cho nhOng nghien cflu tilp theo.

Hinh 1. Hogt tinh ididng B. subtilis Hinh 2. Hogt tinh khdng E. coli cda chung BI cua chung BI

3.2. Bfc diim sinh hgc viphan logi chung Bl

Nudi chung Dl tten MT Czapek d 25°C, quan sdt hinh dsing, mdu sde, bl mft khuan lac, sdc t l tilt vdo MT. Ldm tidu bdn quan sdt hinh dang, mdu sdc khuin ty, dfc dilm CO quan smh sdn. Kit qud dupc trinh bdy trong bdng 2.

Bing 2. Bdc diim phdn logi chung n im sgi Bl

D|c dilm chung Dl

- Khuan 1^ ban dau cd mitu triing, sau chuyin thanh v^g nSu, cuoi cung chuyin thfinh m^u nSu.

- Khu^ ty ph§n nhSnh, khong miu, c6 vfich ngSn.

- Gid bio tiJr tran khong phan nhdnh, c6 phsta dinh phinh ra thinh bpng hinh nia c4u.

- Khdi bio tOr trin d^ng hinh tia tda tr6n.

D$c diem phan lo^i chi Aspergillus theo Bui Xuan Ddng (2004) [1], A. Samson

(1996) m

- Khuin lac thudng phit trien nhanh, c6 miu tring, ving, vai^ nau, niu den ho$c hoi c6 miu l\ic.

- Khuin ty phan nhanh, khdng miu, miu nh^t hoic trong sSm miu, c6 vich ngin.

- Gii bio tu trin khong phin nhinh, khdng c6 ho|c it c6 vich ngin ngang, c6 phin dinh to ra thinh b9ng hinh chuy, hinh elipse ho$c hinh nura cau.

- Kh6i bio tu trin co thi c6 cic d^ng hinh cgt, hinh ciu ho$c Unh tia tda trdn.

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Tgp chi KHOA HOC BHSP TPHCM SS 47 ndm 2013

So sanh kit qua quan sat dupc vd dfc diem ttong khda phdn loai cua Bui XuSn Ddng (2004), Samson A. R. (1996), chiing tdi kit lufn chung Bl thudc chi yJ pergiV/m.

Hinh 3. Khudn lgc chung BI Hinh 4. Caquansml^ar^hdngBI

De djnh danh den lodi ehiing Aspergillus Dl, chung tdi gfli chung ndy din cdng ty xet nghifm Nam Khoa gidi trinh ty gen 28S rRNA, kit qud nhu sau:

GAAAGGGAAGCGCTTGCAACCAGACTCGCTCGCGGGGTTCAGCCGGG CTTCGGCCCGGTGTACTTCCCCGCGGGCGGGCCAGCGTCGGTTTGGGCGGC CGGTCAAAGGCCTCCGGAATGTAGCGCCCTTCGGGGCGCCTTAATGCCGGC GGTGCAATGCGGCCAGCCTGGACCGAGGAACGCGCTTCGGCACGGACGCT GGCATAATGGTTGTAAACGACCCGTCTTGAAAC

So sdnh vol ngdn hang gen NCBI, trinh ty gen 28S rRNA cua ehung Aspergillus Dl ed dp hrong ddng 100% vdi lodi Aspergillus terreus. Kit luan: chflng Dl thufc loai Aspergillus terreus.

3.3. Khio sdt inh hu&ng cia cic dieu ki^n MT lin khi nang sinh trudng vi sinh khing sinh

Cdc dieu kifn nudi ciy nhu thdnh phin MT, ngudn carbon, nguIn nittogen, pH MT, nhift df nudi cay,... cd dnh hudng rit ldn din sy sinh ttirdng cung nhu kha ndng sinh khdng sinh cfla VSV ndi chung, NS ndi rieng. Do dd viec nghien curu tim ra cac dilu kifn nudi ciy thich hpp cho sinh tdng hpp CKS la dilu ein thilt. 3.3.1. Anh hudng cua MT nuoi cdy

Nudi chung Asp. terreus Dl tten cdc MT khac nhau (MTl din MTS nhu da ttinl bay ttong phin 2.1.), sau 6 ngdy thu hoat tinh ddi khang vdi B. subtilis, kit qua duoc tiinh bdy trong bdng 3.

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T9P chl KHOA HOC DHSP TPHCM Trdn Thi Minh Binh vi tgk

Bing 3. Anh huang cua MT nuoi cdy len khd ndng sinh tnrang

vd sinh khdng sinh cua chiing ndm sgi Asp. terreus Bl

STT

1

2

3

4

5

6

, .1

8

Moi trudng

MTl

MT2

MT3

MT4

MTS

MT6

MT7

MTS

Hoft tinh doi khdng, D-d (cm)

1,38 ±0,04

1,38 ±0,04

1,41 ±0,03

0,32 ± 0,02

0,68 ±0,15

3,20 ±0,05

1,85 ± 0,06

1,81 ±0,10

Sinh khii (g)

0,605 ± 0,026

0,100 ±0,012 •

0,840 ±0,064

0,855 ± 0,003

0,350 ±0,006

0,365 ± 0,003

0,185 ±0,020

0,680 ±0,000

Kit qud eho thiy chung Asp. terreus Dl cd hoyt tinh dii khang vdi B. subtilis manh nhit khi nudi ciy trong MT6. 6 MT4, chiing Asp. terreus Dl sinh trudng raynh nhit nhung lyi ed boat tinh ddi khdng vdi B. subtilis ylu nhdt. Dilu nay cho thiy khd ndng sinh trudng vd sinh khdng sinh ciia ehiing Asp. terreus Dl Id khdng dong nhat vdi nhau. So vdi cdc MT khdc, MT6 cd thdra 2 lo?i khoang Id Ca (dudi dang CaCl2) va Zn (dudi d^g ZnS04). Theo N.S. Egorov (1985) [4], Fe vd Zn cin thilt cho sy sinh tdng hpp mpt so chit khdng sinh. Cd thi 2 loai khodng ndy cd tdc dyng tich cyc din qud trinh sinh tong hpp chit khdng sinh eua ehiing.,4jrp. terreus Dl.

Hinh 6. Hogt tinh khdng sinh cda chung Asp. terreus Dl trinMT4

Hinh 5. Hogt tinh khdng sinh cug chung Asp. terreus Bl trenMTd

3.3.2. Anh hudng cua ndng do mudi

Df mfn ciia MT Id rapt ttxmg nhOng ylu td dnh hudng quan ttpng din khd ndng sinh ttudng vd sinh khdng sinh eua NS. Do dd, chiing tdi da tiln hdnh khdo sdt dnh hudng efla nong dp raudi NaCl ttong MT bdng cdch nudi chung Asp. terreus Dl tten MT6 thay dii ndng dp mudi tfl 0-6%, sau 6 ngdy khdo sdt hoyt tinh ddi khdng vdi B. subtilis, kit qua dupc trinh bdy ttong dl thj 1.

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T9P chl KHOA HOC DHSP TPHCM S6 47 n&m 2013

Bd thf 1. Anh huang cua ndng d$ NaCl len hogt tinh Ichdng khuSi chiing Asp. terreus Bl

4.00

3.50

1.00

•

^ » - « . 1 . 3 7 . „ „

TP BS

0.900 0.800 0.700 g

0500 5 0.400 1 0.300

0.200

1.5 2 2.5 3 3.5 4 4 5 5 5 5 6

N n g d NaCI(%)

Kit qua cho thdy chung Asp. terreus Dl c6 ho^t tinh khing sinh manh trong khoing n6ng dp mudi NaCl tir 0-1%. Khi ndng dO mu6i >1%, ho^t tinh khang sinh ciia chung niy giam. Din n6ng dO NaCl 6% chung Asp. terreus Dl khdng cdn ho?t tinh khang sinh. Khi nang sinh trudng cua chiing nay ting dan trong khoing nong dd NaCl tir 0-5,5%. Dilu nay chiing td chiing Asp. terreus Dl li chung ua m§n.

Hink 7. Hogt tinh /didng sinh cua chung Hinh 8. Hogt tinh khdng sinh cua chung Asp. terreus Dl & ndng dd mudi 1% Asp. terreus Dl d nong do muoi 6%

3.3.3. Anh hu&ng cua nguon carbon

Nudi chung Asp. terreus Dl tren MT6, 1% NaCl, thay ddi ngatun carbon bing cic loai dudng glucose, maltose, galactose, tinh bot, lactose, ri dudng, fructose, sucrose, sau 6 ngiy khio sit hoat thih d6i khing vdi B. subtilis, kit qua dugc trinh biy trong bing 4.

Bdng 4. Anh huang ciia ngudn carbon len hogt tinh KS chimg Asp. terreus Dl

Neuin carbon Glucose Maltose

Galactose Tinh bpt Lactose

Ridudng Fructose Sucrose •

Ho$t tfnh dii khdng. D-d (cm)

3,0«±0,14 2,42 ±0,11 1,13 ± 0,26

3,08 ±0,14 2,88 ± 0,06 2,87 ±0,07 2,97 ±0,04 2,42 ±0,12

Sinh khii (e) 0,74 ±0,04

0,73 ± 0,01 0,49 ± 0,04 0,78 ±0,05 0,84 ± 0,16 0,56 ±0,01 0,74 ±0,01 0,91 ±0.03

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'mp chl KHOA HOC DHSP TPHCM Trdn Thi Minh Binh vi tgk

Kit qud cho thiy chflng Asp. terreus Dl cd hoyt tinh khdng smh tten tit ed cdc nguon carbon nghidn cuu. Trong do, chflng ndy cd hoyt tuih khdng sinh rit raanh (D-d > 2,5 cm) ttdn cdc nguon carbon Id glucose, tinh bpt, lactose, ri dudng va fructose. Tten 2 nguon carbon la glucose vd tinh bf t chiing nay cho hoat tinh m ^ nhit.

Hinh 9. Hogt tinh khdng sinh cua cftung Asp. terreus Bl trinMTcd ngudn carbon

Id glucose

Hinh 10. Hogt tinh khdng sinh cda citung Asp. terreus Bl trin MT cd ngudn carbcm

la maltose

3.3.4. Anh huang cda ngudn N

Nudi chflng Asp. terreus Dl ttdn MT6, 1% NaCl, nguIn carbon la glucose, thay dii nguIn nittogen, sau 6 ngdy khdo sat hoyt tinh ddi khdng vdi B. subtilis, kit qua dupc trinh bdy trong bdng 5.

J 5. Anh huang cua ngudn nitrogen lin hogt tinh khdng sinh

cua chung Asp. terreus Bl

Nguon nitrogen

0,5g bft dfu tinmg -1- 2,5s cao nam men

3g cao thjt

3g cao nim men

3g bft dfu tuong

l,5g peptone + 1,5 g cao thjt

.3gNH4CI

,3g(NH4)2S04

3gNH4N03

2,5g bft dfu tuong -i- 0,5 g eao nim men

3gNaNO,

3g peptone

Hoat tinh dii khdng. D-d (cm)

3,54 ±0,05

2,20 ±0,05

3,00 ±0,06

1,70 ±0,09

1,55 ± 0,04

1,64 ±0,07

1,68 ±0,03

1,70 ±0,05

3,33 ± 0,05

1,85 ±0,00

2,38 ±0,07

Kit qud cho thiy chiing Asp. terreus Dl cd hoyt tinh khdng sinh trdn tit ed edc nguon nitrogen nghidn cflu. Trong dd, chflng ndy cd hoyt tmh khdng sinh m ^ ttdn nguon nittogen Id cao nara raen vd cao nim raen + bft dfu tuong. Khi MT chi ed nguIn nitrogen Id bf t ddu tuong thi boat tinh khdng sinh cua chflng ndy khdng m ^ . Khi MT chi cd nguon nittogen Id cao nam men, thi chiing nay cho ho^t tinh khdng sinh

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Tap chi KHOA HOC DHSP TPHCM Sd 47 nam 2013

rit manh. Khi MT cd ca 2 ngudn nitrogen l i cao nim men va diu tuong thi chung nay cho hoat tinh khing sinh manh hem ca. Chung nay cho hoat tinh khing sinh mgnh nhil khi trong MT cd 0,5g bot dau tuong va 2,5g cao nam men.

Hinh 11. Hogt tinh khdng sinh cua chiing Asp. terreus DJ trong MT co nguon nitrogen id 0,5g bot dgu tucmg vd 2,5g cao ndm men

Hinh 12. Hogt tinh khdng sinh dm chung Asp. terreus Dl trong MT cd nguon nitrogen Id 2,5g bot dgu tucmg vd 0,5 g cao ndm men

3.3.5. Anh hu&ng ciia nhiet dp

Nudi chung Asp. terreus Dl tren MT6, 1% NaCl, ngudn carbon la glucose, ngu6n nitrogen gdm 0,5 g bpt dau tuong va 2,5g cao nim men, d cac dieu kidn nhidt dd Ididc nhau. Sau 6 ngay khio sit hoat tinh ddi khing vdi B. subtilis, kit qua duoc trinh bSy trong bang 6.

Bdng 6, Anh hu&ng ciia nhiet dp len hogt tinh khdng sinh chiing Asp. terreus Dl

Nhift df (°C)

20

25

30

35

40

Hojt tinh dii khdng, D-d (cm)

3,24 ±0,09

3,83 ± 0,17

3,39 ± 0,06

2,64 ± 0,06

1,26 ±0,06

Sinh khoi (g)

1,31 ±0,01

1,30 ±0,05

1,61 ±0,06

1,21 ±0,07

1,31 ±0,01

Ket qua cho thay chung Asp. terreus Dl co hoat tinh khing sinh d tit ci cic diSu kien nhiet do nghien cuu. Trong do, chung nay cho hoat tinh rit manh trong khoang nhiet dp tir 20-35^, manh nhit d 25°C.

Hmh 13. Hogt tinh khdng sinh ciia chung Asp. terreus Dl & 25°C

Hinh 14. Hogt tinh khdng sinh cua chiing Asp. terreus Dl &35°C

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T?p chl KHOA HOC DHSP TPHCM Tran TM Minh Binh va tgk

3.3.6. Anh hudng cuapH ban ddu trong MTnudi cdy

Ndng dd ion H* bode OH" lam thay ddi dp phan li cua cac chit ttong MT do dd pH cd anh hudng quan ttpng din sinh trudng, ttao dii chit cua VSV. pH edn anh hudng quan ttpng din hoyt tinh enzyme, din san phim trung gian, din sy phan li, su hda tan,... do do anh huong din sinh tdng hpp khdng sinh. Vi vay, chiing tdi tiln hanh khao sat anh hudng eua pH len hoat tinh khdng sinh cila ehiing Asp. terreus Dl. Nudi chung nay tten MT6, 1% NaCl, ngudn carbon la glucose, ngudn nittogen gdm 0,5 g bot dau tuong + 2,5g eao nim men, d 25°C, vdi pH MT dupc dilu chinh frong khoang tfl 4 - 7 bdng NaOH IN va HCl IN. Sau 6 ngay, khao sat hoat tinh ddi khang vol B. subtilis, kit qua dupc trinh bay trong dd thi 2.

Bd thi 2. Anh hu&ng cua pH len hogt tinh ichdng sinh chung Asp. terreus Bl

4.50 4.00 3.50

r 3.00 [ 2.50 ' 2.00

1.50 1.00 0.50 0.00

i 4 , - 1 3 - - -4ca-- — --4oe

4 4.5 5 5 5 6 6.5 7

Ket qua cho thdy chflng Asp. terreus Dl cd hojt tinh khdng sinh rit manh d tit ca cdc dieu kifn pH nghien ciiu. Trong do, chung nay cd hoat tinh khang sinh manh nhdt d pH 5,5.

Hinh 15. Hogt tinh khdng sinh cua chung Hinh 16. Hogt tinh khdng sinh cda chung Asp. terreus BI trin pH = 4,5 Asp. terreus DI tren pH = 5,5

3.3.7. Bong thdi len men

Nudi chiing^jp. terreus Dl tten MT6, 1% NaCl, ngudn carbon la glucose, ngudn nittogen 0,5 g bpt dfu tuong + 2,5g eao nim men, d 25°C, pH MT 5,5. Sau 12 gid khao sat hoyt tinh ddi khdng vdi B. subtilis, do pH dich ldn men va can sinh khii khd, kit qud dupe ttinh bdy ttong dl thi 3.

129

Tgp chi KHOA HOC BHSP TPHCM SO*/ nam 2013

Bo thi 3. Bgng thdi qud trinh lin men

450 400

i "° ; I -1 5 200

« ;" 050 000

/ \ , .-A—

\

_/^___ 24 36 48 eo 72 84 96 10e 120

Th Iglantfll }

-•-D-d(cm) -fc-Slnhkh

132 144 15S

1(9)1

166 160

4500 4000 3500 3000 g Z500 '

1500 • 1.000

0000

Kit qud cho thiy, sinh khdi ciia chiing Asp. terreus Dl tdng din tfl 24h din khodng 84h, sau dd d?t tt^ng thdi In dinh din khodng 132h vd gidm nhp. pH MT giam ttong thdi gian diu, t i pH ban diu cua MT Id 5,5 gidm din 3,84 sau 48h nudi ciy.

Hoat tinh khdng suih ciia chflng nim spi nghidn citu tang din sau 24h, dat mflc tk manh sau 96h nudi ciy vd dat eye dai d 108h, sau dd gidm nh? vd giu tt-ang thdi In ^ cho din i68h. Hoat tinh khdng smh In dinh d mflc manh ttong thdi gian kha dai (72h, til 96h-I68h) tao dilu kifn thufn lpi cho vifc thu chit khang sinh tfl ehiing NS ndy.

Hinh 17. Hogt tinh khdng sinh Hmh 18. Hogt tinh khdng sinh cug chung Asp. terreus BI sgu 48h cug chung Asp. terreus Bl sau lOSh

Nhu vfy, sau khi chung tdi khao sat tim cdc dilu kifn nudi ciy thich hpp cho hoat tinh khang sinh ciia chflng Asp. terreus DI, boat tinh ddi khdng cfla chiing ndy vdi B. subtilis tang len dang kl tit 2,74 ± 0,01 cm din 4,37 ± 0,03 cm (tdng 1,59 lin). 4. Kit lufn

Qua qua ttinh tiln hdnh dl tdi, chflng toi da thu dupe nhflng kit qud nhu sau;

Da phan ldp dupc 58 chflng NS tii RNM Cin Gio vd tiiyln chpn dupc chiing Bl cd hosit tinh dii khdng manh vol B. subtilis.

- Da dinh danh chiing nim spi Dl cd boat tinh khdng sinh m?mh thufc loSi Aspergillus terreus.

Da khdo sat vd lira ra cdc dilu kien nudi ciy thich hpp eho chiing Asp. terreus Bl sinh khdng smh manh nhit nhu sau: glucose 30 g, bf t ddu tuong 0,5 g, cao nim men 2,5 g, KH2PO41 g, MgS04.7H20 1 g, NaCI 0,5 g, Ca/Ch-lUiO 0,5 g, FeCl3.2H20 2nig, ZnS04.7H20 2 mg, nude cit I lit; pH 5,5; nhift dp nudi ciy 25°C; thdi gian nudi ciy 108 gid.

130

T SP chi KHOA HOC DHSP TPHCM Trin Thi Minh Binh vi ^k

TAI U f u THAM KHAO 1. Bfli Xudn Dong (2004), Vi ndm dung trong cong nghe sinh hgc, Nxb Khoa hoc va ki

thuft, tt. 154-160

2. Brakhage A. A., Seheper T. (2004;, Molecular Biotechnology of Fungal p-Lactam Antibiotics and Related Peptide Synthetases Advances in Biochemical Engineering/Biotechnology, Vol. 88, Springer Berlin Heidelberg New York, pp. 47-49.

3. Carsten Christophersen, Oscar Creseente, Jens C. Frisvad, Lone Gram, Joan Nielsen, Per Halfdan Nielsen, Lisa Rahbaek (1999), "Antibacterial activity of marine-derived 6ia^", Mycopathologia, 143(3), pp. 135-138.

4. Egorov N.S. (1985), Antibiotics - A scientific approach, MIR, pp. 76-340.

5. Gareth Jones E. B. (2011), "Fiily years of marine mycology". Fungal Biversity, 50, pp. 73-112.

6. Masahira Nakagawa, Arika Hirota, Heiichi Sakai (1982), "TerrecycHc acid A, A new antibiotic from Aspergillus terreus". The Journal of Antibiotics, 35(7), pp. 778-782.

7. Pehir W. Dalsgaard, Thomas O. Larsen, Carsten Christophersen (2005), "Bioaetive Cyclic Peptides from the Psychrotolerant Fungus Penicillium algidum". The Journal of Antibiotics, 58(2), pp.141-144.

8. Robert A. Samson, Ellen S. Hoekstta, Jens C. Frisvad (2004), Intruduction to food and airborne ficngi, Centtaalbureau voor Schimmelculture_Uttecht, pp. 52-76.

9. Simone Roehfort, Joanne Ford, Simon Ovenden, Soo San Wan, Samantha George, Howard Wildman, R. Murray Tait, Barbara Meurer-Grimes, Susan Cox, Jonathan Coates, David Rhodes (2005), "A Novel Aspochalasin with HIV-1 Integrase Inhibitory Activity from Aspergillus fiavipes". The Journal of Antibiotics, 58(4), pp. 279-283.

10. Soo-Jin Choo, Hae-Ryong Park, In-Ja Ryoo, Jong-Pyung Kim, Bong-Sik Yun, Chang-Jin Kim, Kazuo Shin-ya, Ick-Dong Yoo (2005), "Deoxyvenucosidin, a Novel GRP78/BiP Down-regulator, Produced by Penicillium sp.". The Journal of Antibiotics, 58(3), pp. 210-213.

(Ngay Tda so?n nhin duvc bai: 05-6-2013: ngiy phin biin dinh gii: 20-8-2013:

ngiy chip nhin ding: 21-6-2013)

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