nghien cltu tuyen chon chung vi khuan lactic co kha nang

Tran Van Chi vd Dtg Tap chi KHOA HOC & CONG NGHE 177(01): 4 3 -

NGHIEN CLTU TUYEN CHON CHUNG VI KHUAN LACTIC CO KHA NANG

SINH B A C T E R I O C I N CAO TU" CAC SAN P H A M LEN MEN TlT NHIEN

Tran Van Chi, Pham Thj Tuylt Mai' Trieang Dai hoc Nong Ldm — DH Thai Nguyen

TOM

T A T

Vi_ khuan lactic da dupe tim thiy va sd dung nhieu trong cac san phim len men truyen thing, theo dieu kien ty nhign. Nhieu nghign ciiu gin dSy da chiing minh vi khuan lactic cd kha nang sinh ra bacteriocin (mflt logi khang sinh sinh hpc cd ban chat protein). Muc dich ciia nghien cuu nay la phan lap va tuyin chon dupe chung vi khuan lactic efl kha nSng sinh bacteriocin cao tir cac san pham Ign men ty nhign. Tir 6 san pham len men ty nhien da phan lgp dupc 14 chiing vi khuin sinh axit lactic va xac djnh dupc d^c diem sinh hoc, sinh ly va hda sinh chiing. Nhiing chung da phSn lap duoc da khao sit kha nang sinh bacteriocin thi hien d kha nang khang vi khuan kiem djnh Staphylococcus aureus, trong do co 8 chiing the hign tinh khang khuan tich cyc (hieu sfl dudng kinh vdng trdn khang khuan Idn hem 10 mm). Tir 8 chung qua so loai da chpn ra dupc chiing SC3 CO kha nSng sinh bacteriocin cao nhat, the hien hoat tinh khang vdi ca 3 chung kiem dmh Staphylococcus aureus, E.coli, Samonella vdi hieu sfl vong khing khuan tir 19,5 den 24 mm. BSn cgnh do, nghien ciiu da chimg minh dupc tdc dp cua qua trinh ly tam d l tach sinh khdi v i tap chat nhim thu hfli bacteriocin tho khdng anh hudng den hogt tinh khang khuan raa chi anh hirong den dd sgch cua che pham, toe do ly tam 13000 vong/phiit la phu hpp nhat.

Tu'khda; Bacteriocin, khdng khudn, lactic, len men, vi khudn D A T V A N DE

Vi khuan lactic ( L A B ) d u g c sii dung nhieu trong thyc pham Ign men n h y d u a muoi, ca mudi, len men r u a u , s u a chua, nem chua, phomai, bia. Chiing cd vai trd quan trpng trong ddi song nhu tang cudng mien dich, lie che cac vi sinh vgt cd hai, bo sung cac vi sinh vgt cd lgi, tac dung khang khuan [1]. Dgc tinh nay la do vi khuan lactic sinh axit hflu c a va sinh bacteriocin khang khuan, khang nam [10].

Bacteriocin la chat khang khuan cd ban chat la protein d u g c tdng h g p d riboxom tii cac chudi peptit hogc protein d ca vi khuan Gram am v a Gram d u a n g [5], Bacteriocin cd kha nang tieu diet cac vi khuan do s u tao thanh cac kenh lam thay doi tinh t h i m cua mang te bao, nhilu logi bacteriocin cdn cd kha nang phan giai D N A , R N A va tan cong vao peptidoglycan lam suy yeu thanh te bao cua vi k h u i n gdy hai [3], Vi vay bacteriocin cd rat nhieu iing dung trong san x u i t my p h i m , x u li mdi trudng, che b i i n thiic an chan nudi, trong y hpc va trong c6ng nghe thyc pham [2].

Ngoai ra, bacteriocin kh6ng gay ra phan img

Tel: 0965 051220, Email: phamthHuyetmai@iuaf edu vi

di ling cho con ngudi v a van de ve siic khde, do chiing bj phan hiiy nhanh bdi enzym proteinase, lipase [9] H a n niia, chiing hoat dpng trong khoang p H r6ng, ch6ng Igi nhieu vi khuan gay h u hdng t h y c pham, c o che hoat dpng d u a tren tfnh tham cua mang tg bao vi khuan nen khdng gay ra tinh de khang cheo vdi cac khang sinh va d u g c ma hda bdi cac gen n i m trgn plasmid nen thuan tien cho thao tac di truygn [7].

N G U Y E N / V A T LIEU v A PHU'ONG PHAP N G H I E N C U U

N g u y e n / v a t lieu

Cac san pham len men t y nhien: Ca phao mudi, me, dua chudt mudi, bap cai mudi, d u a cai mudi - mua tai chg trung tam thanh ph6 Thai Nguygn, siia tuai lay tgi trgi chan nu6i cua Khoa Chan nudi Thii y - Tru'dng Dgi hgc Ndng Lam Thai Nguyen de len men chua ty nhien.

Mdi trudng thach M R S (g/l)

M6i trudng MPA nu6i vi sinh vat kiem dinh ( ^ ) Vi sinh vat kiem dinh: Staphylococcus aureus, Samonella, E.coli - lay tai ngan hang gidng vi sinh vat ciia Khoa C N S H - C N T P - trudng D H Ndng Lam Thai Nguyen.

43

Tran Van Chi vo Dig Tgp chi KHOA HQC & CONG NGHE

177(01): 43-48

Phirong phap nghien ciru

- Phuang phap phan lap va tuyin chgn vi sinh vat [1],

- Phuong phap xac djnh hoat tinh catalase:

Nhd tryc tiep vai gipt H2O2 3% len bl mat khuan lac vi khuan lactic da dupc nudi sau 48 gid trong mdi trudng MRS d 37''C. Quan sat sy xuat hien bpt khi bang mit thudng, Neu thay cd xuat hien bpt khf thi phan iing la duong tinh (+), ngugc Igi khdng xuat hign bpt khf tiic la phan ung catalase am tinh (-) [8], - Phuong phap xac djnh kha nang di dpng cua Vl khuan: Chiing vi khuan dupc ciy vao ong nghiep chiia mdi trudng MRS dac theo phuang thing diing, nudi 37°C trong 48 gid.

Neu vi khuan mgc lan ra xung quanh dudng cay chiing td vi khuan cd kha nang di dpng (+). Ngu vi khuan mpc thang dung theo dudng cay thi vi khuan khdng cd kha nang di dpng(-)[8].

- Phuong phap nhugm Gram: Diing que cay v6 triing lay mpt it vi khuan tii thgch (sau khi cay 48 gid) hoa vao I gigt nude cat d giiia phign kinh, lam khd trong khdng khi. Ho nhanh vet boi trgn ngpn lira den cdn 2-3 lan.

Nhupm bang dung djch tim ket tinh trong I phut, riia nude, tham khd. Nhupm lgi bing dung dich lod trong I phut, riia nude, tham kh6. Nhd djch tSy mau, giQ' khoang 30 giay (cho den khi vira thay mat mau), nia nude, tham khd. Nhupm bd sung bang dung djch Safranin trong 2-3 phiit, rira nude, dg khd trong kh6ng khi. Soi kinh: Dung vat kinh diu lOOx. Kit qua: Vi khuin Gram duang (+) bit mau tim. Gram am (-) bit mau dd.

- Khao sat va chpn Ipc cac ddng vi khuan lactic sinh bacteriocin cd tinh khang khuin cao bing phuang phap khuyich tan tren gieng thach [6],

- Phuong phap due 16 thach dl nghien ciiu kha nang khang khuan cua chl phim

bacteriocin tho: Chiing sinh t6ng hop bacteriocin dugc nu6i tang sinh tren moi trudng MRS i6ng d nhiet dp 37°C sau 48 gid, chuyin 1 ml vao ong Eppendoff roi dem ly tam (5.000-14.000 vdng/phut) frong 10 phut de logi bd sinh khoi, thu djch ndi. Hiit 10 jil djch bacteriocin th6 bom vao gigng thach da due Id vdi kich thudc nhu nhau (dTa thgch da chang vi sinh v|t kilm dinh dl khd). Tiln hanh u cac dta trong 48h d 37°C, tien hanh quan sat va ghi nhgn kfch thudc vdng khang khuan xuat hien quanh khuan lgc vi khuan.

KEY QUA VA T H A O L U A N

Ket qua phan lap chung vi khuan lactic Da phan lap dugc 14 chimg vi khuan tir 6 mau thyc pham Ign men ty nhien. Ket qua cho thay: Dac digm sinh hgc, sinh ly va sinh hoa cda cac chimg rat da dang va diu cd kha nang sinh axit lactic khi thu nghiem trgn mdi trudng MRS. Nhung khuan lac do duac iam thuan bang each cay chuygn nhieu lan roi dupc giS giong trong dng thach nghigng bao quan trong tii Ignh 3 - 5°C, sau 14 ngay cay chuygn giir gi6ng mot lan.

Ket qua phan lap cho thay dac diem hinh dang te bao cua cac chdng tap trung d 3 dgng:

Que ngin, cau va elip va hinh thai khuan lac rat phong phii, kich thudc cac khuan Igc tii nhd khoang 1 mm den Idn 12 mm mpc rigng re, cac chiing dgu cd kha nang sinh axft lactic, am tfnh vdi phan iing catalase, khdng cd kha nang di dpng va diu Gram duang, Su dung 14 chiing LAB phan lgp dugc de tien hanh tuyen chpn chiing cd kha nang sinh bateriocin cao nhat.

Ket qua tuyen chpn chung LAB sinh

bacteriocin cd tinh khang khuan cao

Ket qua khao sat hogt tinh khang khuan ciia

cac chiing LAB da phan lgp dugc vol vi

khuan kiem djnh Staphylococcus aureus dupc

the hign trong bang 2.

Tran van Chi vd Dtg Tgp chi KHOA HQC & CONG NGHE 177(01): 43- Bang L Dgc diim sinh hgc, sinh ly vd sinh hda cda cdc chung LAB da phdn lgp dugc Nguon Ki

phan higu lap chiing

Htnh dang te

b^o

Kha nSngdi

dgng

w. , „ ^ Kha nang Boat tinh . . .= : , smh axit

catalase , .. lactic

Hlnh thai khuan lac

phao CPM2 Que n Tron, nhan

bong, tring n 3

4 5 6 7 8 9 10 II

Me Dua chupt mudi Bip cai

muoi

Dua cai muoi

CPM3 Ml M2 DChMl DaiM2 BCMl BCM2 DCMl DCM2

Elip Que ngin Que ngin c i u c i u Elip Que ngan

c i u Elip

+ + + + + + + + +

+ +

± + + + + + +

Tron, tron bdng, tring nga Tron, tron bdng, tring siJa Tron, tron bdng, tring stta Tron, nhin bong, trang nga Tron, tron bflng, trang nga Tron, tron bdng, tring sQa Trfln, tron bdng, tring stta Tron, tron bdng, tring sffa Trdn, ria gpn, tring siia 12

13 14

SOa

tuai de

ien

men chua tu

nhien

Tron, ria deu, tring nga Trdn, trcm bong, trang nga

SC3 Que ngan Trdn, tron bflng,

tring si!a Bang 2. Hoat tinh khdng Staphylococcus aureus cua cdc chung LAB thu nghiem Chjing khao sat

Dirdng kinh vong khang

k h u i n (R-r)(mm) Chiing khao sat

Dudng kinh vdng khing k h u i n (R-r) (mm) CPMl

CPM2 CPM3 Ml M2 DChMl DChM2

BCMl BCM2 DCMl DCM2 SCI SC2 SC3

14 chiing vi k h u i n da dupc k i l m tra tfnh khang k h u i n , cd 8 chiing t h i hien tinh khang tfch cyc (hi?u so d u d n g kfnh vdng trdn khang khuin >10 mm), Trong 8 chung cho k i t qua tich cyc cd hai chiing: SC2, SC3 t h i hien tinh khang khuan cao nhit, vdi higu so d u d n g kinh vdng khang khuin lan lugt la 20 mm va 24 mm nen d u p c chpn de tiln hanh cac khao sat t i i p theo, Dg chgn ra chiing cd kha nSng khang k h u i n tot nhit, thf nghigm t i i p theo dupc tiln hanh n h i m khao sat kha nang khang da khuan cua SC2, SC3 dugc thyc hipn vdi chung kilm djnh la Samonella.

Dya vao ket qua nghien ciiu t h i hign tren hinh I cho thay chiing SC3 (cho dudng kinh vdng khang k h u i n 19,6 mm) cd kha nang khang lai vi sinh vgt kiem dinh Samonella tot hon SC2 (cho dudng kinh vdng khang k h u i n 9,5 mm) nen SC3 dugc tuyen chpn d l tien hanh cho cac nghien cuu tiep theo.

Tran Van Chi vd Dtg Tgp chl KHOA HQC & C 6 N G NGHE 177(0!): 4 3 - 4 8

Chung

Hlnh 1. Khd ndng khdng khudn cita chimg SC2 vd SC3 doi v&i Samonella Bang 3. Khdo sal dieu ki4n tdch bacteriocin lu chiing nghiin cicu

Tdc do ly tam

(v6ng/phiit) 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000 Ghi C/IH: Vong khang khuSn

Vong khang khuSn

,S. aureus 24,1' 24,2' 24,2' 24,2' 24,3"

24,3' 24,3' 24,4' 24,4*

24,4*

v§n con khuin lac

V6ng Vii&tiz khuan (K- E co«

20,4' 20,4"

20,4' 20,5' 20,4' 20,5' 20,5' 20,6' 20,6*

20,6*

nghien cliu phat trien (*) khong con khuan lac nghien cuu phat trign (+) NghiSn c u u k h a n a n g k h a n g

p h a m bacteriocin t h o

khuan ciia

r) (mm) Saittanella

19,6' 19,6' 19,6' 19,5"

19,5' 19,5"

19,5' 19,5' 19,5*

19,5*

che va thtrc hien a nhiet dQ IS^C trong thoi gian 10 phiit.

Cdc thi nghi?m a hai ngi dung tren da chiing ininh kha nang khang khuan cua djch nuoi cay SC3. Tuy nhien, day moi chi la ket qua nuoi cay t6ng hop giiJa chiing SC3 va chiing kiem djnh. DS tach bacterocin ra khoi djch nuoi ziy nhSm kiem tra kha n5ng khang khuan ciia djch nuoi cay sau khi da lo^i bo sinh khoi vi sinh v|t va tap chat, phuang phap thong thuong dugc sir dung nhit la ly tSm roi thu djch tren, vi vgy thi nghiem nghien ciju anh huong cua toe dp ly tam tai dp sach ciia dich bacteriocin tho va kha nang khang khuan ciia no da du(?c thyc hiSn v6i qua trinh ly tam khao sat theo tflc dp tii 5000-14000 vong/phut

Hlnh 2. Hoat tinh khdng khudn ciia SC3 v&i cdc chiing kiem dllnh

Ket qua tir bang 3 va hinh 2 cho thay toe dp ly

tajn hau nhir khong anh hirdng den tinh khang

khuan ctia djch nuoi cSy chting vi khuin SC3

Tran Van Chi vd Dtg Tap chi KHOA HQC & CONG NGH$ 177(01): 4 3 - 4 sau ly tam, nhung anh h u d n g d i n dd tinh sach

ciia dich chiia bacteriocin, T h y c nghigm cho t h i y , dich sau ly tam vdi toe dp tu 5000 - 12000 vong/phiit khi nho vao g i l n g tren dTa nu6i cay k i l m nghiem van xuat hien khuan lgc cua chung nghign ciiu S C 3 . Tuy nhien, vdi tdc dp 13000 - 14000 vdng/phiit thi khdng cdn thay cd sy xuat hien k h u i n lgc ciia chung nghien cuu. Digu nay chiing td v a i toe dp ly tam Idn hon 13000 vdng/phiit sinh khoi va cac tgp chat ben trong djch nudi c i y da bj loai h i t ra khdi dich thu d u g c sau khi ly tam.

Vay toe dp ly tSm cho viec t^ch bacteriocin tii djch cay dg logi bo sinh khdi va tap c h i t la

13000 vdng/phdt.

Cung tir bang 3 cho thay kha nang khang khuan cua djch bacteriocin thd la khac nhau vdi cac chiing kigm dinh khac nhau. Trong thi nghigm d u g c bo trf sii dung 03 chiing kilm dinh Staphylococcus aureus, Samonella, E.coli, thi kha nang khang Staphylococcus aureus tot n h a t tiep den la E.coli va kha nang khang Samonella thap n h a t Cy the, higu so d u d n g kinh vdng khang khuan ciia djch nudi SC3 sau ly tam vdi cac chiing kiem djnh Staphylococcus aureus, E.coli, Samonella lin lugt la: 24,4 m m ; 20,6 mm va 19,5 mm.

K E T LUAN

T d cac san p h i m len men ty nhien da phan lap d u p c 14 chiing vi khuan sinh axit lactic va xac dinh dugc dgc d i l m sinh hgc, sinh iy va hda sinh, trong dd cd 8 chiing the hign hoat tfnh khang khuan tich cyc. Tii 8 chung tich cyc da chpn ra d u g c chiing SC3 c6 kha nang khang khuan tot nhat, the hien hoat tinh khang vdi ca 3 chiing kigm dinh Staphylococcus aureus, E.coli, Samonella vdi hieu sd vdng khang khuan tir 19,5 den 24 mm. Toe dg ciia qua trinh ly tam d l tach sinh khoi va tgp chat khac nham thu hoi bacteriocin tho khong anh h u d n g den hogt tinh c h l pham ma chi anh h u d n g den kha dp

sach ciia c h l pham, toe dd ly tam 13000 vdng/phut la phu hgp nhat.

TAI LIEU T H A M K H A O 1 Nguy§n LSn Dflng, Doan Xuan Mupn, Nguyen Phiing Tiln, Pham Van Ty (1972), Mgt s6 phuang phdp nghiin cuu vi smh vdt hgc, tap 1, Nxb Khoa hpc K^ thugt Ha Ndi.

2. Nguyen Lan DOng, Nguyen Dinh Quyen, Pham Van Ty (1997), Vi sinh vdt hoc, Nxb Giao due. Ha Npi.

3. Bing Hing Lam, Nguyin HOu Hlgp (2015),

"Phan lgp va tuyen chon vi khuin lactic chju nhigt sinh bacteriocin tir thuc phim Ign men truyin thong", Tgp chi Khoa hgc Tru&ng Dgi hgc An Giang, so l,tr.45-5\.

4. Le Thj Hong Tuylt (2004), Nghiin cuu bcateriocin sdn xudt b&i Lactobacillus acidophilus NrrlB - 2092, Luan van thac sT Sinh hoc, DHQG TPHCM.

5. Dobson A., Cotter P. D., Ross R. P., Hill C.

(2012), "Bacteriocin Production: a Probiotic Trait". Applied Environmental Microbiology, 78(1), pp. 1 - 6 .

6. Huhne K., Axelsson L., Hoick A,, Krockel L, (1996), "Analysis of the sakacin P gene cluster from Lactobacillus sake Lb674 and its expression in sakacin-negative Lb. sake strains".

Microbiology (Reading, Engl.), 142(6), pp. 1437- 1448. PMID 8704983.

7. James G, C. and Natalie (2002). Microbiology- A Laboratory Manual, Nine"' Edition, Pearson Education, Inc., Sanfransisco, CA.

8. Juan C. Oscariz, Antonio G. Pisabarro (2010),

"Classification and mode of action of membrane- active bacteriocin produced by gram positive bacteria". Int. Microbiol., 4, pp. 13-19, DOI 10.1007/s 101230100003, published online 25 August 2001,

9. Katla T., Moretro T., Sveen I., Aasen I. M., Axelsson L,, Rorvik L. M., Naterstad K. (2002),

"Inhibition of Listeria monocytogenes in chicken cold cuts by addition of sakacin P and sakacin P- producing Lactobacillus sakei", Journal Applied Microbiology, 93(2), pp, 191-196, DOI I0.1046/j.l365-2672,2002,0l675.x PMID 12147066.

Trin Van Chi va Dig Tap chi KHOA HQC & CONG NGHE

S U M M A R Y

R E S E A R C H ON SELECTION OF LACTIC ACID B A C T E R I A WITH ABILITY T O P R O D U C E H I G H B A C T E R I O C I N F R O M N A T U R A L F E R M E N T E D P R O D U C T S

Tran Van Chi, Pham Thi Tuyet Mai' TNU — University of Agriculture and Forestry Lactic acid bacteria (LAB) have been found and used in many fraditional fermented products in natural conditions. Recent researches have proved that LAB are able to produce bacteriocin (a biological antibiotic with nature of protein). The aim of this study was to isolate and select LAB capable of producing high bacteriocin from natural fermented products. From six natural fermented products, we isolated 14 LAB strains and identified their biological, physiological, and biochemical characteristics. From the isolated strains, we tested the ability of bacteriocin production which is expressed in the antibacterial activity with control bacteria Staphylococcus aureus, 8 of which exhibited positive antibacterial activity (substraction of antibacterial circle diameters greater than 10 mm). From the 8 above strains, we selected SC3 strain having the highest bacteriocin production, showing activity against all tested strains Staphylococcus aureus, E. coli, Samonella with subsfraction of antibacterial circle diameters ranged from 19,5 to 24 mm.

In addition, research has shown that speed of centrifiigation to separate biomass and impurities for recovery of crude bacteriocin does not affect the antimicrobial activity but only affect on purity of the product, the centrifuge speed of 13000 rpm is best suitable.

Key words: Bacteriocin, antibiotic, lactic, fermentation, bateria

Ngdy nlidn bdi: 24/10/2017; Ngdy phdn bi^n: 16/12/2017; Ngdy duy$t ddng: 30/01/2018 Tel: 0965 051220, Email:phamthituyetmai@tuaf edu.vn