KET QUA NGHIEN CifU KH-CN CUA VEEN CONG NGHE SAU THU HOACH NAM 2001
PHAN LAP VA TUYEN CHON MOT SO CHUNG VI KHUAN LACTIC SINH CHAT DIET KHUAN SINH HOC BACTERIOCIN
Nguyin Huong Tra, Nguyen Thi Hong Ha, Nguyen Thiiy Chau Bd mdn Vi sinh vqt, Vien Cdng nghe sau thu hoach
Hien nay, nhieu hoa chat dugc sir dung trong bao quan thirc pham nhu: formaldehyt, nitrat, nitrit v.v. Song cac hoa chat nay gay doc doi voi ngudi va dong vat kinh te. Do do, viec sir dung cac tac nhan sinh hoc de bao quan thuc pham da thu hdt su chii y ciia nhifiu nha khoa hoc.
Vi khuan lactic dugc coi la tac nhan phong chdng sinh hoc an toan. Cac vi khuan lactic diroc xep chung vao ho Lactobacteriaceae (theo bang phan loai ciia Becgey. Vi khuin lactic co kha nang sinh bacteriocin. Bacteriocin la mot nhom chat khang su phat trien cua vi khualn co quan he gan gui hoac cac vi khuSn khac.
Voi muc dfch phan lap, tuyen chon cac chiing vi khuan lactic sinh bacteriocin co tac dung irc che cac vi khuiin gay benh duong ruot. Chung tdi da thu dirge nhSng ket qua sau: Da phan lap dugc 186 chiing vi khuain lactic va da tuyen chgn dugc 4 chiing co kha nang sinh bacteriocin cao ky hieu la BC4, BC5, CB3 va ST20 tir cac thirc phiim len men truyen thdng khac nhau.
Bacteriocin san sinh tir chiing BC4 va ST20 da u'c che manh su phat triSn ciia cac vi khuain nhu:
Escherichia coli. Salmonella, Staphylococcus aureus. Bacillus cereus. Vibrio ogawa va Vibrio inaba. Chung BC4 co nhiSu dac diem gan gidng vai hg Lactobacillacae. Chung ST20 co nhieu dac diem gidng voi chi Pediococeus cua ho Sireptoeocacea e(theo khoa phan loai ciia Becgey).
I. DAT VAN DE
Trong cdng nghiep thuc phaim, viec bao quan cac thuc phaim tuoi sdng nhu; rau qua, thjt, trung, sua vii cac thuc pham thuy san nhu: tdm. ca, sd. muc. \ .\'. la rat can thiet. Cac san phaim nay rat dd bj thdi hdng trong qua trinh chuyen chd va bao quan ddng thdi cQng rat dd bj nhidm cac vi sinh vat gay benh nhu: Escherichia coli. Salmonella. Khi con ngudi tiep xuc hay sir dung cac thuc pham da bj nhidm cac vi sinh vat gay benh hay ddc td cua chung se dSn den bj ngd ddc thuc phaim cap tinh nhu sdt cao, suy tim mach va rat de mac phai mgt sd benh truyen nhiem ve dudng tieu hoa cd thd dan den tir vong. Theo thd'ng ke, ngay dau nam 1998 da xay ra hang loat vu ngd ddc thuc pham, ma dien hinh la vu ngd ddc thirc an d trudng mam non 1 1A Thanh phd Hd Chi Minh vdi hang tram chau be phai viio benh vien. Nguyen nhan chii yeu gay ra cac vu ngd ddc la do phuong thuc bao quan va che bien thuc an khdng hgp ve sinh, lam cho thuc phaim bj nhiem Ciic vi sinh vat gay benh. • .
Hien nay, nhidu hoa chat dugc sir dung trong bao quan thuc pham nhu : iormaldehy, nitrat,
My, Nhat, Trung Qudc. Vi khuan lactic dugc coi la tac nhan phdng chdng sinh hgc an toan. Cac vi khuan lactic dugc xep chung viio hg Lactobacteriaceae (theo bang phan loai cua Becgey. Vi khuain lactic cd kha nang sinh bacteriocin. Bacteriocin cd ban chat la peptit, do mgt nhdm cac vi khuan lactic sinh ra trong qua trinh sinh trudng va phat tridn , nd cd kha nang ire che su phat tridn CLia cac vi khuain cd quan he gin gui hoac cac vi khuain khac.
Vdi muc dich phan lap, tuyen chgn cac chung vi khuan lactic sinh bacteriocin cd tac dung iJc che cac vi khuan gay benh dudng rudt chiing tdi tien hanh nghien ciiai dd tai: "Phan lap va tuyen chgn mgt sd chung vi khuan lactic sinh chat diet khuan sinh hgc bacteriocin."
IL VAT LIEU VA PHUONG PHAP 2.1. Vat lieu nghien curu
Cac mau thuc pham len men nhu; nem chua, sira chua, dua mud'i, sira tuoi lay d cac chg Ha Ndi. Cac vi khuin gay benh nhu; Salmonella, E. coli, Staphylococus aureus, Bacillus cereus.
Vibrio owaga, Vibrio inaba do Vien ve sinh dich td hoc Trung uong cung cap.
2.2. Phuong phap nghien ciiru
2.2.1. Phuong phap lay mliu de phan lap vi khuan lactic
Tie'n hanh theo phuong phap ciia phdng thi nghiem Vi sinh - Vien Cdng nghe Sau thu hoach;
51 mau dua bap cai va 45 mku dua cai be, 20 mau sira chua, 50 mau sira tuoi da dugc lay d quanh khu vuc Ha Ndi. Djch mau sira tuoi va mSu dua cai dugc lay bang xi lanh, hut mgt it djch cho vao tui nilon ghi ky hieu mSu, ten mau, dja didm lay mlu d ben ngoai.
Hai muoi miu nem chua dugc thu thap tir cac chg d Ha Ndi.
2.2.2. Phuong phap phan lap vi khuan lactic
Tie'n hanh theo phuong phap ciia phdng thi nghiem Vi sinh vat, Vien Cdng nghe Sau thu hoach. Hut khoang I ml mau cho vao dng nghiem chu:a 5 ml nude cat vd triing. Lac cac dng nghiem tren may lac cho djch tan ddu. Phan phdi mdi trudng MRS ra dia petri, hut 20-30 fil hdn djch tren cho vao dia petri cd chii-a mdi trudng MRS, trang deu bang que gat thuy tinh. Nudi cay d nhiet do 37" C, sau 24 gid lay ra quan sat va bat cac khuan lac dac trung cua chiing vi khuan lactic.
Ddi vdi mSu nem chua, Ig mSu dugc nghien nat mgt each vd trung rdi pha loang bang nude cat vd triing vdi ty le 1/ 10 va diing phuong phap pha loang dd phan lap cac vi khuan lactic tren mdi trudng MRS.
2.2.3.Phuong phap len men vi khuan lactic tao bacteriocin
Len men cac chiing vi khuan phan lap dugc tren mdi trudng canh thang MRS d che do tinh tai 37" C, cir I gid lac mgt lan. Len men d cac khoang thdi gian 12, 18, 24, 30, 36, 42 va 48 gid.
Djch len men dugc ly tam 7000 vdng/ phiit trong 45 phut, loai bd can va thu djch ndi.
2.2.4. Phuong phap thiJr boat tinh iirc che cua bacteriocin sinh ra tiir chiing vi khu^n lactic len sir phat trien ciia vi khuan E. coli
Hut 1 ml djch vi khuan E. coli cho vao binh tam giac cd chira 350 ml mdi trudng thach thudng da dun tan va de ngudi den 45" C. Phan ra hop petri, diing khoan nut due Id tren dia thach. Hut vao mdi Id 600 ^1 djch ly tam cd chira djch bacteriocin. nudi d nhiet do 37" C. Sau 16- 24 gid lay ra dgc ket qua, boat tinh ire che vi khuain ciia bacteriocin san sinh tir vi khuan dugc danh gia gian tiep bang dudng kinh vdng ire chd khuain lac E. coli nudi cay tren mdi trudng thach thudng.
2.2.5. Phuong phap xac djnh kha nang di ddng ciia vi khuan
Tie'n hanh theo phuong phap ciia James [11]: chiing vi khuan dugc cay chgc thang vao mdi trudng MRS thach dung, nudi cay d 37"C. Sau 48 gid lay ra xem ket qua. Neu vi khuan chi mgc thing dung theo dudng cay chung td vi khuan khdng cd kha nang di ddng. Neu vi khuan mgc lan ra xung quanh dudng cay chiing td vi khuain cd kha nang di ddng.
2.2.6. Phuong phap xac dinh boat tinh catala/a
Tie'n hanh theo phuong phap ciia James [11]: Chiing vi khuiin lactic dugc nudi tren dug thach nghieng MRS d 48 gid. Mau ddi chimg khdng bd sung HjO, 3%, mau thi nghiem dugc nhd 2ml HjO, 3% len be mat ciia dng vi khuain. Nudi cay d 37'C. Sau 48 gid lay ra xem ket qua. Neu d mau thi nghiem xuat hien cac bgt khi thi la catalaza duong tinh, neu khdng xuat hien cac bgt khi thi la catalaza am tinh.
2.2.7. Phuong phap xac djnh kha nang sinh khi CO, ciia vi khuain
Tien hanh theo phuong phap ciia James [11]: Cho dng Durham vao dng nghiem cd chira 6ml mdi trudng MRS Idng. Hap d lat trong 30 phiit. Cay chimg vi khuain can thir vao dng nghiem va nudi cay d 37"C. Sau 48 gid lay ra xem ke't qua. Neu dng Durham ndi len tren be mat mdi trudng thi chung td \'i khuiin tao khi CO,. Neu dng Durham chim trong mdi trudng churng td vi khuan khdng tao khi CO,.
in. KET QUA VA THAO LUAN
3.1. Tuyen chgn chung vi khuain lactic sinh bacteriocin co kha nang lic che su phat trien ciia E. coli
Hoat tinh ire che vi khuain ciia bacteriocin san sinh tir vi khuain lactic dugc danh gia gian tiep bang dudng kinh vdng ire che £. coli nudi cay tren mdi trudng thach thudng cd bd sung 600 |.il djch ndi cd chira bacteriocin. Tir 51 mau dua bap cai, chung tdi da phan lap dugc 51 chimg vi khuan lactic, nhung chi cd 2 chiing vi khuain lactic cd hoat tinh manh uc che E. coli.
Ket qua d bang 2 cho thay: trong 45 chimg lactic phan lap dugc tir dua cai be, cd 1 chiing vi khuain cd boat tinh ire che cao (ky hieu la CB3). the hien qua duifng kinh vdng uc che la 3 cm.
Ke't qua d bang 3 cho thay: trong 50 chimg lactic phan lap duoc lir sua luoi. cd 1 chimg \i khuan cd hoat tinh uc che kha cao (ky hieu la ST20). the hien qua dudng kinh vdng uc ciie hi 2.9 cm.
Bang 1. Kha nang iJc che sir phat trien E. coli cua djch ndi chiira bacteriocin san sinh tir cac chiing vi khuan lactic phan lap tu dua bdp cai muoi Dudng kinh vdng ire die E. coli ciia
mau ddi chimg (cm)
fXrimg kinh vdng uc chd E. coli ciui mau thir phan theo cac muc (cm)
Sd chimg vi khuain lactic trong mau thir cd dudng kinh vdng irc che tuong ung
Tdng so chimg vi khuain lactic thir nghiem
0 0-1.5
42
1.6-2
6
2.1-2.5
1
2.6-3
1
>3
1
51
Bang 2. Kha nang uc che sir phiit trien E. coli cua dich ndi chua bacteriocin san sinh tir ciic chiing vi khuain lactic phan lap tir dua cai be muoi Dudng kinh vdng u'c chd E. coli ciia
mau ddi churng (cm)
Dudng kinh vdng ire che E. coli ciia mau tliir pliaii tlia) cac mire (cm)
Sd chiing vi khuain lactic trong mau thir cd dudng kinh vdng irc che tuong I'rng
Tdng so chiing vi khuan lactic thir nghiem
0 0-1.5
38
1.6-2
3
2.1-2.5
3
2.6 -3
0
>3
1
45
Biing 3. Khii nang uc che sir phat tridn E. coli ciia djch ndi chira bacteriocin Siin sinh tir cac chiing vi khuan lactic phan lap tir sira tuoi
:*!
Duimg kinh vdng irc che E. coli ciia mau ddi churng (cm)
Dudng kinli vdng ire che /:". < oli ciia niiiu thir phiui theo cac mUc (cm)
Sd chimg vi khuan lactic trong mau thir cd dudng kinh vdng uc die luong irng
Tdng so chimg vi khuan lactic thir nghiem
0 0-1.5
44
1.6-2
3
2.1-2.5
2
2.6 -3
1
>3
0 50
3.2. Hoat tinh urc che cua djch ndi chiifa bacteriocin san sinh tir chiing BC4 va ST20 ddi vdi 5. typhy, E. coli, S. aureus, B. cereus, V. ogawa va V. inaba.
Ke't qua d bang 4 cho thay; tren mdi trudng thach thudng khdng bd sung bacteriocin, dudng kinh vdng ire che S. typhy. E. coli, S. aureus, B. cereus, V. ogawa va V. inaba bang 0 cm. Tren mdi trudng thach thudng bd sung 0.6ml djch ndi chiira bacteriocin san sinh tir chung ST20, dudng kinh vdng irc che 5. typhy, E. coli, S. aureus, B. cereus, V. ogawa va V. inaba la 3.1cm, 3.5cm, 3.0cm, 3.9cm, 2.4cm va 2.5cm, theo thii: tu.
Tren mdi trudng thach thudng bd sung 0.6ml djch ndi chiira bacteriocin san sinh tir chung BC4, dudng kinh vdng iirc che 5. typhy, E. coli, S. aureus, B. cereus, V. ogawa va V. inaba Ik 2.9cm. 3.1cm, 2.3cm, 2.1cm, 3.0cm va 2.7cm, theo thu" tu. Le Thanh Binh va cdng sir [I] da xac djnh dugc kha nang uc che cua bacteriocin ddi vdi vi khuan Bacillus subtilis, B. cereus, Sarcina luteria, tuy nhidn bacteriocin nay lai khdng cd kha nang irc che £. coli. Ebenezer, R. va cdng sir [7] da tien hanh phan lap vi khuan lactic tir sira tuoi, dua mud'i va da tuyen chgn dugc chiing Lactococcus lactic LL-2 cd kha nang iirc che Pediococeus pentosaceus FBB63C, Bacillus subtilis RM125, Lactobacillus casei 326, tuy nhien bacteriocin niiy khdng cd kha nang ire che £. coli, S.
aureus.
Ket qua nay cho thay, djch ndi chira bacteriocin ma chung tdi thu nhan dugc hoan toan cd thd img dung trong viec bao quan thuc phaim de tranh su nhidm vi khuan 5. typhy, E. coli la vi khuan gay benh dudng rudt thuong han va phd thuong han va S. aureus. B. cereus la vi khuan cd kha nang tao ddc td dudng rudt Staphylotoxin va rat cd kha nang nhidm tren nhieu loai thuc phaim.
Ket qua nghien cihi vd cac dac diem hinh thai va dac diem sinh vat hoa hgc ciia chiing vi khuan BC4 va ddi chieu vdi khoa phan loai vi khuain ciia Bergey, chiing tdi nhan thay chiing nay CO nhieu dac didm ciia hg Lactohacillaceae. Chiing ST20 cd nhieu dac didm gidng vdi chi Pediococeus cua hg Streptococaceae. Tuy nhien can lam them nhung phan ung sinh vat hoa hoc khac nira bao gdm nhung phan ung len men vdi nhieu loai dudng khac nhau va dac biet la sir dung ky thuat sinh hgc phan tir mdi hieu qua nhu xac djnh trinh tu ciia 16S rADN de cd the phan loai chinh xac ten loai ciia chiing BC4 va ST20. Cdng vice nay can dugc tien hanh trong khudn khd ciia nhung cdng trinh nghien cihi dai hon.
Bang 4. Hoat tinh irc che ciia djch ndi chiira bacteriocin san sinh
tir chiing BC4 va ST20 ddi vdi S. typhy, E. coli, S. aureus, B. cereus, V. ogawa va V. inaba 0.6 ml djch ndi chira
bacteriocin siin sinh tir cac chiing vi khuain Mau ddi churng khdng bd sung bacteriocin
ST20 BC4
Dudng kinh vdng u'c chd (cm) S. typhy
0 3.1 2.9
E. coli 0 3.5 ' 3.1
S. aureus 0 3.0
2.3
B. cereus 0 3.9 2.1
v. ogawa 0 2.4 3.0
V. inaba 0 2.5 2.7
3 J. Quan sat hinh thai ciia chiing vi khuan BC4 va ST20
Bang 5. Dac diem hinh thai ciia chung BC4 va chiing ST20 Chi tieu
Khuan lac - Hinh dang - Kich thudc - Mau sac Te bao - Hinh dang - Sap xep - Ki'ch thudc
Chiing BC4
- Trdn, Idi, d giira mep mdng va tron
- Rat nhd, dudng kinh< 1mm - Vang kem, vang nhat - Hinh que, gay - Tao chudi
- 0,6-0,7 X 0,1-0,2 ^m
Chung ST20
- Trdn, Idi d giira, mep mdng va mong
- Rat nhd, dudng kinh< 1mm - Vang kem,vang nhat - Hinh cau - •-"«
- Xep thanh timg cap - 0 , 4 - 1,25 nm
Bang 6: Mot sd phan iirng sinh vat hoa hgc ciia chiing BC4 va ST20 Chi tieu
Nhudm Gram Kha nang tao bao tir Kha nang di ddng Hoat tinh catalaza Sinh khi COj
Kha nang sinh axit lactic Phat tridn; -1" =37"C
-PH = 6 , 5 - 6 , 8 Phd khang khuan; + E. coli
+ S. typhy + S. aureus + B. cereus + V. ogawa + V. inaba
Dac diem ST20
Gram duong - - - - +
-1- +
+ + + + +
BC4 Gram duong
- - - -
-1-
-1- -t--1- -1- + 4- -1- -(-
IV. KET LUAN
1. Tir 186 mlu thu thap tir cac thuc phaim len men truyen thdng khac nhau, chiing tdi da phan lap dugc 186 chimg vi khuain lactic va da tuydn chgn dugc 4 chimg cd kha nang sinh bacteriocin cao ky hieu lii BC4, BC5, CB3 va ST20.
2. Tir 600 pi djch ndi chiira bacteriocin san sinh tir chung BC4 cd thd iirc che dugc 1.6x10'' khuan lac E. coli, 1.9x10'' khuain lac Salmonella, I xIO'^ khuain lac S. aureus, 8.6x10' khuan lac B. cereus, 1.7x10'^ khuain lac V. ogawa \a. 1,4 x 10'' khuain lac V^ inaba.
3. Vdi cac dac diem vd hinh thai, tinh chat sinh vat hoa hgc ciia chung BC4, chiing tdi thay chimg BC4 cd nhieu dac diem gan gidng vdi hg Lactobacillacae. Chimg ST20 cd nhieu dac didm gidng vdi chi Pediococeus cua hg Streptococaceae.
TAI LIEU THAM KHAO
1. Bergey Manual of systematic bacteriology, 1986. William & Wilkins, Vol. 2.
2. Holo. H, Nilssen, and 1. F. Nes 1991; Lactococcin A, a new bacteriocin from Lactococcus lactic subspcremoris; isolation and characterization of protein and its gene. J. Baeteriol 173;
3879-3887.
3. James, G. C , Natalie, S. Microbiology - A Laboratory Manual. Fourth Edition.
4. Klaenhammer, 1988. Bacteriocin of Lactic acid bacteria. Biochemie 70: 337-349.
5. Klaenhammer T.R, 1993. Genetics of bacteriocin produced by lactic acid bacteria. FEMS Microbiol Rev, 12: 38-86.
6. Peter A. Vandenbergh, Michael J. Piieci, Blairs Kunka, Bradenton Ebenezer R. Vedemuthu.
patent 4.929.445, 1990. Method for inhibiting Listeria nionocitogenes using a bacteriocin.
7. Pot, W. Ludwig, K. Kerster and K.H Shcleifer, 1994. Taxonomy of lactic acid bacteria, p. 13 -89. In L. de Vuyst and E.J Van damme (ed), bacterioeins of lactic acid bacteria.
Backie Academic & Professional, New York.
8. Schillinger. U and F. K. Liicke, 1989. Antibacterial activity of Lactobacillus aske isolated from meat. Appl. Environ. Microbiol 55: 1901-1906.
9. Schillinger. U and F. K. Liicke, 1990. Lactic acid bacteria as protective cultures in meat product. Frdschwirtschaft 70: 1296-1299.
ISOLATION AND SCREENING OF ACID LACTIC BACTERIA STRAINS PRODUCING BACTERIOCIN
Nguyen Huong Tra, Nguyen Hong Ha, Nguyen Thuy Chau Division of Microbiology, Postharvest Technology Institute
We isolated 186 acid lactic bacterial isolates from 186 samples of traditionally fermented products and received 4 isolates namely BC4, Bc5, CB3 and ST20 having high activity to produce bacteriocin as antibacterieidal substances. The antibaeterieidal effect of bacteriocin produced by BC4 and ST20 isolates were investigated. These bacterioeins gave high antibacterieidal activity to Salmonella. Escherichia coli. Staphylococcus aureus. Bacillus cereus.
Vibrio owaga and Vibrio inaba. The observation on morphological characteristics and study on biochemical properties of the lactic bacterial strain BC4 producing wide bactericidal spectrum and indicated that the strain is closely related with Lactobacillacae family. ST20 strain was also observed in morphological characteristics and indicated that this strain belongs to the Pediococeus genus.
