Ti4u ban: CONG NGHE SINH HOC BI^N VA MOI TRU'ONG 5 3 7
DAC DIEM SINH HOC MOT SO CHUNG VI KHUAN LACTIC PHAN LAP Tir MAU CA BIEN DO SOTN - HAI PHONG
Nguyen The Trang, Tran Dinh Man
Vien Cong nghe Sinh hpc, Vien Khoa hoc va Cong nghe Viet Nam
I. M O DAU
Vi khuan lactic co mgt vi tri dac biet trong he thong vi sinh vat. Mgt diem quan trgng ciia vi khuan lactic la sir chuyen hoa nguon cacbon hydrat chii yeu thanh axit, cac chat c6 ti'nh khang khuan da dan den nhimg img-dung to lan, rgng rai ciia no trong cac ITnh vyc: Cong nghiep dugc pham, cong nghiep thyc pham, cong nghiep hoa chat, vat lieu, nong nghiep, va mai day trong bao ve sue khoe, bao ve moi truang.
Trong nhirng nam gan day do nhu"ng thanh tyu mai cua khoa hgc cong nghe, ciia sinh hgc phan tir da tao ra nhung buac ngoat mai trong nghien ciru ve cong nghe sinh hgc noi chung, cong nghe sinh hgc ciia vi khuan lactic noi rieng. Nha nhuTig ket qua nghien ciru he thong hoa, tien hoa ciia gen 16S rRNA, gen co tinh bao thu nhat da phat hien them cac chi mai. Den nay da CO tai 11 chi vi khuan lactic dugc phat hien so vai 4 chi vao nhirng nam 60 eua the ky truac.
Ciing vai viec phat hien cac chi mai la cac chat trao doi mai, chu trinh chuyen hoa mai, tinh nang mai [3,10]. Cac vi khuan lactic da dugc nhieu tac gia khac phan lap tir thuy san hoac cac san pham len men truyen thong tir ca [2, 4 - 9 ] . Trong khu he vi sinh vat, vi khuan lactic thuong CO mat vai so lugng khac nhau phu thugc vao moi truong sinh song. Trong ca vi khuan lactic la nhan to probiotic giiip cho ca chong lai mgt so vi sinh vat gay benh, chinh nhimg vi khuan lactic nay dong vai tro chii yeu trong san xuat cac san pham len men tir ca theo phuang phap truyen thong. Bai viet nay de cap dac diem sinh hgc ciia mgt so chiing vi khuan lactic sinh tong hgp axit
lactic cao dugc phan lap tir ca tap nuac man Do San - Hai Phong.
II. VAT LIEU VA PHlTOfNG PHAP
1. Vat lieu nghien ciru
Ca tap nuac man loai nho dugc mua a chg ca Do San, Hai Phong. Moi truang phan lap va xac dinh dac dilm sinh hgc MRS (g/l): Pepton - 10; Cao thjt - 10; Cao men - 8; CHjCOONa - 5;
K2HPO4 - 2; Glucoza - 20; Ammonium citrat - 2; Mgs64.7H20 - 0,2; MnS04.4H20 - 0,04;
Tween 80 - 1 ml, agar - 20, CaCOj - 5.
2. Phuong phap nghien cuu
Chiing vi khuan dugc phan lap tren moi truang dac hieu MRS, xac djnh kha nang sinh truang bang phuang phap do OD, a.xit lactic theo phuang phap chuan do, phan loai theo Bergey's va Kit chuan sinh hoa API 50 CHL [1; 9].
538 Tuyen lap HQi nghi KHOA HOC T O A N Q U O C V £ SINH HOC B I E N V A PHAT TRI^N B £ N VCTNG
ra. KET QUA VA THAO LUAN
L Mqt so dac dilm sinh ly, sinh hoa ciia cac chung vi khuSn lactic phan lap tir ca tap fi^
Son - Hai Phong
i-'tijiXir mlu ca tap a khu vyc D6 SOTI - Hai Phong, chiing toi xac djnh dugc so lugng vi khuan lactic dao dgng tir lO'-lO^ te bao/gam ca. Dya tren dac diem hinh thai khuan lac da tach dugc 14 chung vi khuan lactic ky hieu DS01-DS14. Cac chiing vi khuan lactic nay dugc nuoi tren moi truang MRS dl xac djnh kha nang sinh truang cung nhu sinh tong hgp axit lactic a cac th6i diem 24, 36, 48, 60 va 72 gia (hinh 1 va 2).
0 24h
• 36h D48h D60h
• 72h
DSOl OS02 OS03 BS04 DS05 DS06 OS07 DS08 DS09 DS10 OSl 1 BS12 0S13 DS14 Ky h i ^ u Chung
Hinh 1. Anh h i r v n g cua t h a i gian (h) d§n sinh tripang (OD620) cua cac c h u n g vi khu4n lactic
J
n24h
• 36h D 4 8 h DBOh
• 72h
OSOl OS02 OS03 OS04 OS05 DS06 DS07 DSOB DS09 DS10 0S11 DS12 0S13 DS14 Ky hieu chung , . ;
Hinh 2. Anh hu-ang cua tho-l gian (h) d§n sinh t d n g h a p axit lactic (g/l) cua vl khuan lactic
Tir hinh 1 va 2 cho thay cac chiing vi khuan lactic phan lap dugc tir mau ca tap co kha nang sinh truang cung nhu sinh t6ng hgp axit lactic rat khac nhau. Chiing DSOl, DS04, DS06, DS08, DS09, DSI 1 va DS14 co kha nang sinh truang cao hon cac chiing con lai, trong khi do kha nang
Tieu ban: CONG NGHE SINH HOC B I ^ N VA iVIOl TRU'ONG 539
sinh tong hgp a.xit lactic cao la cac chimg DSOB, DS08, DS09 va DSI 1, sau 24 gia kJia nang len men lactic ciia chimg DSOB dat 17g/l, chiing DS09 dat 16g/l. Sau 48 gia kha nang sinh tong hgp axit lactic rat cao nhu chung DS03 dat 19g/l, chimg DS08 dat 19,8g/l, chung DS09 dat 19,8g/l va chiing DSI 1 sau 60 gia dat 19,2g/l.
Nghien ciru anh huang ciia nhiet do den sinh truang cua cac chiing phan lap (hinh 3) cho thay, hau het cac chiing vi khuan lactic phan lap dugc co khoang nhiet do sinh truang rgng.
OSOl DS02 OS03 DS04 DS05 0S06 BS07 BS08 OS09 OS10 0S11 0512 0S13 0S14 Ky hieu Chung
Hinh 3. Anh hu'crng cua nhiet do ( C) den sinh tru'crng (OD620) ciia cac chung vl khuan lactic
Tien hanh lya chgn 9 chiing vi khuan lactic co kha nang sinh tong hgp axit lactic cao han ca cho nghien ciru tiep anh huong ciia nong do NaCl den kha nang sinh truang ciia cac chiing nay.
Ket qua trinh bay a hinh 4 cho thay, tren moi truang MRS, a moi thi nghiem bo sung nong do NaCl khac nhau la 0%, 1,5%, 2,5% va 3,5%). Anh huang ciia nong do NaCl rat ro ret vai vi khuan lactic phan lap tir ca tap bien, nhung kha nang chju muoi cung chi a khoang 2,5%). Vai nong do 3,5%) tat ca cac chiing deu phat trien yeu.
OSOl DS02 BS03 DSM BS06 DS08 DS09 0S11 0S14 Ky hieu chiing
Hinh 4. A n h hu'crng ciia NaCl (%) ddn kha nang sinh tru'dng (OD620) ciia cac chung v l k h u i n lactic
540 Tuyen tgp Hgi nghi KHOA HOC T O A N Q U 6 C V £ SINH HOC BI^N vA P H A T T R I ^ N B S N VCPNG
Ket qua trinh bay a hinh 5 cho thay, anh huang pH moi truang ban dau cung khac nhau giua cac chiing, 7 chiing vi khuan lactic nghien ciru DSOl, DS02, DS04, DS06 va DS14 phat trien tot a pH ban dau la 5,5, hai chiing DS08 va DS09 phat triln t6t a pH ban dau la 6,5. Rieng chiing DS03 va DSI 1 phat trien tot a pho pH ban dau kha rgng tir 5,5-^7,5.
1.1 1 0 0.9
CD
§ 0-8 i 0.7 1 0.6
^ 0.5 0.4 0.3
L
To pH 5.5 I j « p H 6 . 5
| D P H 7 . 5
BS01 DS02 DS03 OS04 OS06 OS08 Ky hi$u Chung
0309 e S I I DS14
Hinh 5. Anh hu'crng ciia pH d§n kha nang sinh tru'dng (OD620) cua cac chung vi khuin lactic
Sy CO mat ciia cae chiing vi khuan lactic trong ca tap la c a sa de phat triln cac san pham len men truyen thong (khong bo sung vi khuan lactic tir ben ngoai vao). Tir nhirng kit qua tren, 4 chung vi khuan lactic DS03, DS08, DS09 va DSI 1 co kha nang sinh t6ng hgp axit lactic cao nhat dugc lya chgn cho nghien ciru dac diem hinh thai, sinh ly, sinh hoa, dac dilm phan loai.
2. Dac diem phan loai ciia 4 chiing vi khuan lactic tuyen chpn
Hinh dang, kich thuac t l bao, sap xep te bao dugc quan sat duai kinh hien vi dien tir dugc trinh bay a hinh 6. Hai chung vi khuin lactic DS03 va DSI 1 c6 te bao hinh cau, hai chimg DS08 va DS09 CO te bao hinh que ngin.
DS03(x 10.000) DS08(x 10.000) DS09(x 10.000) DSll (x 10.000)
Hinh 6. Hinh dang t§ bao vi khuan lactic nghien CI^U
Dac diem hinh thai, sinh ly sinh boa dugc trinh bay a bang 1 cho thiy, ca 4 chiing vi khuan lactic tren deu thugc loai Gram duang, khong sinh bao tOr, khong co boat tinh catalaza va oxidaza, cac chung deu sinh tong hgp DL-lactic axit. Kit qua cho thiy chung vi khuin DS03 va DSll CO dac diem dien hinh ciia vi khuin lactic Pediococcus pentosaceus typ 1 va chiing vi
Tieu ban: CONG NGHE SINH HOC B I ^ N VA MOI TRU'ONG 541
khuan DS08 va DS09 cua vi khuan Lactobacillus plantarum typ 1. De xac dinh den loai vai cac dac diem phan loai tren la chua dii, vi vay 4 chiing vi khuin lactic DS03, DS08, DS09 va DSI 1 dugc phan loai bing Kit chuin sinh hoa API 50 CHL.
B a n g 1 . D a c d i e m h i n h t h a i , s i n h l y , s i n h h o a c u a 4 c h u n g v i k h u a n l a c t i c t u y e n c h p n
D^c diem Ky hi$u c h u n g vl khuJin lactic
0 S 3 0 3 8 0 S 9 D S l l
Te bao:
- Hinh dang - Kinh thuac - s i p xep te bao - Nhugm Gram Khuan lgc:
- Hinh dgng - M a u s i c -Du'ang kinh Sinh tru'dng 6':
25°C 35°C 45°C
Sinh tru-anq g pH:
5,5 6,5 7,5
Sinh proteaza Hoat tinh catalaza Sinh khi tif glucoza Kha nang di dpng Ho hap
Kha nang sinh bao tie Sinh acetoin
Kha nang chiu NaCl:
0%
1,5%
2,5%
3,5%
Lactic axit
Tron, ovan 0,5-1,0 pm Xep doi, bon Gram (+)
Tron loi T r I n g su'a d < 1,5 mm
+ + . +
+ ++ "
+
Tuy tien
+ + +
± DL
Que n g l n 1,0-2,0 pm Xep doi Gram ( t )
Tron loi T r I n g d < 2 mm
+
+ ++
+
Tiiy tien
+ + +
± DL
Que dai 1,0-3,0 pm
Xep doi Gram (+)
Tron loi T r I n g sOa
d < 2 mm +
•+
+ ++
+
Tiiy ti^n
+ + +
± DL
Tron, ovan 0,5-1,0 |jm Xep doi, b6n
Gram (+) Tron loi T r I n g su'a d < 1,5 mm
+ + + + ++
+
Tiiy tien
+ + +
± DL
Chu thich: +: co kit qua, -: khong co kit qua
B a n g 2 . M u ' c d p S I P d u n g cer c h i t t h e o K i t A P I 50 C H L c i i a c h u n g l a c t i c n g h i e n c i i ' u STT
0 1 2
Cc chat
Control Glycerol Er/thritol
Kha nang si> dgng cc chSt ciia cac chiing DS03
24 h
-
48 h
-
- j - i
DS08 24 h
- - -
48 h
- - -
DS09 24 h
- - -
48 h
- - -
D S l l 24 h
- - -
48 h
- - -
542 Tuyen tap HOi nghi KHOA HOC T O A N Q U 6 C V ^ SINH HOC BI^N v A P H A T T R I ^ N B ^ N VONG
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 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
Ribose D-Xylose L-Xylose Adonitol P Methyl-xyloside Galactose D-Glucose D-Fmctose D-Mannose L-Sorbose Rhamnose Dulcitol Inositol Mannitol Sorbitol
a Methyl-D-mannoside a Methyl-D-glucoside N Acetyl glucosamine Amygdaline
Arbutine Esculine Salicine Cellobiose Maltose Lactose Melibiose Saccharose Trehalose Inuline Melezitose D-Raffinose Amidon Glycogene Xylitol P Gentiobiose D-Turanose D-Lyxose D-Tagatose D-Fucose L-Fucose D-Arabitol L-Arabifol Gluconate 2 ceto-gluconate 5 ceto-gluconate
+
- - - -
+ + + +
- - - - - - - -
+ + + + + + +
- - - - - - - - - -
+
- -
+
- - - -
+
- -
+
- - - -
+ + + +
-
+
- - - - - -
+ + + + + + +
- - - - - - - - - -
+
- -
+
- - - -
+
- -
+
- - - -
+ + + +
-
+
- -
+ + +
-
+ + + + + + + + + + +
-
+ +
- - -
+ +
-
+
- -
+
-
+
-
+
+ 1 +
- - - -
+ + + +
-
+
- -
+ + +
-
+ + + + + + + + + + +
-
+ +
- - -
+ +
-
+
- -
+
-
+
-
+
- - - -
+ + + +
-
+
- -
+
-
+
-
+ + + + + + + + + + +
-
+ +
- - -
+ +
-
+
- -
+
-
+
-
+
+
- - - -
+ + + +
-
+
- -
+
-
+
-
+ + + + + + + + + + +
-
+ +
- - - "
+ +
-
+
- -
+
-
+
-
+
+
- - - -
+ + + +
-
+
- - - - - -
+ + + + + + +
- - - - - - - - - -
+
- -
+
- - - -
+
- -
+
. . . -
+ + + +
.
+
- . - - - -
+ + + +
+
+
+ - -, - - - - - - - - + - -
+
- - - -
+
- Chii thich: -: khong co phdn img; +: co phdn ung; *: phdn imgyiu
Tir bang 2 cho thay, chiing vi khuin lactic DS03 va DSI 1 thugc Pediococcus pentosaceus typ 1 vai ID = 99,3% va T = 0,5; chiing vi khuin lactic DS08 thugc Lactobacillus plantarum typ 1 vai ID = 99,9 va T = 0,8; Chung vi khuin lactic DS09 thuoc Lactobacillus plantarum typ 1 vai ID = 99,9 vaT = 0.59.
Tieu ban: CONG NGHE SINH HOC B I ^ N VA MOI TRU'ONG 5 4 3
IV. KET LUAN
Tir cac mau ca thu thap tai Do San - Hai Phong da phan lap dugc 14 chiing vi khuan lactic dugc ky hieu tir DSOl den DSM. Cac chiing nay da dugc nghien ciru mgt so dac diem sinh ly va sinh boa nhu giai nhiet do va pH phat trien, kha nang chiu muoi NaCl va kha nang sinh axit lactic. Dac diem hinh thai te bao cua 4 chiing co ky hieu DS03, DS08, DS09 va DSI 1 co kha nang sinh tong hgp axit lactic cao nhat dugc xac dinh, trong do te bao ciia 2 chiing co hinh cau va 2 chiing co te bao binh que. Sir dung khoa phan loai eua Bergeys va kit chuan API 50 CHL cho thay chiing vi khuan lactic DS03 va D S l l thugc Pediococcus pentosaceus typ 1 vai do tuang dong 99,3%), chiing DS08 va DS 09 thugc Lactobacillus plantarum typ 1 vai do tuang d6ng 99,9 %.
V. TAI LIEU THAM KHAO
1. Nguyen Lan Dung va cgng sy (1977). Mgt so phuang phap nghien ciru vi sinh vat, tap 1.
Nxb Khoa hgc va Ky thuat. Ha Ngi.
2. Nguyen The Trang, Tran Dinh Man (2007). Sir dung vi khuan lactic Pediococcus pentosaceus HN02 de san xuat che pham bao quan ca. Tap chi Khoa hgc va Cong nghe. Tap
45 (4): 35-42.
3. Axelsson L. T. (1993), Lactic acid bacteria: Classification and physiology, hi: Salminen, S.
and von Wright, A. (Eds.), Lactic acid bacteria. Marcel Dekkers Inc., New York, pp. 1-65.
4. Ahamed J, Mahendrakar NS (1997) Chemical and microbial changes in fish viscera during fermentation ensiling at different temperatures. Bioresource Technology ISSN 0960-8524, V o l 59(1), 45-46.
5. Christine Paludan-Muller, Hans Henrik Huss and Lone Gram (1999). Characterization of lactic acid bacteria isolated from a Thai low-salt fermented fish product and the role of garlic as substrate for fermentation Intemational Journal of Food Microbiology. Volume 46, issue 3, 18Fer 1999, 219-299
6. Franfois-Joel Gatesoupe (2008). Updating the Importance of Lactic Acid Bacteria in Fish Farming: Natural Oceiorrence and Probiotic Treatments. Journal of Molecular Microbiology and Biotechnology, Vol 14(1-2) 107-114.
7. L. J. Y I N , C . L . PAN, S. T . JIANG (2006) New Technology for Producing Paste-like Fish Products using Lactic Acid Bacteria Fermentation. Journal of Food Science, Vol 67, Issue 8, 3114-3118.
8. Parvathy Seema Nair and Pufhuvallil Kumaran Surendran (2005), Biochemical Characterization of Lactic acid bacteria Isolated from fish and prawn. Journal of Culture Collections, Vol 4, 48-52.
9. Vijai Pal, Marilingappa Jamuna and Kadirvelu Jeevaratmam (2005). Isolation and characterization of bacteriocin producing lactic acid bacteria from a South India special Dosa (appam) batter. Journal of Culture Collections, Vol 4, 53-60.
10. John G. Holt, Noel R. Krieg, Peter H. A. Sneath, James T. Staley and Stanley T. Wiliams (1986) Bergey s manual of Systematic Bacteriology, 9th ed., 2.
5 4 4 Tuyen tgp HQi nghi KHOA HOC TOAN Q U 6 C V £ SINH HQC BifeN VA PHAT T R | £ N B £ N V O N G
BIOLOGICAL CHARACTERISTICS OF SOME LACTIC ACID BACTERIA STRAINS ISOLATED
FROM FISH AT DO SON - HAI PHONG SEAPORT
Nguyen The Trang and Tran Dinh Man
SUMMARY
Fourteen lactic acid bacteria strains were isolated from fish at Do Son - Hai Phong seaport.
The temperature, pH and NaCl concentration for optimum growth were 30°C, 6,5 and 0-2,5%
respectively. Based on lactic acid producing abilities, 4 strains were selected to study their cell morphology and classification characteristics. The figures under electronmicroscopy showed that cell shape of strains DS03 and DSll were cocci, then strains DS08 and DS09 were rods.
Identification by kit API 50 CHL showed that, the strains DS03 and DSll belong to species Pediococcus pentosaceus and the strains DS08 and DS09 belong to species Lactobacillus plantarum.
