Nghiên cứu quá trình sinh tổng hợp bacteriocin và ứng dụng

I H C QU C GIA THÀNH PH H CHÍ MINH I H C BÁCH KHOA NGUY N TH NGUY T MÃ NGÀNH: 60.42.80 LU TP H CHÍ MINH, tháng 08 C HOÀN THÀNH T I I H C BÁCH KHOA I H C QU C GIA TP H CHÍ MINH Cán b ng d n khoa h c: TS Nguy n Th Bích H ng Cán b ch m nh n xét 1: TS Nguy n H u Phúc Cán b ch m nh n xét 2: PGS TS Nguy Lu ngày 15 tháng cb ov t 2014 ih Thành ph n H (Ghi rõ h , tên, h c hàm, h c v c a H PGS TS Nguy n Ti n Th ng m: ng ch m b o v lu TS Nguy n H u Phúc PGS TS Nguy TS Phan Ng c Hịa TS Hồng Anh Hồng Xác nh n c a Ch t ch H ngành sau lu c s a ch a (n u có) CH T CH H ng Khoa qu n lý chuyên NG KHOA KTHH I H C QU C GIA TP.HCM C NG HÒA XÃ H I CH I H C BÁCH KHOA T NAM c l p - T - H nh phúc NHI M V LU MSHV: 12310742 29/03/1985 Bình Thu n Chuyên ngành: I : 60.42.80 TÀI: Nghiên c u trình sinh t ng h p bacteriocin ng d ng II NHI M V VÀ N I DUNG Kh o sát nghiên c u làm ti th c hi n ch lên men fed- thu bacteriocin có ho t tính cao t Lactococcus lactis Lên men fed-batch ng d ng b o qu n nguyên li u m t hoa d a III NGÀY GIAO NHI M V : 01/2014 IV NGÀY HOÀN THÀNH NHI M V : 06/2014 V CÁN B NG D h Bích H ng 14 CÁN B NG D N (H tên ch ký) CH NHI M B (H tên ch ký) (H tên ch ký) O L IC Tôi xin chân thành c Cơ Nguy n Th Bích H ng nh c hi n lu ng l c r t l quan tâm chia s v i c bi t, tình c m c a Cơ dành cho tơi hồn thành t t lu Tôi xin chân thành c Cô Nguy Hu nh Ng Th y Tiên, Cô t ki n th c cho th i gian h c t p t ng viên an i, quan tâm chia s g p tr ng i, t phịng thí nghi m nh ng góp ý trình làm lu quên g i l i c Nguy n Anh Tu n Tôi xin g i l i c ng, u ki n v thi t b d ng c a i cô Võ Th Ly Tao, ch Tr n Trúc Thanh, b n v d ng c phịng thí nghi m cho tơi n b n h tr nhi i su t th i gian qua Và cu i xin g i l i c tơi tồn tâm q trình h c t p tr ng viên TĨM T T LU tài lu : Nghiên c u trình sinh t ng h p bacteriocin ng d ng N tài: Kh o sát nghiên c u làm ti th c hi n ch lên men fed- thu bacteriocin có ho t tính cao t Lactococcus lactis T ts u ki n nuôi c batch Kh o sát m Kh tiêu th ng thu bacteriocin lên men ng theo th i gian nuôi c y c a ch ng gi ng ng c ch Lên men fed-batch ng d ng b o qu n nguyên li u m t hoa d a K t qu - tài nh danh ch ng gi ng b ng b ng Lactococcus lactis 92% - Trong lên men batch: bacteriocin có th i gian ni c y t ki n nuôi c y t nhi 37 0C, pH 6, t l c y gi ng 10% ng thích h p: sucrose v i n bacteriocin lên men batch 16 gi i u u ki n dinh ng c ch 60 g/l Ho t tính u ki n t t 2180 AU/ml - Trong lên men fed-batch: ti n hành nuôi c y ch ng Lc lactis mơi ng MRS chu n có b sung sucrose ch n th trì n 20 g/l Chúng m b sung sucrose gi giúp trì pha log nhanh, chu k b sung sucrose gi ng th i d u ki n nuôi c y t t 3210 AU/ml sau 12 gi nuôi c y T i th t 3215 AU/ml, t qu ho t tính m 16 gi , ho t tính t t ng h p bacteriocin lên 147% so v i lên men batch - ng d ng b o qu n nguyên li u m t hoa d a: b sung 0,3% d ch bacteriocin v i ho t tính 3210 AU/ml, thu nh n b i trình lên men fed-batch, giúp kéo dài th i gian b o qu n nguyên li u m t hoa d a ngày so v i m s d ng dung d ch bacteriocin u ki n nhi phịng i ch ng khơng ABSTRACT Project: Research of biosynthetic process of bacteriocin and application Research contents: Survey published research articles for further study on collection pathway of highly active bacteriocin from Lactococcus lactis utilizing fed-batch fermentation mode Optimize culture conditions and nutrition for collecting bacteriocin utilizing fed-batch fermentation mode Survey sugar consumption over time by culturing strains and inhibition threshold Fed-batch fermentation Applications in material preservation of coconut nectar Results: - Re-identification of Lactococcus lactis strains by using API 50 CHL Kit: Lactococcus lactis similarity reached 92% - In fed-batch fermentation: bacteriocin has optimal incubation time in 16h; Optimal culture conditions at 370C, pH 6, 10% inoculation rate Appropriate nutrient conditions: sucrose concentration 20g/l, inhibition threshold of 60g/l The bacteriocin activity is 2180 AU/ml - In fed-batch fermentation: Lactococcus lactis cultured strains lactis in MRS medium supplemented with sucrose standards to maintain concentration of 20 g/l The timing of additional sucrose at 6h helped to maintain rapid log phase, additional cycles of sucrose for hours, maintaining optimal culture conditions Results activated bacteriocin reached 3210 AU/ml after 12 h incubation At the time of 16h, activated bacteriocin reached 3215 AU/ml, increasing biosynthesis productivity of bacteriocin up 147% compared to batch fermentation - Addition of 0.3% liquid activated bacteriocin to 3210 AU/ml obtained by the fed-batch fermentation process helps prolong shelf materials of coconut nectar days compared to control samples not use liquid bacteriocin at 30 32oC M CL C M U NG QUAN TÀI LI U 1.1 Vi khu n lactic 1.2 Bacteriocin 1.2.1 m, tính ch t 1.2.2 Phân lo i bacteriocin 1.3 H vi sinh v t trình sinh t ng h p bacteriocin 1.3.1 H vi sinh v t sinh t ng h p bacteriocin 1.3.2 Các c m gen sinh t ng h p bacteriocin 10 1.3.3 Sinh t ng h p bacteriocin 11 1.3.4 u hịa q trình t ng h p bacteriocin 12 1.3.5 ho ng c a bacteriocin 133 t mi n bacteriocin c a t bào ch 15 1.4 ng d ng bacteriocin 16 1.5 Các y u t n trình sinh t ng h p bacteriocin 20 1.6 Nguyên li um t hoa d a 22 1.7 Tình hình nghiên c c 26 1.7.1 Các nghiên c c 26 1.7.2 Các nghiên c c 26 T LI 2.1 Gi ng vi sinh v t 29 Hóa ch t ng 29 2.1.1 Gi ng vi sinh v t 29 2.1.2 Hóa ch ng ni c y 29 2.1.3 Thi t b d ng c s d ng 30 2.2 N i dung nghiên c u 31 2.3 B trí thí nghi m 32 i th , vi th 32 i nh danh gi ng thu n b ng b kit API 50 CHL 32 33 2.3.4 Kh u ki n nuôi c ng lên men batch 34 2.3.4.1 Kh o sát pH t a q trình ni c y .34 2.3.4.2 Kh o sát nhi t nuôi c y t .34 2.3.4.3 Kh o sát t l c y gi ng t 34 2.3.4.4.Kh o sát ng c a ngu n cacbon 34 2.3.4.5 Kh o sát ng c 2.3.5 Lên men Fed ng cacbon 35 batch 35 2.3.6 Kh o sát ng n d n th i gian b o qu n nguyên li u m t hoa d a 35 37 h 37 .39 K T QU VÀ BI N LU N 41 nh danh ch ng gi ng 41 3.2 Xây d ng ch ng Lc lactis 42 3.2.1 Xây d ng chu n 42 3.2.2 Xây d ng 44 3.3 Kh u ki n nuôi c 3.3.1 Kh o sát pH t 3.3.2 Kh o sát nhi ng lên men batch 46 a q trình ni c y 46 nuôi c y t 3.3.3 Kh o sát t l c y gi ng t 49 52 3.3.4 Kh o sát ng c a ngu n cacbon 53 3.3.5 Kh o sát ng c ng cacbon 56 3.4 Lên men fed-batch 59 3.4.1 Phân tích kh o sát làm ti cho lên men fed-batch 59 -batch 62 ii 3.5 K t qu kh o sát ng n d n th i gian b o qu n nguyên li u m t hoa d a 65 3.5.1 Ch tiêu vi sinh 66 3.5.2 Ch tiêu hóa sinh 70 K T LU N VÀ KI N NGH 74 4.1 K t lu n 74 4.2 Ki n ngh 74 TÀI LI U THAM KH O 76 iii CÁC T VI T T T LAB: vi khu n sinh lactic Lc.lactis: Lactococcus lactis TSVKHK: t ng s vi khu n hi u khí TSNM-NM: t ng s n m men n m m c PDA: Potatose Dextrose Agar MRS: Man, Rogosa, Sharpe iv K T LU N VÀ KI N NGH 4.1 K t lu n Qua thí nghi m kh - tài có nh ng k t lu n sau: nh danh ch ng gi ng b ng b ng Lactococcus lactis 92% - Trong lên men batch: bacteriocin có th i gian ni c y t ki n nuôi c y t nhi 16 gi 37 0C, pH 6, t l c y gi ng 10% ng thích h p: sucrose v i n bacteriocin lên men batch i u u ki n dinh ng c ch 60 g/l Ho t tính u ki n t t 2180 AU/ml - Trong lên men fed-batch: ti n hành nuôi c y ch ng Lc lactis mơi ng MRS chu n có b sung sucrose ch n th trì n 20 g/l Chúng m b sung sucrose gi giúp trì pha log nhanh, chu k b sung sucrose gi , ng th i d u ki n nuôi c y t t 3210 AU/ml sau 12 gi nuôi c y T i th t 3215 AU/ml, t qu ho t tính m 16 gi , ho t tính t t ng h p bacteriocin lên 147% so v i lên men batch - ng d ng b o qu n nguyên li u m t hoa d a: b sung 0,3% d ch bacteriocin v i ho t tính 3210 AU/ml, thu nh n b i q trình lên men fed-batch, giúp kéo dài th i gian b o qu n nguyên li u m t hoa d a ngày so v i m s d ng dung d ch bacteriocin u ki n nhi phòng 30 i ch ng không 32oC 4.2 Ki n ngh Kh o sát nghiên c u làm ti th c hi n lên men fed-batch Có th ti n hành kh o sát, t ngu ng khác ng h p bacteriocin v i gi i quy t toán v kinh t s n xu t quy mô l n Lên men fed-batch Kh o sát ho t tính bacteriocin th c hi n b 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batch and fed-batch culture by Lactococcus lactis Chem Technol Biotechnol 80:511 514 [61] Zhu Wenmiao c ng s (2000) Effect of various nutrient sources on nisin production by lactococcus lactic subsp Lactis SM526 Chin J Appl Environ biol, ISSN 1006 687X 82 PH L C ng chu nh n ng s d 1.2 y = 0.242x - 0.089 R² = 0.992 0.8 0.6 0.4 0.2 100 200 300 400 500 OD 540nm ng c a Lactococcus lactis B ng pH, m Th i gian (gi ) 10 12 14 16 18 20 22 24 26 28 30 t ng sót theo th i gian pH Log CFU/ml 5,48 4,54 4,16 3,88 3,75 3,70 3,64 3,63 3,63 3,66 3,62 3,64 3,64 3,64 3,64 7,47 8,03 8,43 8,56 8,63 8,72 8,70 8,68 8,63 8,62 8,61 8,61 8,57 8,55 8,49 ng sót (g/l) 19,29 16,97 13,09 7,87 5,83 3,60 2,42 2,29 2,24 2,19 2,06 2,00 1,81 1,63 1,57 Kh o sát pH t Kh o sát nhi a q trình ni c y pH OD 0,261 0,395 0,487 0,434 0,350 Ho t tính bacteriocin (AU/ml) 1170 1240 1950 1480 1200 nuôi c y t (oC) Nhi 33 35 37 39 41 OD 0,558 0,583 0,242 0,530 0,189 Ho t tính bacteriocin (AU/ml) 1650 1800 1950 1100 900 Kh o sát t l c y gi ng t T l c y gi ng (%) OD 2% 4% 6% 8% 10% 12% 0,397 0,423 0,445 0,471 0,498 0,480 Ho t tính bacteriocin (AU/ml) 1200 1420 1500 1560 1950 1775 Kh o sát Kh o sát ng c a ngu n cacbon Lactose (g/l) 10 15 20 25 30 Glucose (g/l) 10 15 20 25 30 Fructose (g/l) 10 15 20 25 30 ng c OD 0,477 0,568 0,644 0,603 0,598 0,363 0,521 0,593 0,625 0,587 0,468 0,696 0,653 0,635 0,619 ng cacbon (sucrose) Sucrose (g/l) OD 10 15 20 25 30 40 50 60 70 80 90 0,410 0,655 0,706 0,632 0,573 0,425 0,406 0,377 0,377 0,369 0,360 Ho t tính bacteriocin (AU/ml) 1250 2100 2180 2150 2075 2020 1850 1720 1700 1650 1650 Kh o sát trình lên men fed-batch: Lên men fed-batch erlen lít Th i gian (gi ) OD 10 12 14 16 18 20 0,291 0,349 0,414 0,491 0,482 0,454 0,436 0,431 Ch tiêu vi sinh: m TSVK HK(CFU/ml) vi sinh v t Th i gian theo dõi (ngày) Nghi m th c (t l bacteriocin %) 0,1 0,2 0,3 0,4 0,5 u 5,5.102 Ho t tính bacteriocin (AU/ml) 2550 2762 2820 3210 3210 3215 2970 2970 m u kh o sát 4,6.103 8,6.102 7,6.102 5,8.102 5,7.102 5,6.102 5,8.104 6,7.103 5,1.103 5,7.102 5,7.102 5,7.102 9,1.105 1,1.104 9,8.103 6,1.102 6,0.102 6,0.102 7,6.106 2,5.105 2,8.104 4,5.103 4,1.103 4,0.103 4,8.107 5,8.105 1,1.105 1,8.104 8,7.103 8,4.103 1,2.108 3,2.106 7,9.105 2,1.105 7,2.104 7,1.104 TSNM NM (CFU/ml) Th i gian theo dõi(ngày) Nghi m th c (t l bacteriocin %) 0,1 0,2 0,3 0,4 0,5 u 20 6,4.103 5,3.102 3,8.102 1,1.102 102 102 7,5.104 1,4.103 1,2.103 1,2.102 1,1.102 1,1.102 50 25 25 25 25 25 1,5.102 1,1.102 1,0.102 30 30 30 4,2.102 1,2.102 1,1.102 30 30 30 8,2.104 9,8.103 6,5.103 4,5.102 3,8.102 3,5.102 5,0 5,0 5,0 5,1 5,2 5,2 4,8 4,8 4,9 5,1 5,1 5,1 4,5 4,6 4,65 5,1 5,1 5,1 4,3 4,5 4,5 4,75 4,7 4,7 4,25 4,35 4,3 4,6 4,6 4,6 4,1 4,15 4,2 4,5 4,5 4,5 115 125 125 125 125 127 98 115 117 120 125 125 76 98 99 120 122 125 69 88 92 115 115 118 61 81 85 98 110 110 54 74 75 86 96 98 Ch tiêu hóa sinh: pH Th i gian theo dõi (ngày) Nghi m th c (t l bacteriocin %) 0,1 0,2 0,3 0,4 0,5 u 5,2 ng t ng (g/l) Th i gian theo dõi (ngày) Nghi m th c (t l bacteriocin %) 0,1 0,2 0,3 0,4 0,5 u 128 NGUY N TH NGUY T 29/03/1985 206/15 Bình Qu - 09/2004 - 05/2008 - 09/2012 n ng 28, Qu n Bình Th nh, TP HCM m Q TRÌNH CƠNG TÁC - n 09/2009 d y h c t ng Trung H c Ph Thông Nguy n Th Di u - n d y h c t Xuân ng Trung H c Ph Thông Bùi Th ... 1.2 Bacteriocin 1.2.1 m, tính ch t 1.2.2 Phân lo i bacteriocin 1.3 H vi sinh v t trình sinh t ng h p bacteriocin 1.3.1 H vi sinh v t sinh t ng h p bacteriocin. .. m gen sinh t ng h p bacteriocin 10 1.3.3 Sinh t ng h p bacteriocin 11 1.3.4 u hịa q trình t ng h p bacteriocin 12 1.3.5 ho ng c a bacteriocin 133 t mi n bacteriocin. .. nuôi c y Thành ph ng n s sinh t ng h p bacteriocin Sinh t ng h p bacteriocin b i ch ng S mutans s n trypticase m , s hình thành butyricin 7423 ng ph thu c nt Quá trình sinh bacteriocin c a ch ng