Phân lập, tuyển chọn các chủng probiotic từ heo rừng có khả năng ức chế escherichia coli và salmonella typhimurium và đánh giá đáp ứng sinh miễn dịch của chúng trên mô hình tế bào caco 2

TR I H C QU C GIA TP HCM NG I H C BÁCH KHOA HU NH CÔNG DUY PHÂN L P, TUY N CH N CÁC CH NG PROBIOTIC T HEO R NG CÓ KH N NGă C CH ESCHERICHIA COLI VÀ SALMONELLA TYPHIMURIUM VĨă ỄNHăGIỄă ỄPă NG SINH MI N D CH C A CHÚNG TRÊN MƠ HÌNH T BÀO CACO-2 Chuyên ngành: Công ngh sinh h c Mã s : 8420201 LU NăV NăTH CăS TP H CHÍ MINH, tháng 07 n mă2022 VIETNAM NATIONAL UNIVERSITY ậ HO CHI MINH CITY HO CHI MINH UNIVERSITY OF TECHNOLOGY HUYNH CONG DUY ISOLATION, SELECTION OF POTENTIAL PROBIOTIC STRAINS FROM WILD BOAR INHIBITING ESCHERICHIA COLI AND SALMONELLA TYPHIMURIUM AND EVALUATING THEIR ABILITY IN MODULATING IMMUNE RESPONSES IN CACO-2 CELL MODEL Discipline: Biotechnology Discipline code: 8420201 MASTER THESIS Ho Chi Minh City, July 2022 i Cơngătrìnhăđ c hồn thành t i: Tr ngă i h c Bách Khoa ậ HQGHCM Trung tâm Công ngh sinh h c TP.HCM Cán b h ng d n khoa h c 1: TS Nguy n Th L Th y Cán b h ng d n khoa h c 2: TS Hoàng M Dung Cán b ch m nh n xét : PGS TS Nguy n Ti n Th ng Cán b ch m nh n xét : TS Ph m Th Kim Trâm Lu năv năth căs ăđ c b o v t iăTr HCM ngày 13 tháng 07 n mă2022 ngă i h căBáchăKhoa,ă HQGăTp.ă Thành ph n H iăđ ngăđánhăgiáălu năv năth căs ăg m: (Ghi rõ h , tên, h c hàm, h c v c a H iăđ ng ch m b o v lu năv năth căs ) PGS.ăTSăVõă ìnhăL Tâm ậ Ch t ch h i đ ng TS Phan Th Huy n ậ Th ăkỦ PGS TS Nguy n Ti n Th ng ậ Ph n bi n TS Ph m Th Kim Trâm ậ Ph n bi n TS Hoàng M Dung - y viên Xác nh n c a Ch t ch H iăđ ngăđánhăgiáăLVăvàăTr ng Khoa qu n lý chuyên ngành sau lu năv năđãăđ c s a ch a (n u có) CH T CH H Iă NG PGS.ăTSăVõă ìnhăL Tâm TR NG KHOA K THU T HÓA H C ii TR I H C QU C GIA TP HCM C NG HÒA Xà H I CH NGH AăVI T NAM NGă I H C BÁCH KHOA c l p ậ T ậ H nh phúc NHI M V LU NăV NăTH CăS H tên h c viên: Hu nh Công Duy MSHV: 1970369 Ngày,ătháng,ăn măsinh:ă02/08/1997 N iăsinh:ăBìnhăD Chun ngành: Cơng ngh sinh h c Mã s : 8420201 ng I.ăTểNă TÀI: Phân l p, n ch n ch ng probiotic t heo r ng có kh n ngă c ch Escherichia coli Salmonella typhimurium vàăđánhăgiáăđápă ng sinh mi n d ch c a chúng mơ hình t bào Caco-2 NHI M V VÀ N I DUNG: M c tiêu c aăđ tài sàng l c, n ch n ch ng vi sinh v t t heo r ng có kh n ngă c ch vi sinh v t gây b nh E coli S typhimurium, đ ng th iăxácăđ nhăđ c tính probiotic kh n ngăđápă ng mi n d ch c a ch ng probiotic mơ hình t bào bi u mô ru t.ă tàiăđ c ti n hành v i n i dung sau: (1) Phân l p ch ng vi sinh v t t ngu n m u heo khác nhau; (2) ánhăgiáăkh n ngă c ch E coli S typhimurium c a ch ng phân l p đ c; (3) Xácăđ nh y u t c a ch ng vi sinh v t ti măn ngăcóătácăd ng c ch lên E coli S typhimurium; (4)ăXácăđ nhăđ c tính c a ch ng ti măn ng bao g m: kh n ngăch uăđ căđi u ki n axit mu i m t,ăđ an tồn, kh n ngăbámădínhătrênă t bào, tính kháng kháng sinh;ă(5)ă ánhăgiáăkh n ngă c ch s bám dính c a E coli mơ hình t bào bi u mơ ru t c a ch ng vi sinh v t ti măn ng; (6)ă ánhăgiáăm c đ đápă ng mi n d ch c a t bào bi u mô ru tăđ i v i ch ng vi sinh v t ti măn ng II NGÀY GIAO NHI M V : 06/09/2021 III NGÀY HOÀN THÀNH NHI M V : Tháng 06/2022 IV CÁN B H NG D N: Cán b h ng d n khoa h c 1: TS Nguy n Th L Th y Cán b h ng d n khoa h c 2: TS Hoàng M Dung Tp HCM, ngày… tháng… n m 2022 CÁN B H NG D N CH NHI M B TR NG KHOA MỌNă ĨOăT O iii L I C Mă N Lu năv năth căs c u n i gi a lý thuy t th c ti n, gi a ki n th c k n ngăđ t lu năv n,ăh c viên có th v n d ng ki n th c k n ngăđ ti p c n gi i quy t v năđ ă âyălàăn n t ng quan tr ngăđ h c viên tr thành m t th căs ăngành công ngh sinh h c sau Lu năv năth căs q trình tìm tịi, n l c, gi i quy t v năđ t nh ng ki n th c n n t ngăđãăđ c h c t b căđ i h c, nh ng ki n th căsâuăh nă b c cao h c, đ n nh ng ki n th c th c t t n iălàmăvi c.ă h c viên cao h c khơng th t mìnhălàmăđ cóăđ c thành qu này, ch c ch n cămàăsongăsongăđóălàăs t nătìnhăgiúpăđ c a th y cô, b n bè,ăđ ng nghi p L iăđ u tiên xin g i l i c mă năđ n tồn th q Th y,ăCơătr ngă i h c Bách Khoa TPHCM nói chung Th y, Cơ b mơn Cơng ngh sinh h c nói riêngăđãăcungăc p nh ng ki n th c quý báu gi ngăđ ng Ti păđ n, xin g i l i c mă năchânăthànhăđ n TS Nguy n Th L Th y, ch v a ng i th yăh ng d n truy năđ t ki n th c, kinh nghi m, k n ng,ăv aălàăng i b năđ ngăhành,ăđ ng viên su t trình th c hi n lu năv nănày.ăBênăc nh đó,ătơiăxinăcámă năKS.ăLongăTh H nh b n Võ Th TrúcăDunăđãăđ ng hành tơi nghiên c uănày.ăTơiăchânăthànhăcámă năcơăTS.ăHồngăM Dungăđãăt n tình h ng d năvàăgiúpăđ th iăgianăquaăđ tơi có th hồn thành lu năv năt t nghi p Ti p theo xin bày t l i c mă năchânăthànhăđ n Phịng Cơng nghê Sinh h c Yăd c - Trung tâm Công ngh sinh h c thành ph H ChíăMinhăđãăt oăđi u ki n t t nh tăđ tơi có th th c hi n lu năv năt t nghi p Cu i cùng,ătôiăc ngăxinăg i l i c mă năt iăgiaăđìnhătơiăậ đâyălàăbànăt a t t nh t choătôiăvàăluônăđ ngăhànhăcùngătôiăchoăđ n th i m hi n t i, c mă nănh ngăng i b năđãăluônăbênăc nh h tr ,ăđ ng viên tơi q trình h c t păc ngănh ăhoànă thành lu năv năt t nghi p TP.ăHCM,ăthángă06ăn mă2022 Hu nh Cơng Duy iv TĨM T T LU NăV NăTH CăS Lu năv n:ă“Phân l p, n ch n ch ng probiotic t heo r ng có kh n ngă c ch Escherichia coli Salmonella typhimurium vàăđánhăgiáăđápă ng sinh mi n d ch c a chúng mơ hình t bào Caco-2” Các ch ng vi khu n lactic (LAB) có vai trò quan tr ng vi c c ch vi sinh v t (VSV) gây b nh, cân b ng h viăsinhăđ hóaăchoăheo.ă ng ru t c i thi năn ngăsu t tiêu c bi t, vi c s d ng ch ngăLABăđ c phân l p t v t ch đãăđ c ch ng minh có hi u qu caoăh năsoăv i ngu n phân l p khác Trong nghiên c u này, 47 ch ng VSV thu năđ c phân l pătrênămôiătr ru t heo r ng heo nhà K t qu sàng l c b ngăph đ ng MRS t m u phân ngăphápăđ aăth ch hai l p thu c ch ng có kh n ngă c ch m nh v i E coli, trongăđóă3ăch ng có kh n ngă c ch m nh v i S typhimurium Y u t axit thành ph n protein d ch nuôi c y c a ch ng ch n l c đ căxácăđ nh có vai trị quan tr ng c ch hai lo i vi khu n gây b nh ch th thôngăquaăph ngăphápăkhu ch tán gi ng th ch K đ n, ch ng ti măn ngăth hi năcácăđ c tính probiotic thơng qua ch tiêuănh ăkh n ngă ch uăđ căđi u ki n axit (pHă4)ăvàăđi u ki n mu i m t 0,3%; ch ngăđ đ nh an toàn qua kh n ngăkhôngălàmătanăhuy t Các ch ng ti măn ngăđ c xác căxácăđ nh Leuconostoc mesenteroides, Pediococcus pentosaceus, ba ch ng Weissella confusa b ngăph ngăphápăgi i trình t vùng 16S rRNA Ch ng W confusa 23 P pentosaceus 27 có kh n ngăbámădínhăt t Caco-2,ăđ ng th i c ch s bám dính c a E coli mơ hình t bào bi u mô ru t Caco-2 Kh n ngăđi u ch nhăđápă ng mi n d ch c a hai ch ngănàyăđ c ch ng minh thông qua kh n ngăgi m bi u hi n c a cytokine ti n viêm IL-6 TNF- t bào Caco-2 b kích thích v i LPS Nh ng ch ng ng c viênăđ đánhăgiáăin vivo cácătácăđ ng có l i c a chúng ti n đ đ phát tri n ch ph m probiotic phân l p t heo r ng có ti măn ngă ng d ng làm ch t b tr cho th că năch nănuôiăheo.ă v SUMMARY Thesis:ă “Isolation,ă selectionă ofă potentială probiotică strainsă fromă wildă boară inhibiting Escherichia coli and Salmonella typhimurium and evaluating their ability in modulating immune responses in Caco-2 cell model” Lactic acid bacteria (LAB) have an essential role in inhibiting pathogenic microorganisms, balancing intestinal microflora, and improving digestive performance for pigs In particular, the use of LAB strains isolated from the host is more effective than other isolates In this study, 47 LAB strains were isolated on MRS medium from intestinal samples of wild boar and domestic pig Screening results using the spot-on-lawn method obtained potential strains with a strong inhibitory ability against E coli and among in strains showed great inhibitory ability against S typhimurium The acidic condition and protein composition(s) in the culture of selected strains were determined to play an important role in inhibiting two indicator bacteria by the well-diffusion method Next, the characteristics for probiotic properties of the potential strains were determined as tolerance to acidic conditions (pH 4) and bile salt conditions (0.3%) as well as non-hemolysis The potential strains were identified as Leuconostoc mesenteroides, Pediococcus pentosaceus, and three strains of Weissella confusa by 16S rRNA sequencing The result of adhesion ability on Caco-2 model showed that W confusa 23 and P pentosaceus 27 strains had a high adhesion ability on Caco-2 Furthermore, they also inhibited the adhesion of E coli on the Caco-2 model Moreover, Moreover, these two strains were able to modulate the immune response by reducing the gene expression of proinflammatory cytokines IL-6 and TNF- in the LPS-treated Caco-2 model Taken together, these strains are candidates for further in vivo evaluation of their beneficial effects and for developing that can be used for swine feed vi L IăCAMă OAN Tơiăxinăcamăđoanăđâyălàăcơngătrìnhănghiênăc u c a tơiăvàăđ cs h ng d n c a TS Nguy n Th L Th y TS Hoàng M Dung K t qu lu năv nănàyăbaoăg m Lu năv năđ i h c “Phânăl p n ch n ch ng vi sinh v t có kh n ngă c ch Escherichia coli Salmonella typhimurium gây b nhăđ Long Th H nh d is h ng ru t heo”ăc a KS ng d n c a TS Nguy n Th L Th y ThS Tr n Trúc Thanh.ă âyălàăm t ph n c aăđ tài thu c Trung tâm Công ngh Sinh h c TP HCM Lu năv năđ c c p tồn b kinh phí th c hi n t iăPhịngăCNSHăYăd c ậ Trung tâm Cơng ngh Sinh h căTP.ăHCM.ăDoăđó,ăk t qu c a lu năv năthu c s h u c a Trung tâm Công ngh Sinh h c TP HCM Các n i dung nghiên c u, k t qu đ u trung th căvàăch aăcơngăb d i b t k hình th cănàoătr b ng bi u ph c v cho phân tích, nh nă xét,ă đánhă giáă đ căđây.ăNh ng s li u c tác gi thu th p t ngu n tài li u khác có ghi rõ ph n tài li u tham kh o N u phát hi n có b t k gian l n tơi xin hồn tồn ch u trách nhi m v lu năv năc aămình.ăTr ngă i h c Bách Khoa Tp.HCM không liên quanăđ n nh ng vi ph m tác quy n, b n quy n tơi gây q trình th c hi n (n u có) vii M CL C DANH M C B NG x DANH M C HÌNH xi DANH M C CH VI T T T xii M U CH NGă1.ăT NG QUAN TÀI LI U 1.1 Probioticătrongăch nănuôi 1.1.1 Khái ni m l ch s hình thành .3 1.1.2 Các ch ng probiotic ph bi n s d ngătrongăch nănuôi 1.1.3 Ngu n phân l p ch ng probiotic 1.1.4 M t cân b ngăviăsinhăđ ng ru t d năđ n b nh đ ng ru t c a heo 1.2 C ăch tácăđ ng c a probiotic 1.2.1 i u ch nh h vi sinh v tăđ 1.2.2 i u ch nh ph n ng mi n d ch c a v t ch 1.2.3 Gi m tiêu ch y ch ngăđ c t 12 1.2.4 C i thi n kh n ngătiêuăhóaăch tădinhăd 1.2.5 M t s tácăđ ng khác 13 1.3 ng .12 Các yêu c u v đ c tính c a probiotic .14 1.3.1 Kh n ngăkhángăaxităvàămu i m t 15 1.3.2 Ho tăđ ng phân gi i mu i m t 15 1.3.3 Kh n ngăbámădínhăv i ch t nh y t bào bi u mô 15 1.3.4 Ho tăđ ng kháng khu năvàăđ i kháng v i vi sinh v t gây b nh 16 1.3.5 Tính an tồn v i v t ch .16 1.4 Gi i thi u v heo r ng .16 1.4.1 1.4.2 1.5 CH ng ru t Khu v c phân b vàăđ căđi m chung c a heo r ng 16 iăt ng ti măn ngăđ phân l p probiotic 17 Tình hình nghiên c uătrongăvƠăngoƠiăn c .18 1.5.1 Các nghiên c u th gi i 18 1.5.2 Các nghiên c uătrongăn NGă2.ăV T LI UăVĨăPH c 19 NGăPHỄPăNGHIểNăC U .21 2.1 V t li u nghiên c u .21 viii 2.1.1 Ch ng VSV 21 2.1.2 T bào Caco-2 21 2.1.3.ăMơiătr 2.2.ăPh ng, hóa ch t s d ng 21 ngăphápănghiênăc u .22 2.2.1 Thu th p phân l p ch ng vi sinh v t .23 2.2.2.ăă ánhăgiáăkh n ngă c ch E coli S typhimurium c a ch ng phân l păđ c 24 2.2.3.ăXácăđ nh y u t c a ch ng vi sinh v t có tác d ng c ch lên E coli S typhimurium .24 2.2.4.ăXácăđ nhăcácăđ c tính probiotic c a ch ng ti măn ng .26 2.2.5.ă nh danh ch ng ti măn ng .28 2.2.6.ă ánhăgiáăm căđ đápă ng mi n d ch c a t bào ru tăđ i v i ch ng vi sinh v t ti măn ng 28 2.2.7 Phân tích x lý th ng kê 30 CH NGă3.ăK T QU VÀ TH O LU N 31 3.1 K t qu phân l p ch ng VSV t ngu n m u khác .31 3.2.ă ánhăgiáăkh n ngă c ch E coli S typhimurium c a ch ng phân l păđ c 33 3.3 Xác đ nh y u t c a ch ng vi sinh v t ti măn ngăcóătácăd ng c ch lên Escherichia coli Salmonella typhimurium .35 3.4.ăXácăđ nhăcácăđ c tính probiotic c a ch ng ti măn ng 41 3.4.1.ă ánhăgiáăđ an toàn 41 3.4.2.ă ánhăgiáăkh n ngăkhángăaxităvàămu i m t .42 3.4.3.ă ánhăgiáăkh n ngăbámădínhătrênămơăhìnhăt bào ru t Caco-2 44 3.4.4.ă ánhăgiáăkh n ngă c ch s bám dính c a E coli lên t bào ru t c a ch ng ti măn ng 46 3.4.5.ă ánhăgiáătínhănh y c m v i kháng sinh c a ch ng ti măn ng 48 3.5.ă 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ng ti măn ng M tăđ ăt ăbƠoă ăpHă4ă(logCFU/mL) Kíăhi uă ch ng L nă1 L nă2 Trung bình ± SD L nă1 L nă2 Trung bình ± SD 12 10.51 10.54 10.53 ± 0.02 8.48 8.73 8.61 ± 0.13 18 10.51 10.5 10.51 ± 0.01 7.78 8.15 7.97 ± 0.19 22 10.76 10.78 10.77 ± 0.01 8.08 8.80 8.44 ± 0.36 23 9.70 9.78 9.74 ± 0.04 7.30 7.90 7.60 ± 0.30 27 10.31 10.42 10.37 ± 0.06 8.41 8.34 8.38 ± 0.04 Ph l c Kh n ngăkhángămu i m t c a ch ng ti măn ng iăch ngăMRS (logCFU/mL) MRSă+ă0,3%ămu iăm t (logCFU/mL) Kí hi uă ch ng L nă1 L nă2 L nă3 Trung bình ± SD L nă1 L nă2 L nă3 Trung bình ± SD 12 10.62 10.4 9.81 10.28 ± 0.35 9.86 9.64 9.28 9.6 ± 0.24 18 10.9 10.76 10.55 10.74 ± 0.15 9.15 9.58 9.45 9.4 ± 0.19 22 10.95 10.77 10.56 10.76 ± 0.16 8.78 8.78 9.08 8.88 ± 0.15 23 10.85 10.41 10.54 10.6 ± 0.19 9.2 9.15 9.09 9.15 ± 0.05 27 10.57 10.40 10.42 10.47 ± 0.08 9.85 9.08 9.59 9.51 ± 0.32 Ph l c Kh n ngăbámădínhăc a ch ng ti măn ngătrênămơăhìnhăt bào Caco-2 Kíăhi uăch ng T ăl ăbámădínhătrênăt ăbƠoăCaco-2 (%) L nă1 L nă2 L nă3 Trung bình ± SD 12 3.97 4.92 4.52 4.47 ± 0.39 18 4.46 5.53 5.69 5.23 ± 0.55 22 2.88 2.43 3.89 3.07 ± 0.61 23 104.22 84.89 101.87 27 81.43 70.03 65.96 96.99 ± 8.61 72.47 ± 6.55 68 Ph l c Kh n ngă c ch s bám dính c a E coli t bào Caco-2 c a ch ng ti măn ng nghi m th c so v iăđ i ch ng E coli khơng x lí (%) T l E coli bám t bào Kí hi u C nh tranh L n L n L n 12 51.2 82.2 26.1 18 92.6 74.3 67.6 22 63.1 83.8 64.5 23 60.1 90.3 32.4 27 36.7 64.6 19.6 Ph l c 6.ă Thay th Trung bình ± SD 53.2 ± 23.0 78.1 ± 10.6 L n L n L n 80.0 75.5 37.2 68.8 95.4 28.6 70.5 ± 9.4 97.7 88.2 71.5 50.8 85.9 28.7 71.1 66.7 11.4 60.9 ± 23.7 40.3 ± 18.6 c ch Trung bình ± SD 64.2 ± 19.2 64.3 ± 27.4 85.8 ± 10.8 55.1 ± 23.5 49.7 ± 27.2 L n L n L n 72.0 85.8 47.4 71.0 82.9 39.2 90.5 81.1 96.9 89.5 ± 6.5 71.5 70.0 51.6 64.3 ± 9.1 76.9 67.3 73.2 72.5 ± 3.9 ng kính vịng vơ khu n (mm) đánhăgiáăb ngăph khu chătánăđ aăgi y kháng sinh Trung bình ± SD 68.4 ± 15.9 64.4 ± 18.5 ngăphápă Kíăhi uăch ng Kháng sinh Hàm l ngă (g) 12 18 22 23 27 Amp 10 20 24 32 18 22 CZ30 30 - 0 IPM 10 10 32 32 26 30 Ci 12 - 14 10 GE 10 22 22 26 20 22 CT 10 16 24 30 16 20 69 Ph l c Trình t nucleotide t k t qu gi i trình t vƠăđ i chi u v i ngân hàng gen b ng ph n m m BLAST Kí hi u ch ng K t qu gi i trình t vùng 16S rRNA 12 CAGTCGACGCACAGCGAAAGGTGCTTGCACCTTTCAAGTGAGTGGCGAACGGGTG AGTAACACGTGGACAACCTGCCTCAAGGCTGGGGATAACATTTGGAAACAGATGC TAATACCGAATAAAACTTAGTGTCGCATGACACAAAGTTAAAAGGCGCTTCGGCGT CACCTAGAGATGGATCCGCGGTGCATTAGTTAGTTGGTGGGGTAAAGGCCTACCAA GACAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACA CGGCCCAAACTCCTACGGGAGGCTGCAGTAGGGAATCTTCCACAATGGGCGAAAG CCTGATGGAGCAACGCCGCGTGTGTGATGAAGGCTTTCGGGTCGTAAAGCACTGTT GTATGGGAAGAACAGCTAGAATAGGAAATGATTTTAGTTTGACGGTACCATACCAG AAAGGGACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTCCCGAGCGT TATCCGGATTTATTGGGCGTAAAGCGAGCGCAGACGGTTTATTAAGTCTGATGTGA AAGCCCGGAGCTCAACTCCGGAATGGCATTGGAAACTGGTTAACTTGAGTGCAGTA GAGGTAAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACA CCAGTGGCGAAGGCGGCTTACTGGACTGCAACTGACGTTGAGGCTCGAAAGTGTG GGTAGCAAACAGGATTAGATACCCTGGTAGTCCACACCGTAAACGATGAACACTA GGTGTTAGGAGGTTTCCGCCTCTTAGTGCCGAAGCTAACGCATTAAGTGTTCCGCCT GGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAG CGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGAC ATCCTTTGAAGCTTTTAGAGATAGAAGTGTTCTCTTCGGAGACAAAGTGACAGGTG GTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAG CGCAACCCTTATTGTTAGTTGCCAGCATTCAGATGGGCACTCTAGCGAGACTGCCG GTGACAAACCGGAGGAAGGCGGGGACGACGTCAGATCATCATGCCCCTTATGACC TGGGCTACACACGTGCTACAATGGCGTATACAACGAGTTGCCAGCCCGCGAGGGTG AGCTAATCTCTTAAAGTACGTCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATG AAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGG TCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTAATGCCCAAAGCCGGTGGC CTAACCTTAGAAGAG 99,71% 18 GGCTCCGAGGTTACCCCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGG CGGTGTGTACAAGACCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACT AGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGACGTAC TTTAAGAGATTAGCTCACCCTCGCGGGTTGGCAACTCGTTGTATACGCCATTGTAGC ACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCC TCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTGAATGCTGGCAACTAGT AATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGAC GACAACCATGCACCACCTGTCACCTTGTCCCCGAAGGGAACGCTCCATCTCTGGAG TTGTCAAGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCA CATGCTCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGT CGTACTCCCCATGCGGAGTGCTTAATGCGTTAGCTGCGGCACTTAAGGGCGGAAAC CCTCAAACACCTAGCACTCATCGTTTACGGTGTGGACTACCAGGGTATCTAATCCT GTTTGCTACCCACACTTTCGAGCCTCAACGTCAGTTACAGTCCAGAAAGCCGCCTTC GCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACT TTCCTCTACTGCACTCAAGTCATCCAGTTTCCAAAGCAATTCCTCAGTTGAGCTGAG GGCTTTCACTTCAGACTTAAATAACCGTCTGCGCTCGCTTTACGCCCAATAAATCCG GATAACGCTTGGAACATACGTATTACCGCGGCTGCTGGCACGTATTTAGCCGTTCC TTTCTGGTAAGATACCGTCACACATTGAACAGTTACTCTCAATGTCATTCTTCTCTT ACAACAGTGTTTTACGAGCCGAAACCCTTCATCACACACGCGGCGTTGCTCCATCA GGCTTTCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTATGGGCC GTGTCTCAGTCCCATTGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCT TGGTAAGCCATTACCTTACCAACTAGCTAATGCACCGCGGGACCATCTCTTAGTGA TAGCAGAACCATCTTTTAAATAACAACCATGCGGTTGTCATTGTTATACGGTATTAG CATCTGTTTCCAAATGTTATCCCCTGCTAAGAGGTAGGTTTCCCACGTGTTACTCAC CCGTTCGCCACTCTTTGCAATGTCCATCGTCATATCTGAGCAAGCTCTTCAAATCAG TTGAACCACAAAGCGTCGACTGCA 99,86% 22 GGCTCCGAGGTTACCCCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGG CGGTGTGTACAAGACCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACT AGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGACGTAC TTTAAGAGATTAGCTCACCCTCGCGGGTTGGCAACTCGTTGTATACGCCATTGTAGC 98,89% t ngă đ ng 70 ACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCC TCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTGAATGCTGGCAACTAGT AATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGAC GACAACCATGCACCACCTGTCACCTTGTCCCCGAAGGGAACGCTCCATCTCTGGAG TTGTCAAGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCA CATGCTCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTAAGTTTCAACCTTGCGGT CGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCGGCACTTAAGGGCGGAAAC CCTCAAACACGGGGGGGGTCGGGGGGGGACGGTGTGGACTACCAGGGTATCTAAT CCTGTTTGCTACCCACACTTTCGAGCCTCAACGTCAGTTACAGTCCAGAAAGCCGC CTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTC CACTTTCCTCTACTGCACTCAAGTCATCCAGTTTCCAAAGCAATTCCTCAGTTGAGC TGAGGGCTTTCACTTCAGACTTAAATAACCGTCTGCGCTCGCTTTACGCCCAATAAA TCCGGATAACGCTTGGAACATACGTATTACCGCGGCTGCTGGCACGTATTTAGCCG TTCCTTTCTGGTAAGATACCGTCACACATTGAACAGTTACTCTCAATGTCATTCTTC TCTTACAACAGTGTTTTACGAGCCGAAACCCTTCATCACACACGCGGCGTTGCTCC ATCAGGCTTTCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTATGG GCCGTGTCTCAGTCCCATTGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCG CCTTGGTAAGCCATTACCTTACCAACTAGCTAATGCACCGCGGGACCATCTCTTAGT GATAGCAGAACCATCTTTTAAATAACAACCATGCGGTTGTCATTGTTATACGGTATT AGCATCTGTTTCCAAATGTTATCCCCTGCTAAGAGGTAGGTTTCCCACGTGTTACTC ACCCGTTCGCCACTCTTTGCAATGTCCATCGTCATATCTGAGCAAGCTCTTCAAATC AGTTGAACCACAAAGCGTCGACTGC 23 TTACCCCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTAC AAGACCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCG ACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGACGTACTTTAAGAGAT TAGCTCACCCTCGCGGGTTGGCAACTCGTTGTATACGCCATTGTAGCACGTGTGTA GCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGT CACCGGCAGTCTCACTAGAGTGCCCAACTGAATGSTGGCAACTAGTAATAAGGGTT GCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATG CACCACYTGTCACCTTGTCCCCGAAGGGAACGCTCCATCTCTGGAGTTGTCAAGGG ATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACC GCTTGTGCGGGTCCCCGTCAATTCCTTTGAGGTTCAACCTTGCGGAAATTGTTTCTT ATTTGGATTGCTTAATGCGTTAGCTGCGGCACTTAAGGGCGGAAACCCTCAAACAC CTAGCACTCATCGTTTTACKGTGTGGACTACCAGGGTATCTAATCCTGTTTGCTACC CACACTTTCGAGCCTCAACGTCAGTTACAGTCCAGAAAGCCGCCTTCGCCACTGGT GTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTTTCCTCTACT GCACTCAAGTCATCCAGTTTCCAAAGCAATTCCTCAGTTGAGCTGAGGGCTTTCACT TCAGACTTAAATAACCGTCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCT TGGAACATACGTATTACCGCGGCTGCTGGCACGTATTTAGCCGTTCCTTTCTGGTAA GATACCGTCACACATTGAACAGTTACTCTCAATGTCATTCTTCTCTTACAACAGTGT TTTACGAGCCGAAACCCTTCATCACACACGCGGCGTTGCTCCATCAGGCTTTCGCCC ATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTATGGGCCGTGTCTCAGTCC CATTGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCATT ACCTTACCAACTAGCTAATGCACCGCGGGACCATCTCTTAGTGATAGCAGAACCAT CTTTTAAATAACAACCATGCGGTTGTCATTGTTATACGGTATTAGCATCTGTTTCCA AATGTTATCCCCTGCTAAGAGGTAGGTTTCCCACGTGTTACTCACCCGTTCGCCACT CTTTGCAATGTYCATCGTCATATCTGAGCAAGCTCTTCAAATCAGTTGAACCACAA AGCGT 98,66% 27 AAAGGTTACCCCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTG TGNANAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCRCRATTACTAGCGA TTCCGACTTCGNGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGTTTTAA RAGATTAGCTTAACCTCGCGGTCTCGCGACTCGTTGTACCATCCATTGTARCACGNG NGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCGTCCCCACCTTCCTCCGG TTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAATAA GGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACRACAA CCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACCTCTAATCTCTTAGACTGTCA GAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCT CCACCGCTTGTGCGGGCCCCCGTCAATTCTTTTGAGTTTCAACCTTGCGGTCGTACT CCCCAGGCGGATTACTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCA ACACTTAGTAATCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCT ACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGACAGCCGCCTTCGCCACT GGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTC TTCTGCACTCAAGTCTCCCAGTTTCCAATGCACTTCTTCGGTTGAGCCGAAGGCTTT CACATTAGACTTAAAAGACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAA CGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTG 99,3% 71 GTTAAATACCGTCACTGGGTAAACAGTTACTCTTACCCACGTTCTTCTTTAACAACA GAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGC GTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCA GTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCACTGCCTTGGTGAGC CTTTACCTCACCAACTAGCTAATACGCCGCGGGTCCATCCAGAAGTGATAGCAGAG CCATCTTTTAAAAGAAAACCATGCGGTTTTCTCTGTTATACGGTATTAGCATCTGTT TCCAGGTGTTATCCCCTACTTCTGGGCAGGTTACCCACGTGTTACTCACCCGTTCGC CACTCACTTCGTGTTAAAATCTCAATCAGTACAAGTACGTCATAATCAATTAACGG AAGTTC Ph l c K t qu BLAST trình t 16S rRNA c a ch ng ti măn ngătrênăngơnăhƠngăgenă NCBI Ch ng 12: Leuconostoc mesenteroides Ch ng 18: Weissella confusa Ch ng 22: Weissella confusa Ch ng 23: Weissella confusa 72 Ch ng 27: Pediococcus pentosaceus Ph l c M căđ bi u hi n tính theo 2-Ct c a gen m c tiêu so v i gen GAPDH c a m u x lí so v i m uăđ ii ch ng khơng x lí M căđ ăbi uăhi năc aăgenăm cătiêuăsoăv iăgenăGAPDHă(2 -Ct) M u L nă L n 1.00 1.00 2.08 41 45 IL6 Trung bình ± SD L nă L n 1.00 ± 0.00 1.00 1.00 2.03 2.06 ± 0.03 2.43 1.13 1.10 1.12 ± 0.01 0.90 0.87 0.89 ± 0.02 41 + LPS 1.64 1.59 1.62 ± 0.02 45 + LPS 0.93 0.91 0.92 ± 0.01 iă ch ng LPS IL8 Trung bình ± SD TNF- Trung bình ± SD L nă L n 1.00 ± 0.00 1.00 1.00 1.00 ± 0.00 5.50 3.97 ± 1.54 2.14 1.91 2.03 ± 0.12 2.22 5.03 3.63 ± 1.41 0.07 0.14 0.11 ± 0.04 1.68 3.81 2.75 ± 1.07 0.28 0.54 0.41 ± 0.13 2.60 5.90 4.25 ± 1.65 0.30 0.56 0.43 ± 0.13 1.8 4.08 2.94 ± 1.14 0.16 0.31 0.16 ± 0.00 73 LÝ L CH TRÍCH NGANG H tên: Hu nh Công Duy Ngày,ătháng,ăn măsinh:ă02/08/1997 a ch liên l c: Khu ph 1,ăph N iăsinh:ăBìnhăD ng M Ph ng c, th xã B n Cát, t nhăBìnhăD ng QUỄăTRỊNHă ĨOăT O B căđƠoăt o ih c Th c s N iăđƠo t o Tr ngăđ i h c Bách Khoa Tp.HCM Tr ngă i h c Bách Khoa Tp.HCM Chuyên môn Th i gian Công ngh sinh h c 2015-2019 Công ngh sinh h c 2019- 2022 (d ki n) Q TRÌNH CƠNG TÁC Th i gian (T n m đ n n m ) 2019-2022 V trí cơng tác Nghiên c u viên T ch c cơng tác Phịng CNSH Yăd c, Trung tâm Cơng ngh sinh h c Tp.HCM a ch T ch c 2374 qu c l 1, Khu ph 2,ăph ng Trung M Tây, qu n 12, Tp H Chí Minh ... 21 ngăphápănghiênăc u .22 2. 2.1 Thu th p phân l p ch ng vi sinh v t .23 2. 2 .2. ăă ánh? ?giá? ?kh n ngă c ch E coli S typhimurium c a ch ng phân l păđ c 24 2. 2.3.ăXácăđ... TP.ăHCM,ăthángă06ăn m? ?20 22 Hu nh Cơng Duy iv TĨM T T LU NăV NăTH CăS Lu năv n:ă? ?Phân l p, n ch n ch ng probiotic t heo r ng có kh n ngă c ch Escherichia coli Salmonella typhimurium và? ?đánh? ?giá? ?đáp? ? ng sinh mi... vi sinh v t có tác d ng c ch lên E coli S typhimurium .24 2. 2.4.ăXácăđ nh? ?các? ?đ c tính probiotic c a ch ng ti măn ng .26 2. 2.5.ă nh danh ch ng ti măn ng .28 2. 2.6.ă ánh? ?giá? ?m