HỌC VIỆN NÔNG NGHIỆP VIỆT NAM KHOA CÔNG NGHỆ SINH HỌC KHÓA LUẬN TỐT NGHIỆP I PHÂN LẬP, TUYỂN CHỌN VI KHUẨN LACTIC CÓ KHẢ NĂNG SINH ỔNG HỢP GAMMA – AMINOBUTYRIC ACID VÀ KHẢO SÁ I U KIỆN LÊN MEN TRÊN CÁM GẠO HÀ NỘI - 2022 HỌC VIỆN NƠNG NGHIỆP VIỆT NAM KHOA CƠNG NGHỆ SINH HỌC KHĨA LUẬN TỐT NGHIỆP I PHÂN LẬP, TUYỂN CHỌN VI KHUẨN LACTIC CÓ KHẢ NĂNG SINH ỔNG HỢP GAMMA – AMINOBUTYRIC ACID VÀ KHẢO SÁ I U KIỆN LÊN MEN TRÊN CÁM GẠO Người thực hiện: hị Vân Anh K63 ng nghệ sinh họ Người hư ng n S PGS hị inh hành S Ngu n S Hoàng Hải Hà HÀ NỘI - 2022 n Gi ng I O N Sinh viên hị i n nh I Ả G N – B viên Sinh viên hị ii n nh MỤC LỤC I O N i I Ả N ii MỤC LỤC iii DANH MỤC BẢNG vi DANH MỤC HÌNH ẢNH, Ồ THỊ vii NH Ụ H I Ắ viii Ắ ix hương MỞ ẦU 11 ặt v n .1 1.2 Mụ 1 ụ hương TỔNG QUAN TÀI LIỆU 2.1 Tổng quan v gamma-aminobutyric acid 2.1.1 Gi i thi u chung 2.1.2 Vai trò c a GABA ổ 2.1.3 ổ 2.2.1 ặ h p GABA vi khu n lactic có kh m hình thái, sinh lý, sinh hóa ổ 2.2 .10 12 ổ sinh 14 a m t s h p ch t th c p cám g o 15 15 iii 2.3.2 Gamma – oryzanol 18 2.3.3 Acid ferulic 19 2.3.4 M t s nghiên c u s n xu t Gamma – Aminobutyric acid từ cám g o .20 hương VẬT LIỆU PHƯ NG PHÁP NGHIÊN ỨU 22 3.1 V t li u nghiên c u 22 3.1.1 ng nghiên c u 22 3.1.2 V t li u thí nghi m .22 m nghiên c u .22 3.2 Hóa ch t thi t b nghiên c u 23 3.2.1 Thi t b 23 3.2.2 Dụng cụ 23 3.2.3 Hóa ch t 23 3.3 N i dung nghiên c u 23 3.3.1 N i dung 1: 23 3.3.2 N i dung 2: 24 3.3.3 N i dung 3: 24 3.3.4 N i dung 4: 25 u 26 .26 26 ặ 27 lactic 29 30 31 iv 35 lý s li u 36 hương K T QUẢ VÀ THẢO LUẬN 37 ặ 37 42 45 1 45 47 hương UẬN 50 I N NGHỊ 54 54 .54 TÀI LIỆU THAM KHẢO 55 PHỤ Ụ 66 v DANH MỤC BẢNG B ng 2.1 S sinh tổng h p GABA b i vi sinh v c phân l p từ n th c ph m lên men 11 B ng 2.2 Vi khu n acid lactic có m t s th c ph m lên men B ụ 22 B ng 3.2 B ng 3.3 Xây d 31 ng chu n từ dung d ch g c GABA 0,2 mg/mL 33 B ng 4.1: ặ B Vi t Nam 12 37 ổ 42 B ổ 46 B ổ 47 vi DANH MỤC HÌNH ẢNH, Ồ THỊ Hình 2.1: Gama- aminobutyric acid Hình 2.2: Sinh tổng h ng 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pyrosequence Nucleic Acids Research 36(18): 120 124 Kandler O and Weis N (2005) Regular nonsporing gramManual of Systematic Bacteriology 4(2): 1208-1231 ‟ 125 Thwe, S M., Kobayashi, T., Luan, T., Shirai, T., Onodera, M., Hamada-Sato, N., and 11 f γ-aminobutyric acid (GABA)-producing lactic acid bacteria from Myanmar fishery products fermented with boiled rice 65 PHỤ Ụ Phụ lụ hủng vi khuẩn đượ ph n lập từ sản phẩm l n men men ti u h đ ng kh hiệu hủng STT nm u ph n lập LTM1.1 21 LTM1.2 22 LTM1.3 23 Kim chi DC2.1 24 DC2.2 25 DC2.3 26 DC2.4 27 DCI3.1 28 DCI3.2 29 DCI3.3 30 DCI3.4 31 DCI3.5 32 DCI3.6 33 DCI3.7 34 KC4.1 35 KC4.2 36 KC4.3 37 hiệu hủng ph n lập KC4.7 KC4.8 KC4.9 KC4.10 KC4.11 ST6.1 ST6.2 SB7.1 SB7.2 SB7.3 MT7.1 SCND8.1 SCND8.2 SCND8.3 SCND8.4 NG9.1 NG9.2 KC4.4 38 Lcs10 19 KC4.5 39 20 KC4.6 40 STT 10 11 12 13 14 15 16 17 18 nm u Lactomin Plus Kim chi 66 Proby Phô mai lên men PM11 Phụ lụ thị đường huẩn G B đượ s ụng thí nghiệm định hàm lượng G B s u l n men tr n ám gạo 1.600 1.400 y = 7,0275x + 0,2212 R² = 0,9913 OD 645nm 1.200 1.000 0.800 0.600 0.400 0.200 0.000 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 Hàm lượng GABA (mg/ml) Phụ lụ r nh tự gen 16S r N ủ hủng 41 CGCCAGTTTATATTCCCCTGATTTTACAATGAAGCGAGTGGCGAACTGG GAGTAACACGTGGGAAATCTGCCCAGTAAGCAGGGGATAACACTTGGAAA CAGGTGCTAATACCGTATAACAACAAAATCCGCATGGATTTTGTTTGAAAG GTGGCTTCGGCTATCACTTCTGGATGATCCCGCGGCGTATTAGTTAGTTGG TGAGGTAAAGGCCCACCAAGACGATGATACGTAGCCGACCTGAGAGGGTA ATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGC AGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAATGCCGCGT GAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACAC CTTTGAGAGTAACTGTTCAAGGGTTGACGGTATTTAACCAGAAAGCCACGG CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCC GGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGA AAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGGAGACTTGAGT GCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATAT ATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTAGTCTGTAACTGACGCT GAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCA TGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCT 67 GCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGA AACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT AATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGCCAAT CTTAGAGATAAGACGTTCCCTTCGGGGACAGAAATGACAGGTGGTGCATG GATTGTCGTCAGCTCGTGTCGGTGAGATGGTTGGG Phụ lụ 4: r nh tự gen 16S r N ủ hủng 71 ATTAACATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAA CACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAG ATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAA GATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAG ATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGA GAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTAC GGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATG GAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTG TTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGG TATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTA ATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAG CGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGA AGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGT GGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACA CCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCG AAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCG TAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTG CAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCCGCAAGGC TGAAACTCAAAGGAATTGACGGGGGCCCCGCACAAGCGGTGGAGCA TGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTTCTTGAC ATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGGACATGG 68 ATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGG GTTAAGTCCCGCAACGA Phụ lụ H nh ạng khuẩn lạ ủ m t số hủng vi khuẩn l ti DC2.1 DC2.2 DC2.4 DC2.3 DCI3.2 DCI3.1 PM11 NG9.2 69 DCI3.3 KC4.4 KC4.6 KC4.9 KC4.11 ST6.1 ST6.2 SB7.1 70 SB7.2 SB7.3 SCND8.2 SCND8.1 NG9.1 LTM1 LTM1.3 NĐVN 71 Phụ lụ Nhu m Gr m m t số ạng t đặ trưng ủ vi khuẩn l ti LTM1.1 LTM1.2 LTM1.3 DCI3.1 KC4.7 SCND8.3 SCND8.2 SB7.2 72