Phân lập, tuyển chọn chủng vi khuẩn lactic có khả năng sinh tổng hợp gamma aminobutyric axit từ thực phẩm lên men và ứng dụng trong lên men trên cám gạo

HỌC VIỆN NÔNG NGHIỆP VIỆT NAM KHOA CÔNG NGHỆ SINH HỌC KHÓA LUẬN TỐT NGHIỆP ĐỀ TÀI: "PHÂN LẬP, TUYỂN CHỌN CHỦNG VI KHUẨN LACTIC CÓ KHẢ NĂNG SINH TỔNG HỢP GAMMA AMINOBUTYRIC AXIT TỪ TH C PHẨM L N MEN VÀ ỨNG D NG TRONG L N MEN TR N C M G O" HÀ NỘI - 2022 HỌC VIỆN NÔNG NGHIỆP VIỆT NAM KHOA CÔNG NGHỆ SINH HỌC KHÓA LUẬN TỐT NGHIỆP "PHÂN LẬP, TUYỂN CHỌN CHỦNG VI KHUẨN LACTIC CÓ KHẢ NĂNG SINH TỔNG HỢP GAMMA AMINOBUTYRIC AXIT TỪ TH C PHẨM L N MEN VÀ ỨNG D NG TRONG L N MEN TR N C M G O" Sinh viên thực : Chu Hồ Lan Anh Kh : K63 MSV : 637103 Giảng viên hƣớng dẫn : TS Lê Th Minh Th nh : TS Ngu n Th nh Hảo HÀ NỘI - 2022 LỜI CAM ĐOAN T ế ế ủ T Hà Nội, ngày 18 tháng 05 năm 2022 Sinh viên thực Chu Hồ Lan Anh i LỜI CẢM ƠN ế ế TS L T ị M V –V T P T C ế G ọ ọ T h C ọ TS N Họ T N H V n Sinh học N sinh học T Họ N V N Họ T ủ M Tôi xin chân thành cảm ơn! Hà Nội, ngày 18 tháng 05 năm 2022 Sinh viên thực Chu Hồ Lan Anh ii M CL C LỜI CAM OAN i LỜI CẢM ƠN ii MỤC LỤC iii DANH MỤC ẢNG vi DANH MỤC HÌNH vii DANH MỤC Ồ THỊ vii DANH MỤC CÁC TỪ VIẾT TẮT viii T M TẮT KH A LU N ix Ph n 11 MỞ Đ U ặ 1 Mụ 1.3 Y Ph n TỔNG QUAN TÀI LIỆU Tổ G -Aminobutyric Acid 2.1.1 G G 212 C ủ GA A 213 C ổ GA A 2.2 Tổ ẩ L 2 Lị 222 -Aminobutyric Acid ặ 223 K ủ Tổ ẩ 10 231 G ị 232 G ị 233 M ủ ọ 10 ủ 11 GA A 13 Ph n VẬT LIỆU VÀ PHƢƠNG PH P TH NGHIỆM 15 31 V 15 311 15 iii 312 V 15 3.1.3 H ế ị 16 3.1.4 M 16 32 N 17 321 N 1: X ị ặ ọ ẩ 322 N ủ ủ 17 2: N ủ ủ ẩ GA A 323 N ế MSG 17 3: N ị ủ 324 N ủ ẩ 17 4: ủ ổ ẩ 325 N 5: GA A ọ 18 ị ủ 33 P ẩ ọ 18 18 331 P ẩ 332 333 X ẩ ủ ị ẩ 18 19 ặ ủ ủ ẩ lactic 19 334 P ẩ ị GABA 21 335 P 21 336 P 337 X ị ị GA A ị 22 ị ủ ủ ẩ lactic 24 338 P ị ẩ 339 P Ph n 41 P 42 K 25 26 K T QUẢ VÀ THẢO LUẬN 27 ị ặ MSG ọ ủ GA A ủ iv ủ ẩ ủ 27 31 43 K ị ủ ủ ẩ ọ 34 44 Ả ủ ế GA A 36 4.4 H GA A 45oC 36 442 H GA A 37oC 37 4.5 ị Ph n ủ ọ 16S RNA 39 K T LUẬN VÀ KI N NGH 43 Kế 43 52 Kế ị 43 TÀI LIỆU THAM KHẢO 44 PHỤ LỤC 50 v DANH M C BẢNG 1: ặ ọ 2: K ổ 3: K ủ ủ ẩ GA A ủ ế ị vi 27 ủ ẩ ủ DCI3 31 KC 35 DANH M C HÌNH H H 1: C ủ GA A 2: K G GA A G ate decarboxylase H 1: H 1: H KC4 (A- H H 2: P H 3: Kế ẩ GA A 24 ẩ ế ẩ ; - Tế ủ ủ G ) 30 ặ ủ GA A ẩ DCI3 TLC 33 ẩ PCR 16S rRNA 40 H 4: C ủ ủ DCI3 Neighbor - Joining 41 H 5: C ủ ủ KC4 Neighbor - Joining 42 DANH M C ĐỒ TH ị 1: H GA A 45oC 36 ị 2: H GA A 370C 37 vii DANH M C C C TỪ VI T TẮT Ch vi t tắt BLAST Ti ng Anh Ti ng Việt Basic Local Alignment Search C ụ ế Tool DNA Deoxyribonucleic acid NCBI National center for T biotechnology information C T ọ Q PCR Polymerase Chain Reaction H K P Polymerase MRS De Man, Rogosa and Sharpe rRNA Ribosomal RNA TLC Thin layer chromatography MSG M M MRS S ( ) PLP P 5‟- photphat GABA Gamma – Aminobutyric acid GAD Enzyme Glutamate dercarbonxylase LAB Lactic acid Bacteria FA Ferulic acid Tris – acid acetic V TAE EDTA viii ẩ +H GA A 14 ủ L KC4 24 12 mg/100g) S 48 (1 78 ế GA A 1,18 mg/100g, ổ ẩ S GA A ế ị42 48 ủ 45oC 36 ụ 4.4.1 37oC KC4 T 72 ặ ủ GA A ủ ủ KC4 + H GA A ủ 24 48 32 (2 32 L ủ 37 100g) T GA A ụ DCI3 24 KC4 T GA A 45oC ủ 37oC 24 ế DCI3 T 24 72 ặ ủ GA A K t luận: C ủ DCI3 GA A 37oC C ủ ủ KC4 KC4 24 ổ 24 GA A ế GA A C ủ S 72 ị 45OC KC4 ế 37oC GA A N ế GA A ị ủ ặ z f 38 ủ GA A 72 ị GA A S ặ ủ z ẩ ẩ (Cui et al., 2020) ủ N N H GA A ủ ổ 48 (2 ) 736 15 Lactobacillus fermentum L K (2 9) 12 ổ MSG GA A S 61 87 ế L ế GA A ế H6 L sakei 2-16 ế n m men MSG ế ụ t i ế ụ ổ ụ GA A ẩ Đ nh d nh chủng tu ển ch n dựa trình tự gen K KC4 T ế 16S RNA củ 02 ặ 16S RNA (27F 15 (H 1492R) 3) 39 ủ S rRNA ọ DCI3 M1kb 10,000 3,000 1,500 1,000 1,500 Hình 3: K t điện di gel g rose sản phẩm PCR đo n gen 16S rRNA i ng b thang ph m hu ch đ i v ng đ i v ng NA chu n Thermo scientific, r NA c a ch ng gi ng n n ph m khu ch r NA c a ch ng C 16S RNA ẽ G ụ N ủ gi ng LAST –J NC I (H ụ ụ 40 4) T 16S ARN ủ Limosilactobacillus fermentum CIP 102980T (NR104927) Limosilactobacillus fermentum NBRC 15885T (NR113335) DCI3.5 Limosilactobacillus equigenerosi NRIC 0697T (NR041566) Limosilactobacillus reuteri NBRC 15892T (NR113820) Limosilactobacillus vaginalis ATCC 49540T (NR041796) Furfurilactobacillus rossiae DSM 15814T (NR112758) Levilactobacillus senmaizukei DSM 21775T (NR114251) Lentilactobacillus farraginis NRIC 0676T (NR041467) Bacillus subtilis IAM 12118 Hình : Câ ph t sinh lo i củ chủng DCI dự theo phƣơng ph p Neighbor - Joining Kế 16S RNA ủ ủ ủ NC I Limosilactobacillus fermentum CIP 102980 ủ ẩ Limosilactobacillus fermentum Kế ủ DCI3 99 02% DCI3 ế ủ 16S ARN ủ 99 ủ KC4 NC I 99 21 Lactiplantibacillus plantarum JCM1149 NRRL -14768T CIP 3151T ủ KC4 Lb pentosus 124 -2 Kế ế Lb plantarum 41 ủ 74 57 Lactiplantibacillus plantarum NBRC 15891T (NR113338) Lactiplantibacillus paraplantarum DSM 10667T (NR025447) Lactiplantibacillus plantarum CIP 103151T (NR104573) Lactiplantibacillus plantarum NRRL B-14768T (NR042394) Lactiplantibacillus pentosus 124-2T (NR029133) 51 40 KC4.5 Lactiplantibacillus plantarum JCM 1149T (NR117813) Hình 5: Câ ph t sinh lo i củ chủng KC4.5 dự theo phƣơng ph p Neighbor - Joining 42 Ph n K T LUẬN VÀ KI N NGH K t luận  25 ủ ẩ ị L H N ị ị ẩ T Q G MRS  S ọ ổ ủ 21/25 GA A T ủ ẩ ủ ế 84 ) DCI3 KC4 GA A ( ủ ổ 2,407 mg/mL) (2,474 mg/mL MRS ổ sung 2% MSG (w/v)  ị DCI3 ( ế ủ 57 14) KC4 47 ( ủ ủ ị ế )  C ủng KC4.5 ủ 45oC DCI3 ủ ẩ DCI3 48 (3 965 L) T GA A 37oC (3,197mg/mL) C ủ  C ủ DCI3.5 KC4 ị ị Limosilactobacillus fermentum Lactiplantibacillus plantarum Ki n ngh T ế ụ th c hi GA A chủng DCI3 KC4 43 ổ TÀI LIỆU THAM KHẢO T i liệu nƣớc Ki u H u Ảnh (2010) G 132 Nguy L D họ NX G H S ng L c Phẩm (1996), Vi sinh v t b o qu n, chế biến th c phẩm, NX G ục, pp 14, pp 41-44, pp.140-144, pp 185-288 Trị H N N N Q ến, Ph V ục, pp 176-189, pp 224-230 NX G ục, pp 116- Ty (1997) G t 11 P p vi khuẩn Lactobacillus sp t ẩ nh Vibrio sp Lu t nghi p cao họ sinh họ i học C T C T H N T ị N Thanh Sang (2021) Kh o b ng Lactobacillus T T ịN ọ T T t học th c phẩ C C V K p H T Q T N N V u ki n thu nh n acid gamma-aminobutyric t dịch K Họ C V N 63(1):42 N Tọ N ẩ ủ ẩ (Oreochromis niloticus) ọ T ọ C T ặ T ịH A (2 16) X ẩ (Streptococcus agalactiae) ế K T ủ : 42:48-55 ị T i liệu nƣớc ngo i Abdel-Aal, E S M., & Hucl, P (1999) A rapid method for quantifying total anthocyanins in blue aleurone and purple pericarp wheats Cereal Chemistry, 76, 350– 354 Amarasinghe, B M W P K., and Gangodavilage, N C (2004) Rice bran oil extraction in Sri Lanka: Data for process equipment design Food Bio-prod Process 82(C1): 54-59 Ardiansyah, Shirakawa, H., Koseki, T., Ohinata, K., Hazhizume, K., & Komai, M (2006) Rice bran fractions improve blood pressure, lipid profile, and glucose metabolism in stroke-prone spontaneously hypertensive rats Journal of Agricultural and Food Chemistry, 54, 1914-1920 Barone, E., Calabrese, V., & Mancuso, C (2009) Ferulic acid and its therapeutic potential as a hormetin for age-related diseases Biogerontology, 10(2), 97–108 https://doi.org/10.1007/s10522-008-9160-8 Boonstra, E., de Kleijn, R., Colzato, L.S., Alkemade, A., Forstmann, B.U, Nieuwenhuis, S., (2015) Neurotransmitters as food supplements: the effects of GABA on brain and behavior Front Psychol 6: 1520 doi:10.3389/fpsyg.2015.01520 PMC 4594160 PMID 26500584 Cellot G., Cherubini, E., (2014) GABAergic signaling as therapeutic target for autism spectrum disorders Front Pediatr 2:70 https://doi.org/10.3389/fped.2014.00070 44 Choi, S I., Lee, J W., Park, S M., Lee, M Y., Ge, J I., Park, M S., & Heo, T R (2006) Improvement of gamma-aminobutyric acid (GABA) production using cell entrapment of Lactobacillus brevis GABA 057 Journal of Microbiology and Biotechnology, 16, 562-568 http://dx.doi.org/10.1007/s10068-011-0057-y D Diana, M., Quilez, J and Rafecas, M.(2014) Gamma-aminobutyric acid as a bioactive compound in foods: a review J Funct Foods 10:407–420 10 Gatesoupe, F J., 1994 Lactic acid bacteria increase the resistance of turbot larvae, Scophthalmus maximus, against pathogenic Vibrio Aquatic Living Resourses 7: 277-282 11 Godber, J S., & Well, J H (1994) Rice bran: as a viable source of high value chemicals Louisiana Agric Goenka, O P (1987) Nutritional significance of RBO In: Handbook on Rice Bran Processing and Utilization of Products Solvent Extractors Association of India, India Grist, D H (1985) Rice (5th ed.) 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m g o Phụ lục 2: Trình tự gen S rARN củ chủng DCI TTTCGACAGCATGCGTACTGCGATGCTTGCACTGATTGATTTTGGT CGCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCC CAGAAGCGGGGGACAACATTTGGAAACAGATGCTAATACCGCATA ACAACGTTGTTCGCATGAACAACGCTTAAAAGATGGCTTCTCGCTA TCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGGTAA CGGCCTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATC GGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCA GCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAAC ACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTA AAGAAGAACACGTATGAGAGTAACTGTTCATACGTTGACGGTATT TAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAAT ACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGAG TGCAGGCGGTTTTCTAAGTCTGATGTGAAAGCCTTCGGCTTAACCG GAGAAGTGCATCGGAAACTGGATAACTTGAGTGCAGAAGAGGGTA 50 GTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGA ACACCAGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAG ACTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTC CATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCGT GAGTGCCGGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACG ACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAG CGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTAC CAGGTCTTGACATCTTGCGCCCAACCCTAGAGATAGGGGCGTTTCC CTTTCGGGAACGCAATGACAGGGTGGGTGCATGGTCGTCGTCA Phụ lục Trình tự gen S rRNA củ chủng KC TACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACC TGCCCTTTGCGGGGGATAACACCTGGAAACAGATGCTAATACCGC ATAACAACTTGGACCGCTGGGTCCGAGTTTGAAAGATGGCTTCGG CTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGG TAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTA ATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAG GCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGC AACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTT GTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGG TATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGG TAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAG CGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCA ACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGA GGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATG GAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACG CTGAGGCTCGAAAGTATGGGTAGCAAACAGATTAGATACCCTGGT AGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCG CCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGTGT ACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCAC 51 AAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCT TACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCC CTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGT GTCGTGAGA T GTT GGGTTAAGTCCCGCAACGAG 52