Nghiên cứu sản xuất chitosan oligosaccharides bằng cellulase quy mô 50 lít

BAN QUẢN LÝ KHU NÔNG NGHIỆP CÔNG NGHỆ CAO TP.HCM TRUNG TÂM NGHIÊN CỨU VÀ PHÁT TRIỂN NÔNG NGHIỆP CÔNG NGHỆ CAO BÁO CÁO NGHIỆM THU TÊN TÀI: NGHIÊN CỨU SẢN XUẤT CHITOSAN OLIGOSACCHARIDES BẰNG CELLULASE QUY MƠ 50 LÍT CHỦ NHIỆM TÀI KS NGUYỄN HOÀNG THẢO LY THÀNH PHỐ HỒ CHÍ MINH THÁNG 12/2015 i TĨM TẮT ề : N ế M ố COS C cellula ế ố ế ế o C), pH (5, 5.5 ế Kế o ế C, pH ố S ế ố ề ữ ề ố ố COS Kế : o C : : H: COS kDa, DD>80% T ề COS ổ ii ề < ổ FTIR ABSTRACT P j S w w f January to December of 2015 The purpose of this research was to determine appropriate conditions for hydrolysis chitosan using cho cellulase to make chitosan oligosaccharide (COS) We carried out affective factors to hydrolysis processing such as dilution ratio of enzyme (50, 75, 100, 125 times), temperature (50, 55, 60, 65oC), pH (5, 5.5, 6, 6.5) and time (from 0.5h to 6h) with laboratory scale Results show that 100 times dilution enzyme, 60°C, pH and hydrolysis time under 5.5 hours were approciate in labotory scale Response surface methodology – RSM was applied with parameters to determine optimum conditions for isolative processing COS at 50 liters scale Experimental results show that COS with molecular weight 80% were isolated at 75 times enzyme dilution, pH 5.5, 60oC, h hydrolysis time FTIR spectrum of COS shows that its structure did not change to compare to chitosan iii MỤC LỤC Trang TÓM TẮT ii ABSTRACT iii MỤC LỤC…………………………………………………………………………… iv DANH S CH C C CHỮ VIẾT TẮT vi DANH SÁCH BẢNG vii DANH SÁCH HÌNH viii THÔNG TIN Ề TÀI MỞ ẦU C : TỔNG QUAN TÀI LIỆU 1.1 Tổng quan chitosan, chitooligosaccharide 1.1.1 Chitosan 1.1.1.1 L 1.1.1.2 C u trúc chitosan 1.1.1.3 deacetyl hóa c a chitosan c sử nghiên c u chitosan 1.1.2 Chitooligosaccharide 1.1.2.1 Gi i thi u chitooligosaccharide 1.1.2.2 C 1.1.2.3 Th y phân chitosan thành chitooligosaccharide b ng enzyme cellulase 10 n chitooligosaccharide t chitosan 1.2 M t số ng d ng c a chitosan, chitooligosaccharide 12 1.3 Những nghiên c C 2.1 Th c 16 : N I DUNG NGHI N CỨU 20 m nghiên c u 20 2.2 V t li u nghiên c u 20 ố ng nghiên c u 20 iv 2.2.3 Thiết b thí nghi m 21 2.3 N i dung nghiên c u 21 2.3.1 N i dung 1: Nghiên c T n COS 21 : K o sát ng c a ho cellulase thích h p cho th y phân chitosan 21 2.3.1.2 T :K ều ki n nhi 2.3.1.3 T :K ều ki n pH thích h p cho th y phân chitosan 23 2.3.1.4 T : K o sát th i gian thích h p cho th y phân chitosan 24 C thích h p cho th y phân chitosan 22 : KẾT QUẢ VÀ THẢO LUẬN 30 3.1.1 Kh o sát ng c a ho cellulase thích h p cho th y phân chitosan 30 3.1.2 Kh ều ki n nhi 3.1.3 Kh ều ki n pH cho th y phân chitosan 32 3.1.4 Kh o sát th i gian tố cho th y phân chitosan 31 y phân chitosan c a cellulose 33 3.2 Tố thu nh n COS 35 3.3 n ph m COS 38 3.3.1 X 3.3.2 X deacetyl hóa b nh khố 38 ng phân tử c a COS b ng sắc ký gel GPC (Gel Permeation chromatography) 40 C : KẾT LUẬN VÀ Ề NGHỊ 51 4.1 Kết lu n 51 4.2 Kiến ngh 51 TÀI LIỆU THAM KHẢO 52 PHỤ LỤC 56 X LÝ SỐ LIỆU 72 v DANH SÁCH CÁC CH VI T TẮT cs COS FTIR GPC UV - VIS DD MW NMR VI T TẮT THU T NG C Chitooligosacchride Fourrier Transformation InfraRed – Gel Permeation chromatography – ắ ý Ultraviolet–visible spectroscopy – quang VIS Degree of deacetylation – Molecular weight – ố Nuclear magnetic resonance – ổ vi ổ ổ FTIR ế UV - DANH SÁCH BẢNG S T ả H 1.1 u Trang Ying cs, 2010) T 2.1 s 21 phân chitosan 2.2 T ề 2.3 T ề 2.4 T 24 2.5 T 26 3.1 H 30 3.2 Ả ề 3.3 Ả ề 3.4 L 22 H 23 31 H ắ gian Ả 33 H 3.5 ế 35 COS H 3.6 3.7 Kế ố ế K ố ố ế COS 36 ANOVA 37 COS 38 3.8 3.9 32 COS vii 39 DANH SÁCH HÌNH S T 1.1 C 1.2 S 1.3 C 3.1 H 3.2 Ả 3.3 ả Trang C COS Jeon Kim (2000) ế 10 11 ắ khác ề Ả 30 32 H ắ 33 3.4 H 3.5 P ổ FTIR COS ữ 3.6 P ổ FTIR COS ữ 3.7 P ổ FTIR COS ữ 3.8 Ả 3.9 K COS ố Sclerotium sp 46 3.10 K COS ố Fusarium sp 47 3.11 Q 3.12 S 34 42 H 43 44 ế COS COS cellulase COS 45 ẻ 48 50 viii THÔNG TIN Tên ề TÀI :“N C ề C : KS N :T T M T N : K H P : 155.015.000 C : ẻ ề Q B N – 12/2015 S S L : ọ ế MỞ ẦU ế Chitin (poly Nù ế ỏ ỏ C ọ K ố ế ố ế ề converting enzyme angiotensin I ế ế ế … K 2005; Tsai cs H ế ố ế ố … ề ế R j C ọ ố ọ ỏ in vitro ; in vivo (Quin cs ề ế Ở ề ố T ố ố C chitooligosaccharides ố ề ế V ề COS ế ế ề ố 8.083/824009 uRIU Detector B Ch1 -1 -2 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 m in 12.5 15.0 17.5 m in CTS-9 7.724/896580 uRIU Detector B Ch1 -1 -2 0.0 2.5 5.0 7.5 10.0 CTS-10 61 7.902/921425 uRIU Detector B Ch1 7.5 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 m in 27.5 m in CTS-11 uRIU Detector B Ch1 7.866/791177 10.0 7.5 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 CTS-12 62 17.5 20.0 22.5 25.0 uRIU 7.772/735016 Detector B Ch1 7.5 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 m in CTS-13 uRIU 8.069/949220 Detector B Ch1 10.0 7.5 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 CTS-14 63 17.5 20.0 22.5 25.0 27.5 m in 15.0 uRIU Detector B Ch1 7.656/958233 12.5 10.0 7.5 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 m in CTS-15 17.5 uRIU Detector B Ch1 7.707/1052856 15.0 12.5 10.0 7.5 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 CTS-16 64 15.0 17.5 20.0 m in 7.5 8.615/922330 uRIU Detector B Ch1 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 m in CTS-17 8.535/908119 uRIU Detector B Ch1 -1 -2 0.0 2.5 5.0 7.5 10.0 CTS-18 65 12.5 15.0 17.5 20.0 m in 9.120/896483 uRIU Detector B Ch1 -1 -2 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 m in CTS-19 uRIU Detector B Ch1 20 8.363/1151776 15 10 0.0 2.5 5.0 7.5 10.0 12.5 15.0 CTS-20 66 17.5 20.0 22.5 25.0 m in 7.596/908187 uRIU Detector B Ch1 7.5 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 m in CTS-21 uRIU 15.0 Detector B Ch1 8.037/911947 12.5 10.0 7.5 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 CTS-22 67 15.0 17.5 20.0 m in 22.5 uRIU Detector B Ch1 20.0 6.694/1079448 17.5 15.0 12.5 10.0 7.5 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 m in CTS-23 uRIU Detector B Ch1 25 7.344/998318 20 15 10 0.0 2.5 5.0 7.5 10.0 12.5 15.0 CTS-24 68 17.5 20.0 22.5 25.0 m in 7.853/895202 uRIU 7.5 Detector B Ch1 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 m in CTS-25 6.656/860772 uRIU Detector B Ch1 7.5 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 CTS-26 69 15.0 17.5 20.0 m in 7.5 7.320/845428 uRIU Detector B Ch1 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 m in CTS-27 uRIU Detector B Ch1 7.964/682921 10.0 7.5 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 CTS-28 70 17.5 20.0 22.5 25.0 27.5 m in 30 uRIU Detector B Ch1 25 20 6.601/479268 15 10 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 m in 17.5 20.0 22.5 25.0 27.5 m in CTS-29 Detector B Ch1 10.0 7.333/974518 uRIU 7.5 5.0 2.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 CTS-30 71 X L S Nộ du 1: T í m 1: Summary Statistics for NONG DO GLUCOSAMIN MAU Count Average Standard deviation M 25 0.2323 0.00138564 M 50 0.2 0.00517397 M 75 0.179833 0.00315331 M 100 0.174867 0.00307463 M 125 0.0905333 0.00318957 Total 16 0.179056 0.0492188 MAU M 25 M 50 M 100 M 125 Total LIỆU Coeff of variation 0.596488% 2.58699% 1.75346% 1.75827% 3.52309% 27.4879% Minimum 0.2311 0.1956 0.1764 0.1728 0.087 0.087 Stnd kurtosis 1.17938 -0.00393505 ANOVA Table for NONG DO GLUCOSAMIN by MAU Source Sum of Squares Df Mean Square Between groups 0.0362189 0.00905472 Within groups 0.00011844 11 0.0000107673 Total (Corr.) 0.0363373 15 F-Ratio 840.95 P-Value 0.0000 Table of Means for NONG DO GLUCOSAMIN by MAU with 95.0 percent LSD intervals Stnd error MAU Count Mean (pooled s) Lower limit Upper limit M 25 0.2323 0.00164068 0.229747 0.234853 M 50 0.2 0.00189449 0.197052 0.202948 M 75 0.179833 0.00189449 0.176885 0.182782 M 100 0.174867 0.00189449 0.171918 0.177815 M 125 0.0905333 0.00189449 0.0875849 0.0934818 Total 16 0.179056 Multiple Range Tests for NONG DO GLUCOSAMIN by MAU Method: 95.0 percent LSD MAU Count Mean M 125 0.0905333 M 100 0.174867 M 75 0.179833 M 50 0.2 M 25 0.2323 Contrast M 100 - M 125 M 100 - M 25 M 100 - M 50 M 100 - M 75 M 125 - M 25 M 125 - M 50 Sig * * * * * Homogeneous Groups X X X X X Difference 0.0843333 -0.0574333 -0.0251333 -0.00496667 -0.141767 -0.109467 +/- Limits 0.00589692 0.00551606 0.00589692 0.00589692 0.00551606 0.00589692 72 Maximum 0.2343 0.2057 0.1826 0.1784 0.0932 0.2343 Range 0.0032 0.0101 0.0062 0.0056 0.0062 0.1473 Stnd skewness 1.25708 0.749024 -0.64266 1.17248 -0.80077 -1.51432 M 125 - M 75 * -0.0893 0.00589692 M 25 - M 50 * 0.0323 0.00551606 M 25 - M 75 * 0.0524667 0.00551606 M 50 - M 75 * 0.0201667 0.00589692 * denotes a statistically significant difference Summary Statistics for OD MAU Count Average M 25 0.097325 M 50 0.0821 M 75 0.0782 M 100 0.0726333 M 125 0.0374333 Total 16 0.075025 MAU M 25 M 50 M 75 M 100 M 125 Total Standard deviation 0.00629199 0.00786893 0.00523737 0.00590028 0.00118462 0.0212854 Coeff of variation 6.46493% 9.58456% 6.6974% 8.12338% 3.16462% 28.3711% Minimum 0.0879 0.0737 0.0736 0.0659 0.0367 0.0367 Stnd kurtosis 1.60976 -0.111633 ANOVA Table for OD by MAU Source Sum of Squares Df Mean Square F-Ratio Between groups 0.00642613 0.00160653 47.77 Within groups 0.000369901 11 0.0000336273 Total (Corr.) 0.00679603 15 Table of Means for OD by MAU with 95.0 percent LSD intervals Stnd error MAU Count Mean (pooled s) Lower limit Upper limit M 25 0.097325 0.00289945 0.0928125 0.101838 M 50 0.0821 0.003348 0.0768894 0.0873106 M 75 0.0782 0.003348 0.0729894 0.0834106 M 100 0.0726333 0.003348 0.0674227 0.0778439 M 125 0.0374333 0.003348 0.0322227 0.0426439 Total 16 0.075025 Multiple Range Tests for OD by MAU Method: 95.0 percent LSD MAU Count Mean M 125 0.0374333 M 100 0.0726333 M 75 0.0782 M 50 0.0821 M 25 0.097325 Contrast M 100 - M 125 M 100 - M 25 M 100 - M 50 M 100 - M 75 M 125 - M 25 M 125 - M 50 Sig * * * * Homogeneous Groups X X X X X Difference 0.0352 -0.0246917 -0.00946667 -0.00556667 -0.0598917 -0.0446667 +/- Limits 0.0104212 0.00974816 0.0104212 0.0104212 0.00974816 0.0104212 73 P-Value 0.0000 Maximum 0.1007 0.0893 0.0839 0.0769 0.0388 0.1007 Range 0.0128 0.0156 0.0103 0.011 0.0021 0.064 Stnd skewness -1.6197 -0.473963 0.638828 -1.09776 1.21493 -1.35069 M 125 - M 75 * -0.0407667 0.0104212 M 25 - M 50 * 0.015225 0.00974816 M 25 - M 75 * 0.019125 0.00974816 M 50 - M 75 0.0039 0.0104212 * denotes a statistically significant difference Multiple Range Tests for OD by MAU Method: 95.0 percent LSD MAU Count Mean M 125 0.0374333 M 100 0.0726333 M 75 0.0782 M 50 0.0821 M 25 0.097325 Homogeneous Groups X X X X X Contrast Sig Difference +/- Limits M 100 - M 125 * 0.0352 0.0104212 M 100 - M 25 * -0.0246917 0.00974816 M 100 - M 50 -0.00946667 0.0104212 M 100 - M 75 -0.00556667 0.0104212 M 125 - M 25 * -0.0598917 0.00974816 M 125 - M 50 * -0.0446667 0.0104212 M 125 - M 75 * -0.0407667 0.0104212 M 25 - M 50 * 0.015225 0.00974816 M 25 - M 75 * 0.019125 0.00974816 M 50 - M 75 0.0039 0.0104212 * denotes a statistically significant difference HO T ENZYME Summary Statistics for HOAT DO ENZYME MAU Count Average Standard deviation M 25 0.0722 0.00052915 M 50 0.124233 0.00323161 M 75 0.167533 0.00295691 M 100 0.217233 0.00379781 M 125 0.140567 0.00493694 Total 15 0.144353 0.0496694 MAU M 100 M 125 M 25 M 50 M 75 Total Coeff of variation 0.732895% 2.60125% 1.76497% 1.74826% 3.51217% 34.4082% Minimum 0.0718 0.1215 0.1643 0.2147 0.1351 0.0718 Stnd kurtosis -0.576538 ANOVA Table for HOAT DO ENZYME by MAU Source Sum of Squares Df Mean Square Between groups 0.0344222 0.00860555 Within groups 0.000116527 10 0.0000116527 Total (Corr.) 0.0345387 14 F-Ratio 738.50 P-Value 0.0000 Table of Means for HOAT DO ENZYME by MAU with 95.0 percent LSD intervals 74 Maximum 0.0728 0.1278 0.1701 0.2216 0.1447 0.2216 Range 0.001 0.0063 0.0058 0.0069 0.0096 0.1498 Stnd skewness 1.03086 0.765972 -0.680943 1.1781 -0.796685 0.056071 MAU M 25 M 50 M 75 M 100 M 125 Total Count 3 3 15 Mean 0.0722 0.124233 0.167533 0.217233 0.140567 0.144353 Stnd error (pooled s) 0.00197084 0.00197084 0.00197084 0.00197084 0.00197084 Lower limit 0.0690949 0.121128 0.164428 0.214128 0.137462 Multiple Range Tests for HOAT DO ENZYME by MAU Method: 95.0 percent LSD MAU Count Mean M 25 0.0722 M 50 0.124233 M 125 0.140567 M 75 0.167533 M 100 0.217233 Homogeneous Groups X X X X X Contrast Sig Difference +/- Limits M 100 - M 125 * 0.0766667 0.00621027 M 100 - M 25 * 0.145033 0.00621027 M 100 - M 50 * 0.093 0.00621027 M 100 - M 75 * 0.0497 0.00621027 M 125 - M 25 * 0.0683667 0.00621027 M 125 - M 50 * 0.0163333 0.00621027 M 125 - M 75 * -0.0269667 0.00621027 M 25 - M 50 * -0.0520333 0.00621027 M 25 - M 75 * -0.0953333 0.00621027 M 50 - M 75 * -0.0433 0.00621027 * denotes a statistically significant difference 75 Upper limit 0.0753051 0.127338 0.170638 0.220338 0.143672 ... Tổng quan chitosan, chitooligosaccharide 1.1.1 Chitosan 1.1.1.1 L 1.1.1.2 C u trúc chitosan 1.1.1.3 deacetyl hóa c a chitosan c sử nghiên c u chitosan. .. pH T cứu: Chitosan, cellulase B trí t í Bả M -Ấ NT1 :P ế ; ề C t u t eo dõ : L 23 ; 6,0; 6,5 2.3.1.4 T í T m 4: K ảo sát t a t ực V Vật u N C P C C N T Hồ C C -Ấ Minh cứu: Chitosan, cellulase. .. DUNG NGHI N CỨU 20 m nghiên c u 20 2.2 V t li u nghiên c u 20 ố ng nghiên c u 20 iv 2.2.3 Thiết b thí nghi m 21 2.3 N i dung nghiên c u