Nghiên cứu công nghệ khai thác tổ hợp polyphenol từ lá chè xanh việt nam và ứng dụng trong sản xuất thực phẩm chức năng

BỘ GIÁO DỤC VÀ ðÀO TẠO TRƯỜNG ðẠI HỌC BÁCH KHOA HÀ NỘI VŨ HỒNG SƠN NGHIÊN CỨU CÔNG NGHỆ KHAI THÁC TỔ HỢP POLYPHENOL TỪ LÁ CHÈ XANH VIỆT NAM VÀ ỨNG DỤNG TRONG SẢN XUẤT THỰC PHẨM CHỨC NĂNG LUẬN ÁN TIẾN SĨ KỸ THUẬT Hà Nội 2011 BỘ GIÁO DỤC VÀ ðÀO TẠO TRƯỜNG ðẠI HỌC BÁCH KHOA HÀ NỘI VŨ HỒNG SƠN ðề tài NGHIÊN CỨU CÔNG NGHỆ KHAI THÁC TỔ HỢP POLYPHENOL TỪ LÁ CHÈ XANH VIỆT NAM VÀ ỨNG DỤNG TRONG SẢN XUẤT THỰC PHẨM CHỨC NĂNG Chuyên ngành: Chế biến thực phẩm ñồ uống Mã số: 62.54.02.01 LUẬN ÁN TIẾN SĨ KỸ THUẬT Người hướng dẫn khoa học: GS.TS Hà Duyên Tư PGS.TS Nguyễn Duy Thịnh Hà Nội 2011 i LỜI CAM ðOAN Tơi xin cam đoan luận án cơng trình riêng tơi Các số liệu cơng bố luận án trung thực, phần cơng bố tạp chí khoa học với đồng ý đồng tác giả, phần cịn lại chưa cơng bố cơng trình nghiên cứu Tác giả luận án VŨ HỒNG SƠN ii LỜI CẢM ƠN Trong thời gian thực cơng trình nghiên cứu này, tơi nhận nhiều giúp ñỡ hỗ trợ thầy cơ, đồng nghiệp, quan, bạn bè, em sinh viên người thân gia đình Trước hết tơi xin bày tỏ lịng biết ơn chân thành đến GS.TS Hà Duyên Tư, người ñã trực tiếp hướng dẫn, bảo, khích lệ tơi tận tình suốt q trình làm luận án Tơi xin bày tỏ cảm ơn chân thành tới PGS.TS Nguyễn Duy Thịnh ñã tận tình hướng dẫn giúp đỡ tơi hồn thành luận án Tơi xin chân thành cảm ơn GS.TS Hồng ðình Hịa giúp đỡ tơi nhiều suốt q trình làm luận án Tơi xin gửi lời cảm ơn chân thành tới PGS.TS Nguyễn Thị Minh Tú đồng nghiệp Bộ mơn Quản lý Chất lượng, thầy cô giáo Viện Công nghệ Sinh học Cơng nghệ Thực phẩm, Trường ðHBK Hà Nội tạo điều kiện giúp đỡ tơi q trình làm luận án Xin gửi lời cảm ơn chân thành ñến ñồng nghiệp thuộc ñợn vị sau: - Trung tâm Giáo dục Phát triển sắc ký, ðHBK Hà Nội - Khoa Vệ sinh thực phẩm-dinh dưỡng, Viện Vệ sinh phịng dịch qn đội - Viện Pháp y qn ñội, Cục Quân y - Tổ thử nghiệm sinh học, Viện Công nghệ sinh học, Viện Khoa học Công nghệ Việt Nam giúp tơi thực nghiên cứu Xin chân thành cảm ơn KS Nguyễn Tiến Huy, KS Hoàng Thanh Nhung sinh viên khóa 47, 48, 49, 50 ngành Cơng nghệ thực phẩm ñã giúp ñỡ hỗ trợ nhiều thời gian làm luận án Tôi xin gửi lời cảm ơn chân thành tới bạn bè, người thân gia đình hai bên nội ngoại, đặc biệt vợ tơi Phương Thu Huyền gái Vũ Phương Huyền Anh ñã ñộng viên hỗ trợ nhiều suốt trình làm luận án iii MỤC LỤC MỞ ðẦU CHƯƠNG TỔNG QUAN .5 1.1 SƠ LƯỢC VỀ CÂY CHÈ 1.1.1 Tên gọi nguồn gốc chè 1.1.2 Phân loại giống chè 1.1.3 Các vùng chè Việt Nam 1.1.4 Tình hình sản xuất chè giới Việt Nam 1.2 HỢP CHẤT POLYPHENOL THỰC VẬT .10 1.2.1 ðịnh nghĩa, phân loại 10 1.2.2 Hợp chất flavonoit .11 1.2.2.1 Phân loại hợp chất flavonoit 12 1.2.2.2 Sinh tổng hợp flavonoit 13 1.2.3 Hợp chất polyphenol chè 15 1.2.4 Hoạt tính sinh học polyphenol 18 1.2.4.1 Tác dụng chống oxy hóa polyphenol 18 1.2.4.2 Tác dụng ñối với enzym 21 1.2.4.3 Tác dụng kháng sinh, chống viêm nhiễm .22 1.2.4.4 Tác dụng ñối với ung thư .22 1.2.4.5 Tác dụng ñối với bệnh tim mạch 25 1.2.4.6 Tác dụng ñối với HIV/AIDS 26 1.3 ỨNG DỤNG HỢP CHẤT POLYPHENOL CHÈ XANH 26 1.3.1 Ứng dụng polyphenol công nghiệp thực phẩm 26 1.3.1.1 Vai trò polyphenol chè xanh ñược bổ sung thực phẩm .27 1.3.1.2 Sự chuyển hóa polyphenol hệ tiêu hóa .30 1.3.2 Ứng dụng polyphenol Y-Dược 33 1.4 CƠNG NGHỆ TRÍCH LY POLYPHENOL CHÈ XANH 34 1.4.1 Các yếu tố ảnh hưởng ñến trình trích ly polyphenol 34 1.4.2 Các nghiên cứu giới cơng nghệ trích ly polyphenol chè xanh 36 1.4.2.1 Các phương pháp trích ly 36 1.4.2.2 Các phương pháp trích ly đại 39 1.4.3 Các nghiên cứu nước cơng nghệ trích ly polyphenol chè xanh 40 CHƯƠNG ðỐI TƯỢNG VÀ PHƯƠNG PHÁP NGHIÊN CỨU 42 iv 2.1 ðỐI TƯỢNG NGHIÊN CỨU 42 2.2 PHƯƠNG PHÁP NGHIÊN CỨU 42 2.2.1 Quy trình trích ly polyphenol 42 2.2.2 Chuẩn bị dịch chiết chè .43 2.2.3 ðịnh lượng polyphenol tổng số - phương pháp Folin-Denis .44 2.2.4 ðịnh lượng flavonoit tổng số 44 2.2.5 Phân tích thành phần catechin HPLC 45 2.2.6 Xác ñịnh hàm lượng cafein 45 2.2.7 Xác định dư lượng hóa chất bảo vệ thực vật 46 2.2.8 Xác ñịnh vết kim loại ICP/MS 47 2.2.9 Xác ñịnh vết dung môi SPME/GC 48 2.2.10 Hoạt tính chống oxy hóa – khả quét gốc tự DPPH 49 2.2.11 Hoạt tính chống oxy hóa - khả kìm hãm q trình peroxy hóa lipit màng tế bào gan chuột theo phương pháp Blagodrov C.G .50 2.2.12 Hoạt tính chống oxy hóa - khả kìm hãm peroxydaza máu người theo phương pháp Xavron E.C .52 2.2.13 Nghiên cứu tác dụng ức chế phát triển tế bào ung thư in vitro 53 2.2.14 Phương pháp xác ñịnh ñộc tính cấp 57 2.2.15 Phương pháp xác ñịnh hoạt lực kháng khuẩn phương pháp khuếch tán thạch (Agar Diffusion) 58 2.2.16 Phân tích cảm quan 59 2.2.17 Quy hoạch thực nghiệm 60 2.2.18 Phân tích thống kê kết thí nghiệm 62 CHƯƠNG KẾT QUẢ VÀ THẢO LUẬN 63 3.1 XÁC ðỊNH ðẶC TÍNH CỦA NGUYÊN LIỆU .63 3.1.1 Hàm lượng polyphenol giống chè 63 3.1.2 Hoạt tính chống oxy hóa – khả quét gốc tự DPPH polyphenol .66 3.2 XÂY DỰNG QUY TRÌNH TRÍCH LY POLYPHENOL CHÈ XANH 69 3.2.1 Khảo sát yếu tố ảnh hưởng đến q trình chiết dịch chè 69 3.2.1.1 Ảnh hưởng dung mơi trích ly .70 3.2.1.2 Ảnh hưởng thời gian trích ly 71 3.2.1.3 Ảnh hưởng pH trích ly 72 3.2.1.4 Ảnh hưởng tỷ lệ dung môi/nguyên liệu 73 v 3.2.1.5 Ảnh hưởng nhiệt ñộ trích ly 74 3.2.2 Tối ưu hóa q trình chiết dịch chè 75 3.2.2.1 Thiết lập mơ hình 75 3.2.2.2 Tối ưu hóa 80 3.2.3 Tối ưu hóa q trình tách, tinh chế polyphenol 81 3.2.3.1 Khảo sát yếu tố ảnh hưởng đến q trình tách, tinh chế polyphenol 81 3.2.3.2 Thiết lập mơ hình 83 3.2.3.3 Tối ưu hóa 89 3.2.3.4 Sấy khô chế phẩm polyphenol 90 3.3 PHÂN TÍCH MỘT SỐ THÀNH PHẦN CỦA CHẾ PHẨM POLYPHENOL 93 3.3.1 Thành phần catechin 93 3.3.2 Hàm lượng cafein 95 3.3.3 Dư lượng HCBVTV 96 3.3.4 Vết dung môi hữu 97 3.3.5 Thành phần hàm lượng kim loại 97 3.4 XÁC ðỊNH HOẠT TÍNH SINH HỌC CỦA CHẾ PHẨM .98 3.4.1 Hoạt tính chống oxy hóa .98 3.4.1.1 Khả quét gốc tự DPPH 98 3.6.1.2 Khả kìm hãm q trình peroxy hóa lipit màng tế bào gan chuột99 3.4.1.3 Khả kìm hãm peroxydaza nhóm máu người .100 3.4.2 Khả kháng khuẩn 101 3.4.3 Tác dụng kìm hãm phát triển tế bào ung thư in vitro 103 3.4.4 ðộ độc tính cấp 105 3.4.4.1 ðộc tính cấp chế phẩm PP1 chuột thí nghiệm 105 3.4.4.2 ðộc tính cấp chế phẩm PP2 chuột thí nghiệm 106 3.4.4.3 Một số tiêu huyết học hóa sinh máu chuột cho uống PP1 PP2 108 3.5 ỨNG DỤNG CHẾ PHẨM POLYPHENOL CHÈ XANH TRONG SẢN XUẤT THỰC PHẨM 110 3.5.1 Cơ sở khoa học cho việc bổ sung polyphenol vào thực phẩm 111 3.5.1.1 Sự dung nạp polyphenol chè xanh .111 3.5.1.2 Ảnh hưởng yếu tố cơng nghệ đến chuyển hóa catechin 112 3.5.2 Ứng dụng polyphenol sản xuất số sản phẩm thực phẩm 113 vi 3.5.2.1 Quy trình sản xuất kẹo dẻo trà xanh 113 3.5.2.2 Quy trình sản xuất bánh cookie trà xanh 117 3.5.3 ðánh giá chất lượng sản phẩm thực phẩm có bổ sung polyphenol 120 3.5.3.1 Khả kìm hãm peroxydaza máu người dịch chiết polyphenol từ sản phẩm 120 3.5.3.2 Khả kìm hãm q trình peroxy hóa màng tế bào gan chuột 125 KẾT LUẬN .127 DANH MỤC CÁC CƠNG TRÌNH ðà CƠNG BỐ CỦA LUẬN ÁN 129 TÀI LIỆU THAM KHẢO .130 vii DANH MỤC CÁC KÝ HIỆU VÀ CHỮ VIẾT TẮT AChE ADN ARN ATCC Acetylcholinesterase Deoxyribonucleic acid (axit deoxyribonucleic) Ribonucleic acid (axit ribonucleic) American type culture collection (bộ sưu tập giống chuẩn Mỹ) BHA BHT Butylhydrxyanizol Butylhydroxytoluen BuChE CCK Butylcholinesterase Cholecystokinin CFU CHI Colony forming unit (số khuẩn lạc) Chalcone isomerase (enzym đồng phân hóa chalcon) CHS CoA Chalcone synthase (enzym tổng hợp chalcon) Coenzyme A COMT Catechol-O-methyltransferase DAD Diode array detector (detector ñiốt phổ rộng) DFR Dihydroflavonol-4-reductase (enzym khử dihydroflavonol) DiMeEGCG Dimethylepigallocatechin gallate DMEM DMSO Dubelco’s modified eagle medium Dimethylsulfoxide DPPH EC ECD ECG EGC EGCG 1,1-diphenyl-2-picrylhydrazyl epicatechin Electron capture detector (detector cộng kết ñiện tử) Epicatechin gallate Epigallocatechin Epigallocatechin gallate F3H FBS FDR FID Flavanone-3-hydroxylase (enzym hydroxyl hóa flavanon) Fetal bovine serum Flavan 3,4-cis diol-reductase (enzym khử flavandiol) Flame ionization detector (detector ion hóa lửa) GC GC/MS GCG Gallocatechin Sắc ký khí/khối phổ Gallocatechin gallate 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