Extraction and characterization of fish protein isolate from yellowfin (thunnus albacares) dark muscle using ph shift method

MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY LAURINE MULE MUENI 60CH300 EXTRACTION AND CHARACTERIZATION OF FISH PROTEIN ISOLATE FROM YELLOWFIN (Thunnus albacares) DARK MUSCLE USING pH-SHIFT METHOD MASTER THESIS KHANH HOA – 2020 MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY LAURINE MULE MUENI 60CH300 EXTRACTION AND CHARACTERIZATION OF FISH PROTEIN ISOLATE FROM YELLOWFIN (Thunnus albacares) DARK MUSCLE USING pH-SHIFT METHOD MASTER THESIS FOOD TECHNOLOGY Major: Topic allocation Decision Decision on establishing the Committee: Defense date: Supervisors: Dr Nguyen Trong Bach (Principal supervisor) Dr Bui Tran Nu Thanh Viet (Co-Supervisor) Chairman: Faculty of Graduate Studies: KHANH HOA - 2020 UNDERTAKING I undertake that the thesis entitled: “Extraction and characterization of fish protein isolate from Yellowfin (Thunnus albacares) dark muscle using pH-shift method” is my own work The work has not been presented elsewhere for assessment until the time this thesis is submitted 26 September 2020 Laurine Mule Mueni iii FUNDING This research was funded by National Foundation for Science and Technology Development - Ministry of Science and Technology of Vietnam in the Nafosted project with No 106.99-2018.42 iv ACKNOWLEDGMENT First and foremost, I wish to thank God Almighty for His grace and strength and how He has been with me throughout my study period in Vietnam I would like to express the deepest appreciation to the Faculty of Food Technology, Nha Trang University in cooperation with the VLIR-UOS program for helping and giving me the best conditions to finish my thesis In addition, I would like to thank the National Foundation for Science and Technology Development - Ministry of Science and Technology of Vietnam in the Nafosted project with No 106.99-2018.42 for financial support to the research My special and deep appreciations go to my supervisors Dr Nguyen Trong Bach and Dr Bui Tran Nu Thanh Viet for their continuous support of my master‟s studies and research, for their patience, motivation, enthusiasm, and immense knowledge Their guidance helped me in all the time of research and writing of this thesis Last but not the least; I would like to thank my family: my guardian, my friends, classmates and colleagues for supporting me spiritually, mentally and physically throughout writing this thesis 26 September 2020, Nha Trang Laurine Mule Mueni v TABLE OF CONTENTS UNDERTAKING iii FUNDING iv ACKNOWLEDGMENT v TABLE OF CONTENTS .vi LIST OF ABBREVIATIONS ix LIST OF TABLES x LIST OF FIGURES xi ABSTRACT xiii GENERAL INTRODUCTION .1 PROBLEM STATEMENT CHAPTER : BACKGROUND 1.1 Tuna fish and its by-products 1.1.1 Tuna productions 1.1.2 The composition of yellowfin dark muscle 1.1.2.1 Myofibrillar proteins 1.1.2.2 Sarcoplasmic proteins 1.1.2.3 Lipids 1.1.3 Tuna by-product 10 1.2 Factors affecting the utilization of TDM 11 1.2.1 The dark colour and Myoglobin oxidation 11 1.2.2 Lipid and protein oxidation 13 1.2.3 Histamine 15 1.3 Protein extraction techniques used and FPI utilization 16 1.3.1 pH –shift method 17 vi 1.3.2 Protein recovery 19 1.3.3 By-products and FPI utilization 20 1.3.4 Fish protein isolate characterization .21 CHAPTER : MATERIALS AND METHODS 24 2.1 Materials collection and preparation .24 2.1.1 Dark muscle 24 2.1.2 Chemicals .24 2.1.3 FPI extraction by pH-shift method .24 2.2 Methods of characterization 26 2.2.1 Fourier transform infrared (FTIR) spectroscopic analysis 27 2.2.2 Zeta potential 28 2.2.3 Light scattering .29 2.2.4 Turbidity measurements .31 2.2.5 Determination of the protein concentration with UV-Visible spectroscopy 31 2.2.6 Confocal laser scanning microscopy 32 2.2.7 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS- page) .33 2.2.8 Viscosity measurement 36 2.2.9 Data analysis 37 CHAPTER 3: RESULTS AND DISCUSSIONS 38 3.1 FPI extraction 38 3.2 Characterization of TDMPI .39 3.2.1 Amino acids 39 3.2.2 Fourier-Transform Infrared Spectroscopy 41 3.2.3 Size-Exclusion Chromatography Analysis of TDMPI 43 3.3 Structure and physical properties of TDMPI solution 44 vii 3.3.1 Protein solubility 44 3.3.2 Light scattering .46 3.3.3 Micro-structure .47 3.3.4 SDS-PAGE 50 3.3.5 Zeta Potential of TDMPI solution 51 3.3.6 Viscosity of TDMPI solution .54 CONCLUSION AND OUTLOOK .57 REFERENCES 59 APPENDICES I Appendix 1: Test report of Amino acid in TDM II Appendix 2: Test report of Amino acid in TDM III Appendix 3: Test report of Amino acid in TDMPI IV Appendix 4: Test report of Amino acid in TDMPI V Appendix 5: Determination of histamine in TDM VI Appendix 6: Determination of histamine in TDMPI VII Appendix 7: Effect of pH (using NaOH) VIII viii LIST OF ABBREVIATIONS AA : Amino acid APS : Ammonium persulfate Cp : Protein concentration DI : Deionised water DLS : Dynamic Light Scattering EAA : Essential amino acid FPI : Fish protein isolate HMW : High molecular weight LMW : Low molecular weight LOD : Limit of detection LS : Light scattering MHC : Myosin heavy chain MLC : Myosin light chain MW : Molar mass NEAA : Non-essential amino acid pI : Isoelectric point Rg : Radius of gyration Rh : Hydrodynamic radius RI : Refractive index RPM :Revolutions per minute SLS : Static Light Scattering TAA : Total amino acid TEMED : Tetramethylethylenediamine TDM : Tuna dark muscle TDMPI : Tuna dark muscle protein isolate TWM : Tuna white muscle UV : Ultraviolet absorbance ix LIST OF TABLES Table 1.1: Proximate composition of the muscle tissue of yellowfin tuna (%, wet basis) .9 Table 1.2: Protein recovery yield from processing trout by-products by the isoelectric solubilisation/precipitation technology 20 Table 2.1: Recipe for stocking gel and resolving gel 35 Table 3.1: Proximate composition of TDM and TDMPI 39 Table 3.2: Amino acid composition of TDMPI powder, and reference proteins (dry basis) .40 Table 3.3: Effect of salt on pI of TDMPI in different ionic strengths 53 x 0014-Z Lee, H J., Park, S H., Yoon, I S., Lee, G W., Kim, Y J., Kim, J S., & Heu, M S 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Appendices Content Page Test report of Amino acid in TDM II Test report of Amino acid in TDM III Test report of Amino acid in TDMPI IV Test report of Amino acid in TDMPI V Determination of histamine in TDM VI Determination of histamine in TDMPI VII Effect of pH ( NaOH) VIII I Appendix 1: Test report of Amino acid in TDM II Appendix 2: Test report of Amino acid in TDM III Appendix 3: Test report of Amino acid in TDMPI IV Appendix 4: Test report of Amino acid in TDMPI V Appendix 5: Determination of histamine in TDM VI Appendix 6: Determination of histamine in TDMPI VII Appendix 7: Effect of pH (using NaOH) pH Yield,% time time time Mean, % SD 6.52 5.83 7.19 6.52 0.55 10.83 9.95 10.54 10.44 0.37 10 24.85 25.37 26.82 25.68 0.83 11 39.82 41.56 42.82 41.40 1.23 12 57.24 56.15 57.81 57.07 0.69 12.6 51.78 51.20 53.29 52.09 0.88 VIII ...MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY LAURINE MULE MUENI 60CH300 EXTRACTION AND CHARACTERIZATION OF FISH PROTEIN ISOLATE FROM YELLOWFIN (Thunnus albacares) DARK MUSCLE USING pH-SHIFT. .. Faculty of Graduate Studies: KHANH HOA - 2020 UNDERTAKING I undertake that the thesis entitled: ? ?Extraction and characterization of fish protein isolate from Yellowfin (Thunnus albacares) dark muscle. .. extract protein isolates from yellowfin tuna dark muscle  To determine the charge of the extracted protein isolates  To determine the size and molecular weight of the extracted protein isolates