Development and quality evaluation of bioactive rich drink powder based on sea buckthorn by Ke Dong BSc (Food Technology), MSc (Public Health) Supervisors Prof Vijay Jayasena Dr Rosalie Durham Dr Binosha Fernando Dr Regine Stockmann A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Science and Health (Nutrition and Food Science) Western Sydney University November 2019 Statement of Authentication The work presented in this thesis is, to the best of my knowledge and belief, original except as acknowledged in the text. I hereby declare that I have not submitted this material, either in full or in part, for a degree at this or any other institution. Signature: ……………………… Date: 16 November 2019 i Acknowledgements I would like to express profound appreciation and gratitude to my principle supervisor Professor Vijay Jayasena for his invaluable support, encouragement, supervision and constructive suggestions carrying me throughout the completion of this project. Also, I am very thankful to Dr Binosha Fernando at Edith Cowan University where I did significant part of my research using cell culture techniques under her supervision. Her professionalism and patience supported me for timely completion of the research. I would like to thank Dr Rosalie Durham at Western Sydney University and Dr Regine Stockmann at Commonwealth Scientific and Industrial Research Organisation (CSIRO) for their valuable guidance and encouragement throughout my entire PhD study. I am also grateful for Dr Pamoda Jayatunga, Dr Florence Lim, Dr Eugene Hone, Dr Prashant Bharadwaj who assisted me for the lab work at Edith Cowan University. I also would like to thank Dr Chen Chen for his support and arrangement of some lab work that was carried out at Shanghai Institute of Technology. Particular gratitude should also be given to Richard Gong and Yolin Qiang who brought me to sea buckthorn industry and helped me in sample collection. I am as ever, especially indebted to my beloved mother for her love and support throughout my study. These thanks are also extended to all my family members and friends for their emotional support. ii Abstract Sea buckthorn berries have been used as an ancient medicine for thousands of years. Exceptional nutritional value and health benefits of sea buckthorn have been disclosed by recent research. Sea buckthorn berries are high in health-enhancing bioactive compounds. The major health benefits of sea buckthorn include anti-oxidation activities, anti-cancer properties, reduction of cardiovascular disease and improvement of immune system. Evidence suggests that diets rich in antioxidants reduce the risk of Alzheimer’s disease (AD) through direct effects on amyloid beta (Aβ, a hallmark of AD) and consequently influence AD pathogenesis. Despite the links between sea buckthorn and known AD risk factors (altered glucose metabolism and elevated cholesterol levels), the relationship between sea buckthorn and Aβ production/ degradation has not been fully characterised. The aim of this study was to develop a bioactive rich functional drink powder incorporating sea buckthorn and address this knowledge gap by administering drink powder and sea buckthorn to an inexpensive in vitro cell system and assess their effects on Aβ levels. Sea buckthorn berries (H.rhamnoides) were collected from 4 different locations (Jianping, Lueliang, Habahe and Datong) and sea buckthorn powder (SBP) was made through convective drying (at 80, 90, 100°C for 12 h respectively), freeze drying ( at – 60°C for 24, 30 and 36 h respectively) and spray drying (inlet air temperature at 180, 190 and 200°C respectively). Freeze drying resulted in a higher quality product compared to convective drying and spray drying as freeze dried SBP had higher nutritional value and higher antioxidant activity. For freeze dried SBP, the average total flavonoid, vitamin C, sugar, fat, vitamin E contents were 641 mg/100g, 112 mg/100g, 32.0 g/100g, 5.8g /100g and 49 mg/100g respectively. The fatty acids composition of freeze dried SBP was 34.88%, 21.58%, 18.50%, 11.82% and 9.77% for palmitic, linoleic, palmitoleic, linolenic and oleic acids respectively. The freeze dried SBP also indicated the highest antioxidant activity which was 48% in terms of DPPH inhibition. Location also impacted the final quality of iii SBP. SBP from Habahe had the lowest nutritional value possibly due to low rainfall. SBP from Jianping and Datong demonstrated similar and better quality than that of Lueliang and Habahe in terms of total flavonoid, vitamin C, sugar, fat and vitamin E contents. However, SBP from Datong showed the best antioxidant activity (49% in terms of DPPH inhibition). Taking both drying method and location into account, SBP under freeze drying at – 60°C for 24 h from Datong (2500 m in altitude) indicated the best nutritional value with the highest antioxidant activity. Therefore, it was selected for drink powder formulation. Goji berry powder (GBP) and turmeric powder which have cognitive improvement abilities were also added into the formulation. Lupin flour, carob powder, malt extract and xanthan gum were introduced into the formulation to improve the nutritional value and sensory properties. Seventy-one formulations of drink powder were prepared and their total flavonoid, vitamin C, sugar, fat, protein, polysaccharide, total phenolic, carotenoid contents and antioxidant activity were analysed. The formulation 68 (45% SBP, 30% GBP, 10% lupin flour, 10% carob powder, 1% turmeric powder, 3.9 % malt extract and 0.1% xanthan gum) had the strongest antioxidant activity which was therefore selected for determination of effect on the degradation or clearance of amyloid beta via cell culture techniques. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H- tetrazolium) assay indicated that drink powder, SBP, GBP and SG (a mixture of SBP and GBP at a ratio of 45:30 by weight as per drink powder formulation 68) significantly increased cell viability in Aβ-induced cells up to 100%. The results of Western blot showed maximum over 50% inhibition of Aβ in drink powder and its major ingredients treated cells compared to control (Aβ only). Enzyme-linked immunosorbent assay (ELISA) demonstrated significantly lower amyloid-β levels in cell lysate which were 45–85% of that in control (cell lysate without treatment of drink powder and its ingredients). It was found that 1.5 μg/ mL concentration of drink powder, SBP, GBP and SG respectively had the highest effect on the reduction of amyloid beta under in vitro condition in neuron cells. The Aβ concentration was 4405, 6672, 8499 and 4533 pg/mL in cell lysate when drink powder, SBP, GBP and SG was at 1.5 μg/ mL respectively. Images of atomic force microscopy (AFM) confirmed the presence of low quantity of amyloid beta in drink iv powder, SBP, GBP and SG treated cells. The combination of SBP and GBP (SG) showed a better effect against amyloid beta than individual ingredients possibly due to synergistic effect. Therefore, drink powder, sea buckthorn and goji berry warrant further investigation as potential therapeutic agents in the treatment of AD. Key words: sea buckthorn, goji berry, drink powder, spray drying, freeze drying, convective drying, Jianping, Lueliang, Habahe, Datong, nutritional value, bioactive compound composition, antioxidant activity, amyloid beta, Alzheimer’s Disease, MTS assay, Western blotting analysis, ELISA, AFM v Abbreviations Aβ amyloid beta or β-amyloid ACE angiotensin-converting enzyme AD Alzheimer’s disease AFM atomic force microscopy ANOVA analysis of variance AOAC Association of Official Analytic Chemists APC Aerobic Plate Count APP amyloid precursor protein APOE apolipoprotein E ATP adenosine triphosphate BCA assay bicinchoninic acid assay BDNF brain-derived neurotrophic factor BHA beta hydroxy acid BHT butyl hydroxyl toluene CFU colony-forming units cAMP cyclic adenosine monophosphate CSF cerebrospinal fluid DASH dietary approaches to stop hypertension DE dextrose equivalence DMEM Dulbecco's Modified Eagle Medium vi DMEM/F-12 Dulbecco's Modified Eagle Medium/Ham's F-12 DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DPPH 2, 2-diphenyl-l-picrylhydrazyl ECE endothelin converting enzymes E. coli Escherichia coli ELIA enzyme-linked immunosorbent assay FAME fatty acid methyl ester FBS fetal bovine serum FCS fetal calf serum FID flame ionisation detector FSANZ Food Standards Australian and New Zealand HDL high-density lipoprotein HFIP hexafluoroisopropanol IDE insulin-degrading enzyme IGF insulin-like growth factor GAE gallic acid equivalents GBP goji berry powder GC gas chromatograph GDP gross domestic product GI glycaemic index GMP good manufacturing practise HBSS Hanks’ Balanced Salt Solution HIF hypoxia-inducible factor vii HPLC high performance liquid chromatography LBG locust bean gum LDL low-density lipoprotein MES 2- (N-morpholino) ethanesulfonic acid MIF migration inhibitory factor MMP matrix metalloproteinase MTS assay 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy- phenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay MUFA monounsaturated fatty acid NEP neprilysin NFTs neurofibrillary tangles PBS phosphate buffered saline PCR polymerase chain reaction PI3K phosphatidylinositol 3-kinase PKA protein kinase A PUFA polyunsaturated fatty acid ROS reactive oxygen species SALX Salmonella express SBP sea buckthorn powder SDS sodium dodecyl sulfate SEC Size-exclusion Chromatography SEM scanning electron microscope SFA saturated fatty acid SOD superoxide dismutase viii