Exploiting natural variation in Plantago seed composition for food and human health applications

This thesis is comprised of nine chapters: a general thesis introduction (Chapter 1), a review of the literature (Chapter 2), two published research articles (Chapters 3 and 4), four unpublished research manuscripts (Chapters 5, 6, 7 and 8) and a final general discussion (Chapter 9). A schematic overview of the thesis structure is presented in Figure 1.2. Chapter 3 is a published research and methodology article describing a method for small-scale rapid fractionation of seed mucilage (Cowley et al. 2020, Plant Methods, 16:20). Most published seed mucilage extraction techniques while simple and robust, were recognised early in my candidature to be less than ideal for screening natural variants.. Previously published methods were often incomplete, time-consuming, and unnecessarily wasteful of precious seed stocks. Instead the new method described in Chapter 3 can be used to extract fractions of mucilage from small quantities of seed, which are still sufficient for chromatographic and other downstream analyses. Chapter 4 is a published research article describing the unique aspects of seed mucilage polysaccharide accumulation, storage, and release in Plantago ovata (Phan et al. 2020, Scientific Reports). Literature describing the polysaccharide biology of most mucilage-producing species is lacking and as such has led to an overreliance on understanding from the well-characterised model species Arabidopsis. In this manuscript, work by Dr Jana Phan (submitted 2018) completed during her PhD candidature showing that in P. ovata, mucilage polysaccharides were deposited and stored in a different fashion to Arabidopsis, was complemented by collaborative work done during my candidature where we spatiotemporally defined P. ovata mucilage release characteristics. This resulted in the formulation of a new model to describe mucilage-related processes in this species, and will be used to guide future work on Australian Plantago species that share some but not all features with P. ovata. Chapter 5 is an unpublished research manuscript where variation in seed composition and structure was compared between twelve Australian Plantago species: commercial psyllium, three naturalised species and eight native species. In addition to an analysis of the functional mucilage polysaccharides, this research showed that Plantago seeds have a substantial endosperm that contains beneficial proteins and fats. Natural variation in these seed constituents is described and discussed in a biogeographic and evolutionary context. Chapters 6 and 7 explore the influence that differences in Plantago seed composition and properties identified in Chapter 5 have on the in-food functionality of whole seed flour (WSF). Chapter 6 is an unpublished research manuscript where changes to the cooking/gelatinisation properties of rice flour and rice starch upon a small addition of Plantago WSF are described. Different WSFs have different influences on the gelatinisation properties with similarity observed between Australian species. Differences were attributed to mucilage content and structure and an explanatory model for a novel polysaccharide-polysaccharide interaction is proposed. In a more applied study presented in Chapter 7, Plantago WSF was added to gluten-free doughs which enhanced the rheology and proofing behaviour and led to improved loaf structure and texture. Differences in the degree of improvement were again attributed, in part, to the interaction described in Chapter 6 and an adapted model is presented. Chapter 8 describes experiments where an aqueous extract was produced from seeds of four Plantago species (deemed to perform well in Chapters 6 and 7) and applied to two intestinal epithelial cell lines in vitro. Plantago seed extracts were found to enhance the viability of these cell lines which has gastrointestinal health and nutrition impacts. It is likely that cell proliferation is enhanced by these extracts and based on characterisation of the extract, the constituents likely to be involved are discussed. The final chapter, Chapter 9, presents a summary and general discussion of the findings of this work and suggests future research directions.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 202