Strategies for extraction, characterization and enzymatic modification of dietary fibre

Abstract

In recent times, a twofold trend is taking hold: on the one hand, the ethical, environmental and economic need to manage wastes and by-products originating from the food supply chain differently and better, and on the other hand the will of consumers to direct their choices toward healthier foods. An intelligent and logical consequence for meeting both needs is the reuse of dietary fibre from unexplored and undervalued biomasses. When it comes to isolating dietary fibre, there are several extraction methods that may be more or less suitable depending on the matrix considered, and that may have as a consequence an impact on dietary fibre’s chemical structure, with the latter which in turn is closely related to the techno/bio-functional properties. This Ph.D. thesis explores the feasibility of reusing dietary fibre from various matrices by employing different approaches, in terms of extraction, characterization, enzymatic modification, evaluation of functional properties, and investigation of the chemical compounds inevitably produced during certain extraction methods. Chapter 1 gives a general overview of dietary fibre, briefly describing some of the most common types and their chemical structures, covering the main extraction methods, the use of enzymes to modify their structure, and the most popular analytical techniques for determining structural information on extracts and hydrolysates. The limitations that all these processes still have are here critically discussed, analysing what will be necessary to be done in the future steps. In particular, a special focus is made on lignocellulosic materials and xylo-oligosaccharides (XOS), which are topics particularly central to this thesis. Chapters 2 and 3 deal with the mild enzymatic assisted extraction and characterization of two types of dietary fibre from very different sources, namely bacterial culture broths and fruit wastes, respectively. Chapter 2, in particular, concerns the use of an official method for dietary fibre quantification (AOAC 991.43) that has been here adapted for the extraction of bacterial exopolysaccharides (EPS), namely from Lactobacillus strains previously isolated from food products. The method is proposed as a universal methodology for the isolation and purification of EPS from complex matrices, allowing the detailed characterization of their molecular structure, extremely variable depending on both the strain and the substrate. Chapter 3 used a similar approach, namely, isolating dietary fibre through a protease-based enzymatic assisted extraction on kernels, seeds, and peels derived from fruits. The purpose was to evaluate whether it was possible, under mild conditions in terms of temperature and pH, to extract at the same time not only a mixture of proteins and peptides, but also a fraction of soluble fibre. The results showed, for some by-products, high extraction yields for both protein and soluble fibres, suggesting the possibility to integrate the recovery of these important plant components. Molecular characterization of isolated fibre showed in most cases the presence of arabinogalactans, with potential technological applications. Chapters 4 and 5 focused on hydrothermal treatment, a harsh extraction method needed to extract hemicellulose from lignocellulosic biomasses, studying in detail its effects on hazelnut shells’ fibre composition. Chapter 4 deals with the evaluation of three analytical techniques, namely 1H NMR, GC-MS, and UHPLC-IM-Q-TOF-MS, to investigate the wide range of degradation compounds, mostly still unknown, that originate following such thermal treatment. NMR analysis identified the main chemical classes present, allowing their quantification. GC-MS detected in more detail the presence of small molecules, while the LC method coupled with ion mobility separation allowed to identify more than 200 compounds belonging to numerous different chemical classes, becoming a candidate as one of the most dominant techniques in the near future for a comprehensive characterization of these extracts. In Chapter 5, the impact of temperature in hydrothermal treatments was evaluated both on the fibre extracted from hazelnut shells and on the pattern of degradation compounds. It was found that lower temperatures yield high molecular weight pectin, while when they get higher xylans and xylo-oligosaccharides gradually become predominant, suggesting the potential for a fractionation approach by means of consecutive treatments. In addition, the formation of degradation compounds was investigated, showing how the total number of molecules is generally positively correlated with temperature, but also how a strong variability occurs at different temperatures in terms of type of compounds belonging to different chemical classes. Chapters 6 and 7 have in common the focus on the enzymatic hydrolysis of xylans, one of the main types of hemicellulose, for the production of XOS. In Chapter 6, in particular, the modelling of this enzymatic hydrolysis was studied by using commercial acetylated xylan as substrates and different enzymes, namely, endo-xylanase and acetylxylan esterase, and evaluating through the application of a Design of Experiments how hydrolysis parameters could influence the product outcome in terms of the degree of polymerization (DP). XOS with DP 2-6 were always present, but some experiments led to mixtures with higher DP, up to 10, expanding the possibility to test specific bioactivities related to the degree of polymerization. The method was also tested on acetylated xylan previously extracted from grape stalks, suggesting the feasibility of the approach even on non-pure agro-industrial samples. Chapter 7 exploited the results of the previous one for the production of XOS mixtures having variegate structures, in terms of DP and degree of acetylation. Tangential ultrafiltration was here performed together with the xylanolytic hydrolysis, enhancing the conversion yield of xylan into XOS, while a purification by preparative gel permeation chromatography allowed to obtain for the first time a highly pure mixture of XOS having DP 6-9. The various XOS mixtures were subsequently tested in terms of in-vitro antioxidant activity and prebiotic properties by testing Lactobacillus brevis and Escherichia coli strains. The results showed a clear influence of XOS’ chemical structure in both tests, suggesting that a higher DP and low substitution degree could have improved functionalities in many cases. Finally, the relevance of this research and its implication for future applications is discussed in Chapter 8.