Insight in protein (micro)structural organisation and consequences for macronutrient in vitro digestion kinetics of plant-based beverages

Plant-based beverages are basically emulsified systems containing macronutrients such as proteins, lipids, and/or carbohydrates and can be consumed by people following any type of diet (e.g. vegans, vegetarians, flexitarians, or lactose-free diets). These products are commercialised as (i) ready-to-drink beverages or (ii) in the form of protein-rich powders that can be dispersed in water before consumption. Therefore, they can be quickly and conveniently prepared and/or consumed. The ready-to-drink beverages typically contain raw materials such as legumes, oil seeds, and nuts, which normally undergo a series of processing steps such as grinding, filtration, thermal processing, mixing, high pressure homogenisation (HPH), etc. Although these processes are essential to ensure a stable and safe product with an extended shelf life, processing may alter the native structure and consequently functionality of macronutrients. HPH for instance, is often applied aiming to alter the microstructural organisation of macronutrients and generate more homogeneous systems. The powdered forms of these beverages, are typically composed of extracted and purified macronutrients (e.g. isolated protein, purified oils). In general, the nutritional composition of these powders depends on the processing conditions and formulation with some commercial products containing more than 85% isolated plant protein. Importantly, the intense processing conditions usually applied to extract macronutrients from plant sources to produce a protein-rich powder, affect the technological and digestion properties of the final product. In this context, thermal processes and extreme pH conditions (pH << pI >> pH) might (partially) denature protein and affect their functionality (e.g. dispersibility).

Despite the great industrialisation of plant-based beverages, there is relatively little information available about their in vitro macronutrient digestion kinetics as affected by processing, (micro)structure, presence of other macronutrients (e.g. lipids), and/or the link with their techno-functionalities (e.g. dispersibility). In the context of this PhD project, a commercially available protein-rich powder was used to prepare dispersions further combined with other components (e.g. lipids) which were subjected to distinct processing steps. These resulting protein containing dispersions will be referred to as ‘plant-based beverages’.

The general objective of this PhD project was to design plant-based beverages using a commercial pea protein isolate (PPI) as the main protein source and to evaluate the effect of different factors on in vitro macronutrient digestion kinetics: (i) different processing techniques and strategies in the presence of lipids, (ii) protein fractions with postulated distinct digestion enzyme accessibility levels (i.e. from a more free form to a more naturally entrapped form), and (iii) distinct HPH intensities and environmental conditions (i.e. temperature and pH-shifting).

To evaluate the influence of processing techniques and strategies on in vitro protein and lipid digestion, plant-based beverages were prepared composed of 6% PPI, 5% high oleic acid sunflower oil (HOSFO), 1% soy lecithin, and 88% water (w/w). Initially, the impact of different processing techniques was studied: (i) only mixing versus (ii) mixing followed by HPH at 100 MPa. Besides, two processing strategies were considered, meaning the manner these techniques were applied, (iii) adding all ingredients together versus (iv) stepwise addition of ingredients. Beverages only mixed (i), consisted of large, irregular particles (1 – 100 µm). Eventually, this resulted in a relatively low in vitro lipid and protein digestion extent after 2 h of gastric digestion (9% and < 1%, respectively). In contrast, beverages that were also subjected to HPH (ii), displayed small, homogeneous particles (< 10 µm). Moreover, lipids and proteins were digested to a high extent in the gastric phase (40% and 10%, respectively). The small intestinal digestion kinetics indicated a significant impact of proteins on lipid digestion kinetics but no significant effect of lipids on protein digestion kinetics. Furthermore, the processing strategies evaluated (iii) and (iv), did not significantly influence the particle size (distribution), (micro)structural organisation, and in vitro macronutrient digestion kinetics of plant-based beverages.

The quantification of multiple lipid species (i.e. TAGs, DAGs, MAGs, FFAs) during in vitro lipid digestion allows gaining insight into the mechanism of lipid hydrolysis. In this regard, quantifying the most abundant lipid species present in highly consumed plant-based oils rich in unsaturated fatty acids and the lipid digestion products derived thereof (e.g. oleic acid, linoleic acid, 1-monolinolein, 1/3-monoolein, 1-linolein, 2-olein, 1/3-diolein, triolein, and 1,2-diolein-3-linolein), allows a better understanding of the biochemical processes occurring during lipid digestion. Hence, an upgrade of a (previously developed and used) reversed-phase high performance liquid chromatography with a charged aerosol detector (RP-HPLC-CAD) method (Method A) was done. The upgraded method (Method B), underwent optimisation of gradient program and detector settings as well as validation. Besides, Method B was demonstrated to be simple, robust, and able to detect all the analytes with high sensitivity and precision. Importantly, Method B allowed faster analysis time and thus higher throughput than Method A.  

The impact of distinct digestion enzyme accessibility levels on macronutrient digestion kinetics was investigated by preparing plant-based beverages based on two protein-rich ingredients. On the one hand, as learned from the first research chapters, protein dispersions were prepared with commercial pea protein isolate (PPI) and further stabilised by HPH, 100 MPa, as a source of free, ‘non-entrapped’ protein. On the other hand, individual pea cell (IPC) suspensions were considered as a source of ‘naturally entrapped’ protein. Besides, IPC were also a source of starch. The two protein structural organisations were blended. The ratio of protein derived from PPI and IPC was modified, yet, the total protein content was kept constant at 6%.

The results showed that IPC contain around 20% protein, therefore, increasing the amount of IPC (5% versus 15%) in the beverage, significantly decreased in vitro protein as well as starch digestion kinetics. For instance, at the end of the simulated small intestinal phase, the protein digestion extent decreased from 75% to 68%, and the starch digestion extent decreased from 63% to 47%. Finally, introducing individual pea cells in the beverages retarded in vitro lipid digestion kinetics. This study showed that altering (the ratio of) protein with distinct digestion enzyme accessibility levels in plant-based beverages can be a manner to modulate macronutrient digestion kinetics.

From the processing techniques applied to prepare plant-based beverages in previous chapters, it was clear that HPH (100 MPa) significantly reduced the size of insoluble protein aggregates and improved in vitro protein digestion kinetics in comparison to mixing only. Therefore, in the last chapter, a more systematic study on the impact of HPH intensities on digestive and technological functionalities was performed. For this, protein dispersions were prepared using the commercially available PPI and applying a range of HPH intensities (0 to 200 MPa) (room temperature, pH 7). Additionally, (other) PPI dispersions were treated at different environmental conditions (40 °C, 60 °C, or 95 °C, at pH 2, 7 or 12). This with the aim of generating dispersions with distinct protein solubility and (micro)structure. Interestingly, HPH intensities and temperature-pH combinations seemed to generate dispersions with similar protein (micro)structures. Nevertheless, these dispersions showed different solubility and/or protein digestion kinetics. Generally, increasing pressure of the homogenisation treatment was linked with decreasing particle sizes, increasing protein solubility, and in turn, faster in vitro protein digestion. More specifically, the dispersion that did not undergo HPH (0 MPa) as well as the dispersion treated at 60 °C, pH 7, had highly similar shell-like (micro)structures, consisting of large irregular particles (10 – 500 µm). Furthermore, even though these two dispersions i.e. 0 MPa and 60°C, pH 7, exhibited low solubility, it was substantially different (around 15% and 28%, respectively), resulting in a limited final in vitro proteolysis in the small intestinal phase (35% and 42%, respectively). In contrast, the dispersion subjected to HPH at 100 MPa and the dispersion treated at 60 °C, pH 12 also had similar (micro)structures with small and homogeneous particles (< 1 µm), and exhibited relatively good solubility (54% and 31%, respectively), which led to enhanced protein digestion levels (87%, and 74%, respectively). Moreover, the latter two protein dispersions presented less intermolecular β-sheets, β-sheets, and α-helices fractions than the former dispersions, which can be related to the enhanced digestibility.

This PhD work proved that besides nutrient composition, there are also other factors that have an impact on macronutrient digestion kinetics of the beverages. Influencing factors are for example, (i) the structure of the macronutrients within the beverage, which at the same time is affected by processing, (ii) the possible interactions among macronutrients, (iii) the accessibility of digestion enzymes to their substrates. All these factors may influence in vitro macronutrient digestion kinetics in plant-based beverages and could be used by producers as a strategy to modulate macronutrient digestion kinetics as well as to target specific physiological responses in particular population groups with specific needs (e.g. elderly).