In vitro protein and starch digestion kinetics of individual chickpea cells: from static to more complex in vitro digestion approaches.

Attention has been given to more (semi-)dynamic in vitro digestion approaches ascertaining the consequences of dynamic in vivo aspects on in vitro digestion kinetics. As these often come with time and economical constraints, evaluating the consequence of stepwise increasing the complexity of static in vitro approaches using easy-to-handle digestion set-ups has been the center of our interest. Starting from the INFOGEST static in vitro protocol, we studied the influence of static gastric pH versus gradual gastric pH change (pH 6.3 to pH 2.5 in 2 h) on macronutrient digestion in individual cotyledon cells derived from chickpeas. Little effect on small intestinal proteolysis was observed comparing the applied digestion conditions. Contrary, the implementation of a gradual gastric pH change, with and without the addition of salivary α-amylase, altered starch digestion kinetics rates, and extents by 25%. The evaluation of starch and protein digestion, being co-embedded in cotyledon cells, did not only confirm but account for the interdependent digestion behavior. The insights generated in this study demonstrate the possibility of using a hypothesis-based approach to introduce dynamic factors to in vitro models while sticking to simple and cost-efficient set-ups.

Pulse seeds as promising and sustainable source of ingredients with naturally bioencapsulated nutrients: Literature review and outlook.

Pulse seeds are nutritious and sustainable matrices with a high level of intrinsic microstructural complexity. They contain high-quality plant-based protein and substantial amounts of slowly digestible starch and dietary fiber. Starch and protein in pulses are located inside cotyledon cells that survive cooking and subsequent mechanical disintegration, hence preserving natural nutrient bioencapsulation. In this context, several authors have explored a number of techniques to isolate individual cotyledon cells from these seeds, aiming to unveil their digestive and physicochemical properties. In recent years, isolated pulse cotyledon cells are also being highlighted as promising novel ingredients that could improve the nutritional properties of traditionally consumed food products. Even more, they could enable to implement a strategy for increasing pulse intake in populations where these seeds have not been traditionally consumed. This review mainly focuses on the reported digestive, physicochemical, and technofunctional properties of pulse cotyledon cells isolated through different techniques, preceded by a descriptive summary of the nutritional properties, structural organization, and traditional process chain of pulse seeds. It also offers an outlook of research directions to take, based on the identified research gaps. All in all, it is clear that isolation of pulse cotyledon cells using diverse techniques constitutes a promising strategy for the development of pulse-based ingredients where natural bioencapsulation of macronutrients is preserved. However, much more research is needed at the level of ingredient characterization to better understand the effect of starting pulse seed material, isolation technique, and isolation conditions on the nutritional and functional properties of the finished product(s) where the isolated cells are (to be) used.

In vitro starch and protein digestion kinetics of cooked Bambara groundnuts depend on processing intensity and hardness sorting.

When pulse seeds from a single batch are cooked, considerable variability of hardness values in the population is usually observed. Sorting the seeds into hardness categories could reduce the observed diversity and increase uniformity. Therefore, we investigated the effect of processing intensity whether or not combined with sorting into hardness categories on the in vitro starch and protein digestion kinetics of cooked Bambara groundnuts (cooking times 40 min and 120 min). The average hardness values were 89 ± 32 N and 42 ± 20 N for 40 min and 120 min cooking time, respectively. The high standard deviation of hardness for each cooking time revealed a high level of diversity amongst the seeds. Individual cells were isolated from (non-)sorted seeds before simulating digestion. The estimated lag phase describing the initial phase of starch digestion was not significantly different despite the processing intensity or the hardness category, implying that cell wall barrier properties for these samples were not majorly different. However, the rate constants and the extents of starch digestion of samples cooked for 40 min were significantly higher for the low hardness (50-65 N) compared to the high hardness (80-95 N) category (0.71 vs 1.02 starch%/min and 63 vs 77%, respectively). Kinetic evaluation of digested soluble protein (after acid hydrolysis of the digestive supernatant) showed that low hardness samples were digested faster than high hardness samples (0.037 vs 0.050 min-1). The faster protein hydrolysis in the low hardness samples was accompanied by faster starch digestion, indicating the possible role of the protein matrix barrier. Individual cells of comparable hardness obtained from the two different processing times had similar starch and protein digestion kinetics. Our work demonstrated that, beyond cooking time, hardness is a suitable food design attribute that can be used to modulate starch and protein digestion kinetics of pulse cotyledon cells.

Pectin and phytic acid reduce mineral bioaccessibility in cooked common bean cotyledons regardless of cell wall integrity.

Common bean cotyledons are rich in minerals (Mg, Ca, Fe and Zn), but they also contain natural barriers that can potentially prevent mineral absorption during digestion. In this study, both the cell wall integrity and mineral chelators/antinutrients (phytic acid and pectin) were investigated as natural barriers in common bean cotyledons. To examine the cell wall integrity as a physical barrier for mineral diffusion, soluble mineral content was determined in a cooked cotyledon sample before and after disruption of intact cell walls. While this study showed that the cell wall in cooked common bean cotyledons does not hinder mineral diffusion, it also demonstrated that the presence of antinutrients decreases mineral bioaccessibility. It was shown that a certain mineral fraction is naturally bound to phytic acid and/or pectin and, by enzymatically degrading these antinutrients, the antinutrient-chelated mineral fraction decreased. Moreover, although pH changes are occurring during simulated digestion experiments, which might affect charge of the antinutrients and thus their chelating capacity for minerals, no difference in mineral distribution over antinutrients was observed due to digestion. In addition, this study showed that mineral bioaccessibility in common bean cotyledons could be potentially increased by degrading antinutrients during digestion in the small intestinal phase.

Effect of process-induced common bean hardness on structural properties of in vivo generated boluses and consequences for in vitro starch digestion kinetics.

In the present study, we evaluated the effect of process-induced common bean hardness on structural properties of in vivo generated boluses and the consequences for in vitro starch digestion. Initially, the impact of human mastication on the particle size distribution (PSD) of oral boluses from common beans with different process-induced hardness levels was investigated through a mastication study. Then the effect of structural properties of selected boluses on in vitro starch digestion kinetics was assessed. For a particular process-induced hardness level, oral boluses had similar PSD despite differences in masticatory parameters between participants of the mastication study. At different hardness levels, a clear effect of processing (P<0·0001) was observed. However, the effect of mastication behaviour (P=0·1141) was not significant. Two distinctive fractions were present in all boluses. The first one was a cotyledon-rich fraction consisting of majorly small particles (40-125 µm), which could be described as individual cells based on microscopic observations. This fraction increased with a decrease in process-induced hardness. The second fraction (>2000 µm) mostly contained seed coat material and did not change based on hardness levels. The in vitro starch digestion kinetics of common bean boluses was only affected by process-induced hardness. After kinetic modelling, significant differences were observed between the reaction rate constant of boluses generated from the hardest beans and those obtained from softer ones. Overall this work demonstrated that the in vitro nutritional functionality of common beans is affected to a greater extent by structural properties induced by processing than by mechanical degradation in the mouth.

Texture and interlinked post-process microstructures determine the in vitro starch digestibility of Bambara groundnuts with distinct hard-to-cook levels.

Particular storage conditions are described to promote the development of the hard-to-cook (HTC) phenomenon for most legumes. However, it is not clearly established whether the HTC phenomenon influences starch digestion kinetics. Therefore, this study explored how the HTC phenomenon influences in vitro starch digestion of Bambara groundnuts, taking into account three distinct HTC levels. Stored Bambara groundnuts required prolonged cooking times. Increasing storage time led to a decrease in the rate constant of texture degradation, signifying the development of the HTC phenomenon. For cooking times of 60 min and 120 min, high HTC level samples exhibited higher rate constants and extents of starch digestion compared to the fresh sample. The higher rate of digestion was attributed to the high hardness that resulted in greater cell rupture and faster access of amylase to starch. Adapting cooking times of Bambara groundnuts with distinct HTC levels to obtain equivalent hardness values and microstructures resulted in comparable starch digestion kinetics. Spectrophotometric analysis overestimated the amount of digested starch, in contrast to the more accurate HPLC analysis, which further provided more insight by quantifying multiple digestion products. This work demonstrates that it is the hardness and interlinked pattern of cell failure (microstructure) that determines starch digestion of Bambara groundnuts with distinct HTC levels.

Cotyledon pectin molecular interconversions explain pectin solubilization during cooking of common beans (Phaseolus vulgaris).

Dynamics of pectin extractability in cotyledons and seed coats were explored for mechanistic insight into pectin changes due to aging and cooking of beans. In addition, changes in mineral distribution during cooking were determined in order to investigate their retention in the matrix. Pre-soaked fresh and aged beans were cooked in demineralized water for different times and the cotyledons, seed coats and cooking water were lyophilized. From cotyledon and seed coat powders, alcohol insoluble residue (AIR) was extracted and sequentially fractionated into water-, chelator- and sodium carbonate-extractable pectin (WEP, CEP and NEP, respectively). Characterization of pectin in AIR and pectin fractions revealed inherent structural differences between cotyledon and seed coat pectin with the latter exhibiting a lower degree of methylesterification (DM) and being more linear. Due to aging, WEP decreased whilst NEP substantially increased and the CEP fraction and DM of pectin in AIR did not change significantly, suggesting a more crucial role of increased covalent bonding than cation-mediated crosslinking in aging-induced hardening of beans. During cooking, some NEP was converted into WEP and no pectin depolymerization was observed from molar mass distribution profiles. Pectin changes due to aging and cooking of beans were more pronounced in the cotyledon compared to the seed coat. Whilst Ca2+, Fe2+ and Zn2+ were largely retained in the bean matrix during cooking, Mg2+ was largely leached from cotyledons into the cooking water. In conclusion, aging-induced hardening of beans and softening during cooking were found to be premised on interconversion of pectin fractions in cotyledons.

Process-induced cell wall permeability modulates the in vitro starch digestion kinetics of common bean cotyledon cells.

The presence of cell walls entrapping starch granules in common bean cotyledons, prevailing after thermal processing and mechanical disintegration, has been identified as the main reason for their (s)low in vitro starch digestibility. Nevertheless, it is unknown if the role of cell walls on starch digestion changes as processing conditions (e.g. time) are modified. In this study, it was hypothesised that cell wall permeability would be differently affected depending on thermal process intensity, giving origin to distinct in vitro starch digestion kinetic profiles. Cotyledon cells were isolated from common beans by applying processing conditions normally found at the household level (95 °C and times between 30 and 180 min (palatable range)). Isolated cells were characterised and subsequently subjected to in vitro simulated digestion. Microstructural properties, the starch gelatinisation degree, and the total starch content were similar among samples. In contrast, a higher diffusion of fluorescently labelled pancreatic α-amylase inside the cells was evident as processing time increased. From the kinetic analysis of digestion products, it was determined that longer lag phases and slower reaction rate constants were present in samples with a lower degree of process-induced cell wall permeability. The qualitative analysis of the remaining pellets showed that cellular integrity was maintained throughout in vitro digestion. A mechanism for the in vitro starch digestion of isolated common bean cotyledon cells as well as an alternative kinetic model to describe this process were proposed. Overall, our work demonstrated that the in vitro starch digestion kinetics of common bean cotyledon cells can be modulated by influencing cell wall permeability through thermal processing time.

Mechanistic insight into softening of Canadian wonder common beans (Phaseolus vulgaris) during cooking.

The relative contributions of cotyledons and seed coats towards hardening of common beans (Phaseolus vulgaris) were investigated and the rate-limiting process which controls bean softening during cooking was determined. Fresh or aged whole beans and cotyledons were soaked and cooked in demineralised water or 0.1 M NaHCO3 solution, and texture evolution, microstructure changes and thermal properties were studied. Fresh and aged whole beans cooked in demineralised water had significantly different softening rate constants and so did fresh and aged cotyledons. The comparable softening rate constants of aged whole beans and cotyledons indicated an insignificant role of the seed coat in hardening during storage. All samples cooked faster in 0.1 M NaHCO3 solution. Disintegration of cooked tissues followed by microscopic examination revealed a transition from cell breakage through a phase of cell breakage and separation to complete cell separation with increased cooking time wherefore texture decayed. Therefore, progressive solubilization of pectin in the middle lamella greatly promoted texture decay. While residual birefringence even after substantial cooking time suggested some molecular order of the starch, calorimetric analyses revealed complete starch gelatinisation before complete cell separation occurred. This implies an insignificant role of starch in texture decay during cooking but its hindered uncoiling into a viscous gel after gelatinisation due to the restricting cell wall could promote its retrogradation. Therefore, we suggest that the rate-determining process in bean softening relates to cell wall/middle lamella changes influencing pectin solubilization.

Carotenoid bioaccessibility and the relation to lipid digestion: A kinetic study.

The micellar incorporation of carotenoids (lycopene, α- and ß-carotene) and lipid digestion products (free fatty acids, FFAs, and monoacylglycerides, MAGs) during in vitro digestion of oil-in-water emulsions was investigated by a kinetic approach. A fractional conversion model could adequately describe the hydrolysis of triacylglycerides, formation of FFAs and MAGs, and micellar incorporation of carotenoids, FFAs and MAGs. The release of FFAs and MAGs from TAGs proceeded faster than their incorporation into micelles. Rate constants of carotenoid micellar incorporation were inversely proportional to their hydrophobicity and dependent on the isomeric configuration, being the incorporation of the cis faster than their all-trans isomers. Furthermore, a positive linear relation was found between the micellar incorporation of carotenoids and lipid digestion products. The isomeric form of carotenoids did not affect such relation. The present kinetic approach can be useful to gain mechanistic insight into carotenoid bioaccessibility as affected by various process- and product-related factors.