Food matrix and disease prevention: Bioprocessing approaches to improve bioactivity and acceptability of pulse seeds

Elisa Di Stefano

Research output: Thesisinternal PhD, WU


Noncommunicable diseases are on the rise, with unhealthy diets being one of the major contributors. Food consumption should therefore be rethought as a strategy to prevent the diseases. Pulses are an excellent candidate for this transition, being a rich source of macro and micro-nutrients, fibers and bioactive compounds, with low fat content. However, the low digestibility of the plant food matrix by humans hinders their nutritional and functional benefits. Alongside, the consumer choice of pulse products remains low in Western counties. Off-flavours attributes such as “beany, green, earthy” are commonly associated with green lentil and yellow pea consumption. Formation of flatus, primarily associated with the high content of raffinose family oligosaccharides (RFOs), is a second major problem. To promote inclusion of pulse products in daily diets, these limitations have to be addressed. In this thesis, we explored the use of natural processing methods, namely germination and bacterial fermentation, to address the major issues involving acceptability and digestibility of pulse seeds, and enhancement of the bioactivity towards improving glucose regulation in humans. Digestibility and bioactivity were the focus of chapter 2. DPP-IV and α-glucosidase inhibitors have been observed in a wide variety of plant-derived foods isolates such as purified protein hydrolysates and phenolic extracts. However, little attention has been placed on the interaction of these bioactive components within the seed matrix, and the related bioaccessibility issues. In this chapter, we investigated the implications of germination and microbial fermentation on seed microstructure and cell wall integrity, on the bioaccessibility of peptides and phenolics, and the in vitro DPP-IV and αglucosidase inhibitory activity. We identified a potential in germinating chickpeas for 3 days, in regard to its significant increase in α-glucosidase inhibition. We also identified a potential in fermenting green lentil flour, in regard to its significantly improved DPP-IV inhibitory activity. Following up on the findings from chapter 2, in chapter 3 we investigated the effect of fermentation of green lentil with L. plantarum on circulating and cell-bound (in situ) DPP-IV inhibitory activity in polarized Caco-2 cells. We observed a dose-dependent inhibition, which however was peaking within the exposure of 500 mg/mL of digested product. Increasing fermentation time from 48 to 72 hours led to an increase in bioactivity. The inhibition was more pronounced in situ than on the circulating DPPIV. We further tested if the bioactivity would extend to the glucose transport enzymes, by measuring the transport of glucose across the cell monolayer. This was not affected, confirming that the beneficial effect of the bioprocessing was centred on DPP-IV inhibition, therefore confirming the observation from chapter 2. An important component potentially involved in the DPP-IV inhibitory activity of pulse-foods are bioactive peptides. Recent studies have emerged, investigating the bioaccessibility and intestinal transport of purified bioactive peptides, however comprehensive studies investigating the proteome of pulse foods and related potential functionalities are still scarce. In chapter 4, we assessed the impact of gastrointestinal digestion and L. plantarum fermentation on the formation of bioaccessible peptides. We further evaluated the transport of such peptides across a Caco-2 cell monolayer. Overall, 16.1 – 24.5 % of the bioaccessible peptides were transported. Interestingly, we observed a size and weight effect on the intestinal transport, as peptides larger than 2000 Da or 22 amino acids were not transported. 63 peptides were formed upon brush border enzymatic activity, and were more negatively charged and less hydrophobic compared to the other peptides identified in the study. Additionally, L. plantarum fermentation led to the formation of more hydrophobic peptides, suggesting higher bioactivity. The amino acids located in P1, P2, P3 and C-terminal of some of the hydrophobic peptides formed by L. plantarum fermentation also matched the ones predicting potential for DPP-IV inhibition. We speculate a role of these peptides in the improved DPP-IV inhibitory bioactivity of fermented lentil samples observed in chapter 3, although further studies are needed to confirm this observation. Lastly, this thesis aimed at addressing two important factors currently limiting consumer choice for pulse foods, which are flavour and intestinal discomfort. These issues were addressed in chapter 5, where we profiled the volatiles present and/or formed during microbial fermentation. We then profiled the major RFOs, to investigate whether it was possible to decrease them while retaining the related potential prebiotic functionality. As a result, we observed that fermentation of the two pulses with L. plantarum was effective in depleting the RFOs, without affecting the short-chain fatty acids profile. The bioprocessing additionally improved the aroma profile of green lentil, while the effect on yellow pea was less clear. The 48 h fermentation of green lentil flour therefore emerged as promising treatment to lower RFOs content and improve aroma. This was tested in an ex-vivo system simulating the fermentation of the product by colonic microbiota over a period of 3 days, to see if the potential prebiotic effect was also retained. An increase in beneficial commensal species was observed in both unfermented and fermented green lentil samples compared to the control, suggesting a potential prebiotic designation for both products. L. plantarum pre-fermentation was not compromising the functionality of the lentil flour on the gut microflora. To conclude, in chapter 6 we discussed the results as a whole, looking at the major findings of the thesis and their implication for the development of novel pulse-based foods with enhanced functionality and improved acceptability. We further discussed the methods used for addressing these issues and their limitations. Finally, we looked at future directions set by this work, and the recommendations for future research on the topic.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • Fogliano, Vincenzo, Promotor
  • Oliviero, Teresa, Co-promotor
  • Udenigwe, C., Co-promotor
Award date17 Jan 2022
Place of PublicationWageningen
Print ISBNs9789464470130
Publication statusPublished - 17 Jan 2022


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