Effect of cell integrity on soybean protein digestion and fermentation: an in vitro study

Mostafa Zahir

Research output: Thesisinternal PhD, WU

Abstract

Plant foods are organized into hierarchical structures that range in scale from centimeter dimensions of plant tissue to the nanometre scale of intracellular macronutrients (starch, lipid, and protein) inside plant cells. This natural encapsulation system may restrict the access of digestive enzymes to macronutrients during gastrointestinal digestion, particularly when the integrity of plant cell walls is preserved after food processing. A better understanding of the triangular relationship between plant food structure, food processing, and food digestion is of great importance and it is essential for designing and developing the strategies that could help to improve plant protein digestibility and utilization by the human body.

In this thesis, we have used soybean particles and intact cells as plant food models to elucidate the effect of cell wall integrity on plant protein digestion and colonic fermentation. Initially, different particle sizes of raw and boiled soybean (as whole cotyledon or flour) were compared for their protein digestibility and cellular integrity. The data showed that decreasing particle size and increasing the proportion of cell rupturing increases the digestibility of protein contained within soybean particles. The cellular integrity of particles was observed by the use of confocal microscopy. The microscopy images showed that the proportion of ruptured cells within particles having the same size was greater for particles prepared from soybean boiled as flour compared to the particles prepared from soybean boiled as a whole cotyledon or raw soybean flour. This data gave insights into the fate of cell wall integrity during food processing procedures (milling before or after boiling) and the influence of cellular integrity of soybean particles in modulating protein digestibility. To investigate the individual contribution of the cell wall of intact cells in modulating protein digestibility, isolated intact cells were digested and the results indicated that cell wall intactness of cells limited protein digestibility, possibly via limiting the access of digestive enzymes to intracellular protein.

The role of cell wall porosity and permeability of soybean cells in regulating the accessibility of digestive enzymes to intracellular protein was therefore further investigated in this thesis. For this, the penetration of different fluorescein isothiocyanate dextrans probes (20, 40, 70, and 150 kDa) into intact cells isolated from processed soybean cotyledon was visualized by using confocal microscopy to first determine cell porosity and permeability. The confocal observations showed that intact cells of soybean cotyledons are not permeable to the fluorescent probe (20 kDa) after only the boiling process. Interestingly, after the combination of fermentation or germination with boiling, soybean intact cells become permeable to all different dextran probes sizes. The diffusion behavior of fluorescently labelled trypsin was also studied to predict the access of protease enzymes into intact cells. The images showed that fluorescently labelled trypsin was able to diffuse through the cell walls and accumulate in the cell space of soybean cells, irrespective of the differences in the treatments applied (boiling alone or in combination with germination, or fermentation). The comparison of the in vitro protein digestibility of the differently pre-treated isolated cells showed that protein digestibility was increased when boiling was combined with fermentation or germination. This is probably due to the pre-digestion of storage proteins and inactivation of trypsin inhibitors which occur during the fermentation and germination processes.

Therefore, this thesis also defined the relationship between and protein digestibility and the role of cell integrity in modulating protein physicochemical change during cooking. Proteins of boiled non-germinated and germinated soybean (as whole cotyledon or flour) were compared for their trypsin inhibitors levels, surface hydrophobicity, secondary structure, thermal denaturation, aggregation, and digestibility. The results showed that proteins of boiled cotyledon of germinated soybeans had distinct physicochemical properties which in turn translated into an increment in protein digestibility compared to non-germinated boiled soybeans irrespective of the boiling as a whole cotyledon or flour. When comparing boiled cotyledons with boiled flour, results indicated that the preservation of an intact cell during cooking contributed to reducing protein physicochemical changes and thus limiting its digestibility during in vitro intestinal digestion. Nevertheless, the effect exerted by cellular integrity on protein physicochemical changes during cooking of whole cotyledons was limited when soybean cotyledons were previously subjected to germination process before boiling. This is most likely due to the role of the germination process in changing the molecular structure of storage proteins due to metabolic reactions naturally occurring during germination. The role of cooking time in modulating protein physicochemical changes and digestibility was also investigated for both boiled germinated and non-germinated soybean either as whole cotyledon or flour. The data showed that the differences in cooking times either among cotyledon or flour samples of both germinated and non-germinated soybean resulted in slight changes in protein physicochemical properties that were accompanied by a limited improvement in the protein digestibility.

Similar to what was observed during small intestinal digestion, the structural attributes of plant cells could also modulate protein fermentability in the large intestine. To test this hypothesis, an in vitro model of colon fermentation was used to study protein fermentation in intact cells, broken cells, and isolated protein was conducted. Results indicated that the fermentability of protein within intact cells decreased by cellular integrity as evident from the difference in BCFAs and ammonia production between intact and broken cells. In a separate experiment, to simulate protein digestion and fermentation processes in humans, intact and broken cells were pre-digested using trypsin and chymotrypsin before incubation with a fecal human inoculum. The findings of this experiment revealed that following the in vitro gastrointestinal digestion, higher BCFAs, and ammonia was produced during the fermentation of intact cells compared to what was found for broken cells. At the same time, intact cells produced low gas, and SCFAs compared to broken cells. To investigate the role of heat treatment in protein fermentability, raw, and heat-treated soybean protein were compared. The time course of BCFAs and ammonia production showed the degradation of raw soybean proteins was lower compared to its heat-treated protein counterpart. This thesis highlights the influence of cellular integrity of plant foods in limiting protein digestibility, but overall suggests that food processing could be instrumental to modulate plant protein digestibility.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Fogliano, Vincenzo, Promotor
  • Capuano, Edoardo, Co-promotor
Award date26 Feb 2021
Place of PublicationWageningen
Publisher
Print ISBNs9789463956444
DOIs
Publication statusPublished - 26 Feb 2021

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