*4.8. Factors A*ff*ecting PD*

Various processing techniques affect legume PD to various extents compared to the PD of raw legumes. Generally, the IVPD increases after processing by different heating methods. The improvement in PD was attributed to protein denaturation. Thus, the increased digestibility was ascribed to the resulting heat-induced denaturation of the proteins, thereby enhancing accessibility of susceptible sites to proteolysis. On the other hand, digestibility can be compromised by protein aggregation due to the thermal treatment [29]. A consequence of protein denaturation is increased opportunity for various intra- and intermolecular interactions, especially disulfide bridges between amino acids containing free thiol groups. Crosslinked, aggregated proteins are less accessible to digestive enzymes because of a different localization of amino acid residues specific for protease action resulting in lack of PD improvement. It is noted that the degree of amino acid reactivity and extent of aggregation differs under various conditions and not all amino acids participate in protein crosslinking irrespective of the processing condition. In addition, the native protein conformation of the primary legume proteins, globulins and albumins, may also affect the PD as such. The albumins have a compact globular structure stabilized by a large number of disulfide bonds, thus possessing an inherent structural hindrance limiting enzyme access [21]. In that respect, it was observed that an increased proportion of globulins and decreased proportion of albumins in pea seeds could contribute to an increased IVPD [21]. This may indicate that the ratio of albumin:globulin also has an influence on the IVPD of legumes. It is generally accepted that the abundance of ANF in plant protein sources contributes to the

lower digestibility compared to typical mammal proteins. Phytic acid, tannins, and polyphenols may interact with protein to form complexes by cross-linking with the proteins, resulting in a decreased protein solubility and making these protein complexes less susceptible to proteolytic attack in the GI tract. Partly elimination of tannins and phytic acid was observed after different thermal treatments (Table S1). The reduction of reactive tannins and phytic acid would result in less protein complexing and creating more space within the matrix, which increased the accessibility of the enzyme resulting in improved IVPD [11]. Trypsin inhibitors may interfere with the action of proteolytic enzymes in the GI tract by forming inactive complexes of trypsin and chymotrypsin. However, since trypsin inhibitors are heat-labile compounds, thermal processing should be an effective method for reducing its activity. Indeed, a marked decrease in trypsin inhibitors after different thermal processing was reported in many of the studies (Table S1) and a complete inactivation was also found for peas and kidney beans [23,25]. However, Embaby found that some treatments resulted in an increased level of anti-nutrients, but still obtained improved IVPD [26]. Thus, he suggested that ANFs are not solely responsible for lowering IVPD, and factors like cell wall rigidity and fiber content may influence the protein digestibility as such.

In conclusion, the improvement of PD of legume proteins for all reported process methods is explained by the structural disintegration of the native protein concomitant with the reduction or even removal of anti-nutrients.
