*3.3. Ileal Digestability (AID, SID)*

The ileal digestibility of the proteins under investigation is based on intervention study. Thus, different protein diets (usually with similar crude protein and trypsin inhibitor activity levels) are fed to animals, usually rats, pigs, or chickens, living under controlled conditions. The feeding trials may run from 10 to 20 days depending on study and animal. Afterwards, the animals are euthanized and the ileal digesta is collected by mildly flushing the digesta with clean distilled water and the amount of crude protein or amino acids is measured. Overall, the ileal protein digestibility is the difference between the amount of protein or amino acid ingested by the animal and the amount of protein or amino acid in the ileal digesta outflow. However, the calculation of the ileal digestibility depends on which amount of the ileal protein/amino acid outflow is used in the actual calculation and can be calculated as apparent (AID), true (TID), or standardized (SID) ileal digestibility. The AID for a certain amino acid is computed by subtracting the total ileal outflow of that specific amino acid from the quantity consumed by the animal. The values for SID are computed similarly to the values for AID, apart from the fact that the basal ileal endogenous amino acid (AA) losses (IAAend) are subtracted from the ileal outflow [17–19].

#### **4. E**ff**ect of Processing on Protein Digestibility**

Legumes are rich sources of proteins (18–41%) and are important raw food materials worldwide. The proteins provide the essential amino acids necessary for maintaining body muscle and growth. However, legumes, and, thereby their proteins, are not similar. Moreover, the nutritional quality of proteins is not solely dictated by the AA composition, but important factors like digestion rate and digestibility in the GI tract, which in turn are determined by the protein structure and enzyme accessibility, are of outmost importance. The protein digestibility is affected by both endogenous and exogenous factors. Endogenous factors relate to the protein as such, that is, protein structural characteristics and how and to what extent food processing may affect this structure. Exogenous factors are related to the food matrix, and include protein interactions with other compounds like carbohydrates, lipids, and especially anti-nutritional factors (ANF). ANFs include the anti-nutritional proteins like trypsin inhibitors and lectins and the anti-nutritional chemicals like tannins, phytates, and polyphenols. Therefore, it may beforehand be expected that both different types of legume protein and different processing may result in a very diverse digestibility. Indeed, Table S1 (Supplementary Materials) shows the difference in protein digestibility of legumes using various processing methods.

#### *4.1. Cooking*

The simplest processing technique, conventional cooking, is the most studied method with 12 studies as seen in Table S1. Four different types of legumes, beans, peas, lentils, and chickpeas, but of various cultivars, were investigated. Most of the investigations were reporting in vitro protein digestibility, while only few studies reported protein content changes [15] and in vivo digestion [16,20]. The protein content in the flour of unprocessed bean, pea, and lentil is rather similar, ranging from 24.8% to 28.7% [15]. However, comparing legume subtypes that underwent cooking, the effect on the protein content differed considerably (Table S1). Unprocessed bean (*Phaseolus vulgaris*) with protein contents of 24.8% (Raba) and 26.2% (Warta) significantly decreased to 23.0% and 21.3%, respectively. The protein content in cooked and uncooked peas (*Pisum Savitum*) yielded quite different results, since the content in the Milwa cultivar increased, while the Medal's protein content decreased [15]. The lentil cultivars (*Lens culinaris*) demonstrated both the highest and a significant increase in protein content after cooking. However, digestibility was not investigated [15]. For all cooked legumes, IVPD increased significantly, as seen in Table S1. For the flour of eight different species of unprocessed peas (*Pisum sativum* L.) the IVPD ranged from 79.9% to 83.5%, while the IVPD of the processed samples varied between 85.9% and 86.8% [21]. The IVPD of 83.61% for unprocessed chickpea (*Cicer arietinum* L.) increased to 88.52% after cooking for 90 min [22]. The time of cooking was found to be important for the IVPD, since Habiba et al. [23] found an increasing IVPD upon increasing cooking time. Interestingly, the increased IVPD was concomitant with a decrease in total protein content. This decrease in total crude proteins was suggested to be a result of leaching of water-soluble proteins during cooking [23]. A similar explanation is likely for the reported decreases in protein content for some of the legumes mentioned above [15]. The cooking resulted in improved IVPD of lentils, chickpea, peas, and soybean, but soaking the legumes prior to heating did not result in consistent significant effects [24]. Similarly, cooking of three different varieties of kidney beans significantly increased the IVPDs, while pre-soaking did not have any major effect [25]. Moreover, soaking in alkaline solution (sodium bicarbonate, pH 8.2) did not improve protein digestibility. However, Embaby [26] found that soaking cooked bitter and sweet lupin seeds for 96 and 24 h, respectively, further improved the IVPD.

In same line, the in vivo digestibility differed according to the lentil type, temperature, and time applied. Digestibility (SID) of unprocessed full-fat soybeans (FFSB) was 46% [16]. Incremental increase of the cooking temperature and duration caused a correlated increase in the soybean's SID. Hence, beans cooked at 80 ◦C for one min had a SID of 52%, whereas FFSB processed at 100 ◦C for six or 16 min had a SID of 73% and 80%, respectively, Table S1. Similarly, the cooking of peas (*Pisum sativum* L.) prior using as a diet resulted in an increase of true digestibility (TD) (79.8%) as compared to raw pea diet with TD of 74.7% [20].

For centuries, prior human consumption legume seeds have been soaked and thermally treated by conventional cooking due to the simplicity in the execution and equipment. However, the drawbacks of cooking are a fairly uncontrolled and non-adjustable process and the potential loss of valuable nutrients like vitamins. Therefore, other processing techniques are investigated in order to optimize the protein digestion by better control of the heating process.
