###### Abstract  
Dietary proteins are no longer just nutritional ingredients in our food. During hydrolysis, some of the released peptides may possess properties that favor the health of the human body. In our study enzymatic hydrolysis of lupin proteins was performed using papain. Three enzyme-to-substrate ratios were set for three different duration times. The SDS-PAGE of the samples was performed. Each hydrolysate was studied for the degree of hydrolysis (DH), acetylcholinesterasecholinesteraseerase (AChE) inhibitory, antimicrobial, and antioxidant activities (AOA, according to four spectrophotometric methods). The DH varied from 9.06 \(\pm\) 0.20 to 27.97 \(\pm\) 0.37%. According to the results, the best AOA was measured by the ABTS method (from 0.76 \(\pm\) 0.03 to 1.15 \(\pm\) 0.46 M TE/100 g protein). All the hydrolyses displayed AChE inhibitory activity (IC50), which varied between 155.58 \(\pm\) 1.87 and 199.63 \(\pm\) 0.41 mg/g protein. To the best of our knowledge, this is the first report of the acetylcholinesterasecholinesteraseerase inhibitory activity of lupin protein hydrolyses. In conclusion, lupin proteins prove to have a high potential to serve as a source of bioactive peptides. lupin; hydrolysis; proteins; peptides; papain; antioxidant peptides; antimicrobial peptides; acetylcholinesterasecholinesteraseerase inhibitory peptides; SDS-PAGE +
Footnote †: journal: Applied  
of structural features [6]. The role of dietary proteins as a source of bioactive peptides in this regard is constantly emphasized [7; 8; 9; 10]. Bioactive peptides are reported to have properties such as: antihypertensive [11; 12], antimicrobial [13; 14], antithrombotic [15], antioxidant [16; 17], anti-inflammatory [18], osteoprotective [19], immunomodulatory [20; 21], and others [22]. There are three approaches to releasing active fractions from the intact protein molecule: microbial fermentation, purified enzyme hydrolysis, and gastrointestinal digestion. While fermentation is associated with a lack of reproducibility, the use of a single, or combination of proteases to digest specific proteins seems to be the better approach. Different proteases, unlike most enzymes, are able to produce different products from the same proteins substrate when used separately or in combination. This opens the doors for many investigations in different dimensions. Papan (EC 3.4.22.2) is a cysteine endopeptidase with plant origin. When isolated from papaya, it is recognized by the US Food and Drug Administration (FDA) as a GRAS (generally recognized as safe) substance [17]. It has a broad pH range of action (3.0-9.0) and that is why it is used more and more in food processing. In addition, the obtained hydrolysates from different substrates show a high potential for the release of bioactive peptides [23; 24; 25]. The species _Lupinus_ and family _Fabaceae_, which include the plant lupin, have more than 4000 years' history of use in agriculture [26]. The plant has been used primarily as animal feed and for soil development. Lupin's seeds are gluten-free, high in fibers (30-41%) and proteins [27]. Lupin is considered safe for human consumption; moreover, de-hulled seeds have a low content of anti-nutrients (e.g., phytic acid, lectins and saponins), are free of phytoestrogens, and possess good technological properties [28; 29]. The protein content may vary in different species but it is approximately 35-40% db and the ratio of albumins to globulins in the seed protein is roughly 1 to 9 [30]. Two recent review papers summarized the reported activities of bioactive peptides from lupin and revealed the significant potential of this source [31; 32]. Compared to established plant protein sources such as soy and pea, lupin proteins are far from being thoroughly investigated. Fermentation and purified proteases (sool or combination) were examined so far.