## 1 Introduction  
Pumpkin (_Cucurbita pepo_; _Cucurbita maxima_; _Cucurbita moschata_) is a traditional culture for Bulgaria with a wide culinary and industrial application. The increased interest in the consumption of healthy and minimally processed foods has also led to the increase in the production and consumption of cold-pressed pumpkin seed oil, which is rich in bioactive compounds such as unsaturated fatty acids (mostly linoleic and oleic acid), tocopherols, and phytosterols [1, 2]. The utilization of the protein-rich residue in the production of this oil has great potential for valorization [3, 4]. Currently, the pumpkin seed cold-pressed oil cake (CPC) is mainly used for animal feed or crop fertilizer, resulting in a great waste of resources [4]. However, due to the high protein content, availability, and low cost, CPC is a valuable alternative raw material for obtaining protein isolates, protein hydrolysates, and bioactive compounds (mainly oligopeptides) that possess a wide range of dietary and therapeutic functions. The major proteins in pumpkin seeds are globulins accompanied by glutelins and smaller amounts of albumins and prolamins [5, 6]. The pumpkin seed protein isolate is rich in essential amino acids and contains proteins of good quality in terms of digestibility and nutritional properties and can be consumed as a dietary supplement or incorporated into other food products [7]. [PERSON] and [PERSON] found that the functional properties of the protein isolate from _Cucurbita moschata_seeds were compatible with those of soy protein [8]. Methods for the separation of proteins from defatted pumpkin seeds meal include extraction at pH 9.0 and fractionation with sodium chloride solutions of different concentrations. Microwave and ultrasonic extraction were also experimented, with the highest protein yield found in alkaline extraction with ultrasonic treatment [9; 10]. On the other hand, a lot of research has shown that pumpkin seed proteins can be considered an innovative source of bioactive peptides with beneficial effects on human health [11; 12]. Enzymatic hydrolysis is preferable because it achieves mild processing conditions and more specific protein cleavage than chemical hydrolysis. As a result of the enzymatic hydrolysis of the pumpkin protein, peptides of small size are obtained, which have been reported to have antioxidant, antihypertensive, and antihyperglycemic activities [11; 12; 13; 14]. The main factors influencing the process are the type of enzyme used, enzyme/substrate (E/S) ratio, pH, temperature, and hydrolysis time. These factors affect not only the degree of hydrolysis, but also the molecular weight distribution of the peptides in the hydrolysate and their bioactive and functional properties [13]. In published studies, the proteases commonly used to hydrolyze pumpkin seed proteins are of animal (pepsin, trypsin, chymotrypsin) or microbial origin (Alcalase, Neutrase, Flavourzyme) [12; 14; 15]. [PERSON] et al. analyzed the influence of four different microbial enzymes (Alcalase, Flavourzyme, Protemax, and Neutrase) on the protein hydrolysis process of Cucurbita moschata seeds [16]. They found that Alcalase was the most suitable of all the enzymes tested to achieve the highest degree of hydrolysis and the highest proportion of low molecular weight peptides in the hydrolysate [16]. Enzymatic hydrolysis of cucurbitin (11S globulin) obtained from CPC (_Cucurbita pepo_ [PERSON] c.v. [PERSON]) with Alcalase, Flavourzyme, and pepsin was carried out under optimal reaction conditions for each enzyme. Alcalase treatment was found to result in a higher degree of protein hydrolysis than pepsin and Flavourzyme. The hydrolysate obtained by Alcalase and pepsin showed antioxidant activity whereas Flavourzyme hydrolysates did not. The authors suggest that this is due to the specificity of the enzymes, which hydrolyze different peptide bonds and, accordingly, the obtained hydrolysate have a different peptide composition [15]. [PERSON] et al.