**1. Introduction**

According to the last Food and Agriculture Organization (FAO) reports, a huge effort must be deployed to meet the future food demand connected to the increasing world population. The world in 2050 will host about 9 billion people, 30% higher than nowadays, and a strong lack of farmland, water, nutrients, and non-renewable energy is expected [1]. Along the food chain, the meat production represents the most impacting field and for these reasons novel food protein sources have been envisioned to meet the future demand [2].

Insects represent a good source of proteins for feed and food applications. In fact, the amount of proteins in insects can range between 30% and 60% on dry matter basis, with a high-quality profile rich in essential amino acids [3,4]. Furthermore, insects, in comparison to the common livestock, are characterized by many environmental advantages, such as less land use, feed, and water requirement, and a high food conversion ratio [5]. In the European Union, the legalization on insect proteins in food and feed applications has been granted by including them in the novel food category and by permitting their use for pet, fur and aquaculture [6–8], respectively.

As future protein source, insects have been studied and different extraction protocols for proteins were explored. For examples, Yi et al. performed a chemical extraction on *Tenebrio molitor* by combining concentrated salt and alkali pH for an overnight extraction [9]. Bußler et al. and Zhao et al. applied an alkali extraction only after defatting the insects with hexane or ethanol [10,11]. Soetemans et al. reported the use of organic acids to obtain protein and lipid enriched fractions from black soldier fly larvae, after a mechanical removal of chitin [12]. Caligiani et al. compared the ability of three different

protocols to fractionate and separate the main components of insect: Chitin, lipid, and protein. The protein fraction of black soldier fly was recovered with chemicals, by following both an alkali extraction and the milder Osborne fractionation, and enzymatic processes [4]. Indeed, the enzymatic method constitutes an essential part of the processes used by modern companies to produce, from complex matrices, a large and diversified range of products for human and animal consumption [13]. The use of exogenous enzymes, instead of chemicals, allows not only to control the process, but also to prevent protein degradation due to milder reaction condition. Proteases are efficient to separate proteins from lipids and insoluble compounds (e.g., fibers) by hydrolyzing peptide bonds and releasing peptides and free amino acids in solution with high efficiency [14]. Peptides, compared to the parental proteins, are characterized by an enhanced in gastro-intestinal digestibility and bio-accessibility [15]. In literature many studies have investigated the bioactivity of protein hydrolysates obtained from insects, such as antioxidant, angiotensin-converting enzyme-inhibitory, antidiabetic, and antihypertensive activity [16]. On the other side, limited publications are available regarding the techno-functional properties of insect protein hydrolysates. Purschke et al. [17] demonstrated the ability of targeted enzymatic hydrolysis to produce protein hydrolysates from *Locusta migratoria* protein flour with tailored techno-functional properties. They observed an increase in protein solubility, emulsifying activity, foam ability, and oil binding capacity in a broad spectrum of pH. Hall et al. [18] proved that Alcalase hydrolysis could represent an efficient biotechnology tool to improve the techno-functionality of cricket proteins. In particular, they demonstrated the ability of this enzyme to enhance the solubility, emulsion and foam capacity of insect protein hydrolysates obtained at different enzyme concentrations and time of hydrolysis.

Enzymatic assisted extraction has been demonstrated to be a valid method to extract proteins from insects in form of peptides. In our previous work, we have tested on lesser mealworm (*Alphitobius diaperinus*, LM) seven different enzymes from microbial, vegetable and animal origins, by performing at a laboratory scale an end-point hydrolysis [14]. The hydrolysates were characterized for the degree of hydrolysis (DH), the yield of extraction and the presence of free amino acids, but for their future involvement as insect-based protein ingredients in food or feed formulations, it is necessary to assess also their techno-functional properties. For the first time, the present work investigates the techno-functional properties of protein hydrolysates isolated from LM, focusing on the effect of the DH.

### **2. Materials and Methods**
