**1. Introduction**

"Scotta", also called "ricotta cheese exhaust whey" (RCEW) or "second cheese whey" (SCW) is the residual liquid by-product of the ricotta cheese production process, obtained by thermal flocculation of whey proteins, and heating the whey at a temperature of 85–90 ◦C and 78–85 ◦C for bovine or buffalo and ovine or goa<sup>t</sup> whey, respectively [1]. Whey composition, the treatments performed during ricotta production process (i.e., adding milk, whey protein extraction method), and ricotta yield (depending on the temperature of protein coagulation, pH, and ionic strength of the whey) are the main factors that affect the physicochemical characteristics of scotta [2].

In Italy, scotta is mainly produced from bovine and ovine milk and, in less quantity, from buffalo and goa<sup>t</sup> milk. During 2019 about 900,000 tons [3] of whey were transformed into ricotta cheese in Italy, representing about 16% of total whey produced, giving rise to more than 750,000 tons of scotta.

The production of scotta from ovine whey is mainly concentrated in Sardinia, which hosts about 22% of Italian dairy ewes [4] and where is produced more than 65% of Italian ovine milk. The Sardinian dairy system produces about 320,000 tons of ovine milk [5], which is almost completely destined for cheese production. About 12,000 tons of ricotta

**Citation:** Cabizza, R.; Fancello, F.; Petretto, G.L.; Addis, R.; Pisanu, S.; Pagnozzi, D.; Piga, A.; Urgeghe, P.P. Exploring the DPP-IV Inhibitory, Antioxidant and Antibacterial Potential of Ovine "Scotta" Hydrolysates. *Foods* **2021**, *10*, 3137. https://doi.org/10.3390/ foods10123137

Academic Editor: Manuel Castillo Zambudio

Received: 23 November 2021 Accepted: 14 December 2021 Published: 17 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

are produced in Sardinia, with a potential production of more than 250,000 tons of scotta. The disposal of scotta poses serious environmental concerns due to its high biochemical oxygen (BOD) and chemical oxygen demand (COD) [6–8]. Therefore, its valorization is becoming crucial for the dairy industry.

For many decades whey, and especially scotta, were underused or treated as waste, due to poor knowledge of their valuable components and the unavailability of adequate technologies to valorize such components. Over the years, several approaches to its valorization have been developed [9–11], since scotta still retains significant amount of useful compounds from whey, such as lactose, minerals, oligosaccharides, vitamins, proteins, soluble peptides, and free amino acids [9]. Scotta has been employed using biotechnological approaches as a growth substrate for some selected Lactic Acid Bacteria (LAB) for the production of lactic acid [12], or yeasts such as *Chlorella protothecoides* for the production of carotenoids, chlorophyll [13], and bioethanol [6,14], as ingredient to fortify ricotta, including bioactive peptides with antioxidant and anti-tyrosinase activities [15], bioactive peptides with angiotensin-I-converting enzyme (ACE)—inhibitory [16] biodegradable bioplastic [17], fermented drink [18] and hydrogen [19,20].

Furthermore, the interest in the application of enzymes of animals or plant sources applied to food matrices has grown during over the years [21,22], with the aim of valorizing by-products and reducing environmental impact [23]. The hydrolysis of whey proteins is a widespread practice, and the hydrolyzed whey protein (WPH) has had an important impact as functional or nutraceutical ingredient. WPH is produced by enzymatic hydrolysis of whey proteins, mainly from whey protein concentrates (WPC) or isolates (WPI), which leads to an increase in solubility and digestibility, reducing whey protein allergenic properties. Peptides obtained by enzymatic hydrolysis of whey proteins have shown interesting biological activities, with potential benefit for human health [24,25]; however, scientific studies specifically addressed to the valorization of scotta proteins and derived peptides are still poor [15].

Recently, several authors studied the potential of whey protein hydrolysates as inhibitors against dipeptidyl peptidase-4 (DPP-IV) [26–29]. DPPI-IV inhibitors play a key role in the treatments of type 2 diabetes (T2D), a worldwide diffused disease that affects 415 million people and in Italy accounts for more than 3 million patients, i.e., about 5% of the population [30].

The DPP-IV inhibitors adopted as oral antidiabetic agents act by promoting glucose homeostasis through the inhibition of the enzyme DPP-IV involved in the mechanism of degradation of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), two key glucoregulatory hormones. DPP-IV inhibitors can reduce glucagon levels and at the same time stimulate insulin release.

The antioxidant potential of peptides derived from milk and whey proteins is well-reviewed [31]. However, up to now, just two works have focused on the antioxidant activity of scotta. Sommella et al. [11] reported bovine scotta peptides with antioxidant activity derived from αs1-casein and β-casein. Monari et al. [15] tested different proteases on bovine scotta, observing in vitro the antioxidant potential of the obtained hydrolysates.

The antibacterial activity of biopeptides encrypted into the protein fraction of ovine milk and whey has been already shown [32–34]. Several peptides of the C-terminal region of the ovine αs2-casein were shown to possess antibacterial activity against Gram-positive and Gram-negative bacteria, with the first group of bacteria more susceptible than the second group [33]. El-Zahar et al. [32] found that the peptic hydrolysates of whey protein inhibited, in a dose-dependent manner, the growth of *Escherichia coli*, *Bacillus subtilis*, and *Staphylococcus aureus*. Regarding scotta, recent work [11,15] showed the presence of several antibacterial peptides in the protein fraction of scotta.

Regarding the available proteases, bromelain and pancreatin were previously applied in the hydrolysis of dairy products and by-products to produce peptides with DPP-IV inhibitory [35] and antioxidant activity [15]. In addition, bromelain was reported to release antibacterial peptides from goa<sup>t</sup> milk and whey [36].

While the literature has been mainly focused on the bioactive properties of peptides obtained from milk and whey proteins, the available data on scotta are limited to the bovine source [9] and, to the best of our knowledge, the present study is the first conducted on the ovine scotta as a potential source of bioactive peptides. Conversely, this matrix could represent an important source of such peptides, due to its higher amount of nitrogen, compared with bovine whey and scotta. The research conducted in this field could lead both to an adequate valorization of this by-product and an improvement of the sustainability of the dairy farms. Therefore, the aim of the present study was to evaluate the possibility of producing peptides with DPP-IV inhibitory, antioxidant and antibacterial activities from ultrafiltered ovine scotta by enzymatic hydrolysis with bromelain (BSPH), and pancreatin (PSPH), and evaluate their biological activity by in vitro tests. Furthermore, an in-depth characterization of peptide mixtures obtained from the hydrolysis with the two enzymes by gel permeation chromatography (GPC) and LC-MS/MS was performed.
