**1. Introduction**

Whey is the main and most polluting by-product obtained from cheese manufacturing processes due to its organic load consisting of lactose, lactic acid, proteins and salts. However, the substantial production of whey worldwide, estimated to be around 180–190 <sup>×</sup> <sup>10</sup><sup>6</sup> ton/year [1], and the consideration that 1 or 2 kg of cheese yields 8 to 9 kg of whey [2] is fostering its valorization. In fact, the discovery of its potential use as a functional food with nutritional applications is transforming it from a waste [3] into an added value product. Numerous studies have attributed several biological actions to these by-products which are important in the medical, pharmaceutical and food industries for their properties with potential benefits to human health [4]. The biological components of whey, including lactoferrin, beta-lactoglobulin, alpha-lactalbumin, glycomacropeptide, and immunoglobulins, demonstrate a range of immune-enhancing properties [5]. In addition, whey has the ability to act as an antioxidant, antihypertensive, antitumor, hypolipidemic

**Citation:** Alfano, A.; D'ambrosio, S.; D'Agostino, A.; Finamore, R.; Schiraldi, C.; Cimini, D. Concentrated Buffalo Whey as Substrate for Probiotic Cultures and as Source of Bioactive Ingredients: A Local Circular Economy Approach towards Reuse of Wastewaters. *Fermentation* **2021**, *7*, 281. https://doi.org/ 10.3390/fermentation7040281

Academic Editor: Alessia Tropea

Received: 8 October 2021 Accepted: 23 November 2021 Published: 26 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**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/).

and chelating agent. A number of clinical trials have successfully been performed using whey in the treatment of cancer, HIV and hepatitis B [6–8]. Moreover, today, whey is a popular dietary protein supplement that may provide antimicrobial activity, immune modulation, improved muscle strength and body composition, and prevent cardiovascular disease and osteoporosis [8,9]. The commercial success of whey proteins has led to the development of high-quality protein-based supplements manufactured as primary products, and not as a by-product, of cheese manufacturing. In fact, currently, proteins are processed with less aggressive treatments, for example under low temperatures and controlled pH, to avoid denaturing their native structures [5].

Due to the high lactose content in addition to proteins, whey, very often in combination with other medium components, was also used as a substrate for the cultivation of diverse microorganisms [10]. Whey-based media were in fact investigated for the production of value-added chemicals (e.g., succinic acid, lactic acid), antimicrobial peptides and probiotic biomasses [11–16].

The use of membrane technologies in order to obtain bioactive molecules from whey is a topic of growing interest. The main advantages are low energy requirement, no need for additives, separation efficiency and easy scale-up, and temperature control that prevents denaturation of recoverable added value products [5]. Several treatment technologies based on membranes were proposed. In particular, four basic types of membrane filtrations present potential applications for the dairy industry, i.e., microfiltration (MF), ultrafiltration (UF), nanofiltration (NF) and reverse osmosis (RO) [17,18]. The application of filtration processes to produce clean effluents thereby reducing wastewater and generating a purified stream (e.g., machinery washing, irrigation) transforms a difficult to manage and polluting effluent into a resource. Moreover, the use of membrane devices with different cut-offs allows the separation of compounds (proteins, peptides and lactose) present in whey into differentially enriched fractions that are, therefore, suitable for different applications.

Cheese manufacturing is one of the main industrial activities in the food sector present in the Campania region. The scope of this study was to promote a locally integrated bio refinery approach fully exploiting discarded whey. Therefore, the permeate and retentate of ultrafiltered whey, both provided by a local dairy factory, were evaluated in this work. The permeate was further processed to investigate a potential downstream approach to obtain reusable water with a low organic load. The retentate was evaluated to identify other potential biotechnological applications of whey from buffalo milk. In particular, it was investigated as the main substrate for the growth of *Lactobacillus fermentum*, a probiotic with several potential biomedical usages [19]. Moreover, it was also assessed for the presence of molecules active on tissue repair induction by using wound healing assays on mammalian cells.
