**1. Introduction**

Nowadays, consumers show increasing preference for foods with additional or enhanced benefits beyond their basic nutritional value. These benefits come from the composition, e.g., bioactive compounds, which may have long-term health effects. There is convincing evidence of the cardioprotective effects for frequent consumption of fruits and vegetables high in fiber, micronutrients, and several phytochemicals. Specifically, the association between flavonoids and an increased cardiovascular health has been proven in berries [1]. Producing berry-based juice generates by-products, comprising peel and seeds, having a high nutritional value because of their polyphenol and fiber content. Berry by-products can be a value-added food ingredient [2–4] and recent studies show that the enrichment of food products with these by-products is feasible [5–7]. In this context, chokeberry (*Aronia melanocarpa*) pomace can be used, as chokeberry is one of the richest plant sources of phenolic phytochemicals, including procyanidins and anthocyanins [8,9] which are related to effectiveness in several pathological conditions where damage is caused by uncontrolled oxidative processes [10].

Previous studies have used dairy products and pomace for the production of yogurts with apple pomace [11–13] or fermented milk with grape pomace [14]. However, in the work of Issar et al. [11] and de Souza et al. [14], polyphenols and fiber were extracted, respectively, and added to milk for product preparation. Wang et al. [12,13] incorporated the apple pomace directly into the dairy matrix. Regarding berry pomace, Ni et al. [15] formulated yogurts with aqueous berry extracts from salal berry

and blackcurrant pomace. In this study, we propose the incorporation of the pomace directly into the milk using high hydrostatic pressure (HPP) to help polyphenols being extracted into the matrix.

HPP is a method to preserve food and has the potential to retain or improve the bioaccessibility and bioavailability of nutritional and antioxidant compounds because of microstructural modifications [16]. HPP retains the nutritional and sensory quality of food products, but there is a concern related to food safety [17]. In this context, high pressures have been effective at inactivating vegetative cells when sufficient intense pressure is applied [18].

Thus, the present study aimed to prepare milkshakes enriched with polyphenols by adding chokeberry pomace to the milk and using HPP to improve polyphenols extraction from the pomace. The effect of high HPP parameters such as time and pressure on total phenolic content (TPC), antioxidant capacity (AC), and the microbiological inactivation in milkshakes with different concentrations of chokeberry pomace will be studied. To define the best processing conditions, response surface methodology (RSM) was used to maximize the TPC and the AC results while minimizing the microbiological survival.

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

#### *2.1. Sample Preparation and HPP Treatments*

Döhler GmbH (Darmstadt, Germany) provided fresh chokeberry pomace. The pomace was dried at 70 ◦C for 3 h and milled in a ZM 100 ultracentrifuge mill (Retsch GmbH, Haan, Germany) at 14,000 r.p.m. using a 0.5 mm sieve [19]. Reconstituted skimmed milk powder (Corporación Alimentaria Peñasanta S.A., Siero, Spain) was selected for chokeberry pomace inclusion.

Different concentrations of chokeberry powder were added to skimmed milk samples to give final chokeberry pomace concentrations of 2.5%, 6.25%, and 10% (*w*/*v*). The samples were poured into 50 mL polypropylene tubes that were introduced into polyethylene bags and heat-sealed (MULTIVAC Thermosealer, Switzerland) before undergoing HPP treatment. HPP treatments were performed in a unit with a 2.35 L vessel volume and maximum operating pressure of 600 MPa (High Pressure Food Processor, EPSI NV, Belgium). The samples were pressurized at 200, 350, and 500 MPa, at 18–22 ◦C, for 1, 5.5, and 10 min, using a compression rate of 300 MPa/min and a decompression time < 1 min, [20,21]. Other parameters, pressure intensity, pressurization time, and temperature were automatically controlled. Once the treatment was completed, the samples were taken from the vessel, immersed in an ice-water bath, and refrigerated (3 ± 1 ◦C) until use. Before each analysis, both microbiological and chemical, the samples were filtered with paper filter previously sterilized using an autoclave. The microbiological cultures and microscopic observations were immediately conducted after the filtration while the samples for the TPC and AC determination were stored by deep-freezing at −80 ◦C until use.
