**1. Introduction**

In the last two decades, there has been an increasing interest of the winemaking sector towards innovative technologies, particularly certain physical, non-thermal processing methods, able to improve process efficiency and reduce the use of chemical inputs. Ultrasounds (US), pulsed electric fields (PEF) or high hydrostatic pressures (HHP) are able to increase the mass transfer rate from grape skins [1], allowing polyphenols extraction and shortening certain wine production steps (e.g., skin maceration) [2]. In addition, they may decrease must and wine microbial populations, facilitating the reduction of sulfur dioxide use [3], an important goal of modern enology, due to the toxicity and allergenic potential of this additive [4]. Currently, in Europe, the use of these technologies are still not allowed at winery scale [5]. However, some of them, e.g., US or PEF, are in the course of evaluation by the International Organization of Vine and Wine (OIV) and presumably, they will be included soon in the OIV International Code of Oenological Practices [6].

Among these novel technologies, high pressure homogenization (HPH) is the least studied and only a limited number of papers have reported scientific results about its use for must and wine processing. Outside winemaking, HPH is utilized in several areas, such as chemical, pharmaceutical, biotechnology, and food industries. Emulsion, dispersion, encapsulation, and mixing are among the traditional reasons for employing homogenization operation during fluid food processing. However, beside these traditional functions, HPH is nowadays claimed to be one of the most promising novel non-thermal technologies that can be applied to improve fluid food safety and quality. It is a matter of fact that different potential applications of HPH have emerged in the last decades from both industrial

and scientific efforts. For this reason, considering the strong leaning towards innovation of modern enology, HPH may represent an interesting perspective for winemaking applications. Today several HPH devices are available for lab scale experiments but also ready for the scaling up at an industrial level to process and design novel foods with improved functionalities.

Based on these considerations, this review will critically discuss the possible applications of HPH in the wine sector, highlighting relevant results reported in literature, advantages as well as possible drawbacks of this technology, particularly considering its application in a rationale of reduction of chemical input and maximization of the quality of wine production.

#### **2. Basic Principles of High Pressure Homogenization and Process Design**

The term "homogenization" refers to the physical process allowing the reduction of the particle size of a polydisperse liquid system. The main result is an increased number of smaller particles of a narrow size range [7]. The most common installations used to this purpose are today based on valve technology: A fluid is forced to pass through the homogenization valve and the mechanical forces suffered by the product lead the disaggregation of particles. Therefore, the total surface area of newly formed particles increases, leading to a significant improvement of product physical stability. Standard homogenization at pressure levels around 20–50 MPa is widely used in chemical, pharmaceutical, biotechnology, and food industries, mainly to reduce the particle size of liquid dispersions, preventing phase separation during storage.

The current development of homogenizers have allowed much higher pressures leading to the development of high pressure homogenization (HPH) technology. This technology is based on the same principle of conventional homogenization, but it works at significantly higher pressures, up to 400 MPa. Depending on the nominal pressure level, the technology is called high pressure homogenization (HPH, up to 150–200 MPa) or ultra-high pressure homogenization (UHPH, up to 350–400 MPa) [8]. Basically, during the HPH process, the fluid is forced to pass through a narrow gap in the homogenizer valve, where it is submitted to a rapid acceleration [9]. Therefore, the suspended materials in the fluid are subjected to grea<sup>t</sup> mechanical forces and elongation stresses, becoming twisted, deformed, and disrupted [9].

The design of the homogenization valve and its geometry is of a special importance to generate the desired effect on processed fluid. Homogenizers are usually equipped with a high pressure homogenization valve that must resist to the intense mechanical stresses generated during the fluid passage and a low-pressure valve allowing stresses reduction before fluid exit (Figure 1). Nowadays, there is a number of commercially available valves with different geometries and made of different materials, as extensively described by Martinez-Monteagudo et al. [10]. Basically, these authors classified HPH valves into counter-jet, radial diffusers, and axial flow valves.

It should not be underestimated that independently on valve geometry, the liquid travelling through the HPH valve is accompanied by intense heating phenomena, causing the liquid temperature increase. The fluid temperature increases linearly with the pressure by 14–18 ◦C per 100 MPa due to shear effects and the conversion of mechanical forces into heat [8]. The temperature changes during HPH should be carefully taken into account during process design to avoid possible undesired thermal damages to the product or, contrarily, to exploit temperature as additional factor, beside pressure, to generate the desired changes to the product.

The treatment intensity and relevant effects can be modulated by applying different operative pressures and number of passes at the selected pressure. To quantify the process intensity, the energy density (E*<sup>v</sup>*, MJ/m3) transferred from the homogenization valve to the sample can be determined as described by Stang et al. [11] and Calligaris et al. [12]:

$$E\_{\upsilon} = \Delta P \cdot \mathbf{n} \tag{1}$$

where Δ*P* is the pressure difference operating at the nozzles and *n* the number of passes at the selected pressure. Basically, process intensity is directly related to the homogenization pressure and number of consecutive passes through the valve.

**Figure 1.** Scheme of a two-stage high pressure homogenizer. Modified From Plazzotta and Manzocco [13].

#### **3. Possible Applications of HPH in the Food Sector**

The intense mechanical stresses suffered by the product during the HPH process can induce different modifications in food constituents. The most widely studied effect of this technology is the cell disruption capacity to induce microbial inactivation and facilitate the removal of intracellular components. However, more recently, HPH has been proposed as an efficient tool to steer the structure and functionality of food biopolymers. For this reason, different food sectors are interested in understanding the feasibility of this technology for specific applications. Table 1 shows the main possible uses of HPH technology in the food sector and relevant recent reviews on this topic. Beside these specific papers, other general reviews have also been published on HPH and UHPH [8,10,14,15]. Below, a brief summary of the main possible application of HPH on fluid foods and beverages is reported.

**Table 1.** Recent reviews on specific applications of high pressure homogenization.


#### *3.1. Emulsification and Nanoemulsification*

The droplet size reduction generated by the application of a homogenization process is of key importance in the production of stable emulsion and, thus, to obtain the desired quality of the final product. The possibility of increasing the pressure level from conventional homogenization to higher pressures has favored the application of this technology to produce nanoemulsions that are heterogeneous systems containing particles with droplets diameter lower than 200 nm [16,17]. During a single pass process, a progressive size reduction can be obtained by increasing the homogenization pressure. However, up to a certain pressure level, which depends on the plant design and emulsion formulation, particle size reduction is no longer expected [8,12,25–27]. Multiple passes through the homogenization valve are eventually applied to further reduce not only the mean particle diameter but also the width of the particle size distribution, improving emulsion stability against coalescence [12,25,26].

## *3.2. Microbial Inactivation*

The majority of the studies deals with the possibility of using HPH to disrupt microbial cells and thus as an alternative technology to conventional heat-treatments [8,19,28]. Good results have been obtained for HPH inactivation of microorganisms and foodborne pathogens present in di fferent food substrates. The capacity of HPH to induce microbial inactivation has been attributed to the high pressure, velocity gradients, shear stresses, turbulence, shocks, and cavitation phenomena occurring during the passage through the homogenization valve. These stresses induce cell membrane permeabilization, followed by the deformation of the cell structure and cytoplasmatic organelles, and leaking of intracellular materials. Moreover, the temperature increase su ffered by the fluid during the process could improve the microbial inactivation e fficacy of HPH [19]. Indeed, additional or synergy effects between mechanical forces and heat have been described by di fferent authors at temperatures higher than 60 ◦C [28–30].

In general, gram-positive bacteria show a higher resistance to HPH than gram-negative ones [31,32]. This e ffect was associated to the thinner cell walls membrane of gram-negative bacteria in comparison to gram-positive. Yeast and fungi exhibited resistance to HPH, intermediate between gram-negative and gram-positive bacteria. In fact, even if they have a thicker membrane than gram-positive bacteria, their larger size as well as di fferent cell wall composition, rich in glucans, mannans and proteins, reduced their resistance to the HPH stresses [33,34]. Finally, spores are the most resistant to high pressure homogenization and their inactivation by HPH or UHPH is still a challenge. Literature data have pointed out that spore inactivation can be obtained only by defining a proper combination of pressure, temperature, and additional chemical hurdles [35–37].

It can be concluded from literature that HPH can be reliably applied for microbial inactivation, and has been proposed as alternative to food pasteurization or, in some cases, sterilization [15]. Promising results have been obtained with cow milk [30], vegetable milks [38], and fruit juices [28,39,40]. Overall, the reduction of microbial count is pressure dependent, showing better inactivation as homogenization pressure increased. However, other process parameters such as pressure level, inlet temperature of processed fluid, number of recycling passes, should be accurately defined, taking into account not only the target microbial strain and level of inactivation but also food characteristics in term of chemical composition and physical properties.

#### *3.3. Cell Disruption and Recovery of Intracellular Components*

Mechanical cell disruption by HPH is routinely exploited in pharmaceutical and biotechnological industries to disrupt bacteria and yeas<sup>t</sup> in the attempt to recovery bioproducts [22]. Today it is claimed to be an e fficient tool for cell disruption also in the food sector and it can be potentially exploited as pre-treatment to facilitate the recovery of valuable components from vegetable wasted material [13,41,42] or from microalgae [21,43]. The cell disruption degree is reported to be highly dependent on matrix characteristics and HPH intensity in term of operating pressure and number of passes through the homogenization valve [44,45].

#### *3.4. Physical and Physical-Chemical Modifications of Food Biopolymers*

Beside the direct e ffect of HPH on cells, a wide number of papers deal with the possibility of applying HPH treatments to modify the physical and physical–chemical characteristics of food components, mainly proteins and polysaccharides.

Regarding proteins, it has been reported that protein structure can be modified during the intense mechanical stresses su ffered by the product passing through the homogenization valve. Because of protein conformational changes induced by HPH, protein functionalities, in terms of solubility, emulsifying, gelling, or foaming capacity, could deeply change. HPH treatments were able to affect the physicochemical properties of a number of proteins including milk proteins [46], soybean proteins [47,48], myofibrillar proteins [49], peanut proteins [41], mussel proteins [50], and egg white proteins [51].

Similarly, HPH can induce severe changes in polysaccharides dispersions, such as mass, hydrodynamic radius, and viscosity. On this regards, studies on carboxymethlycellulose, guar gum, hydroxymethylcellulose [52], starch [53], methylcellulose [54], inulin [55], and pectin [44] are available in literature.
