*3.5. Enzyme Inactivation*

Since as previously reported, HPH can modify protein structure, this technology has been proposed as tool to reduce the activity of indigenous enzymes. According to literature, HPH can increase or decrease enzyme activity depending on the enzyme source, processing conditions as well as matrix characteristics [24]. The modification of enzyme activity has been attributed to the changes of protein native structure associated to the mechanical forces and temperature increase during HPH process. In fact, protein unfolding and dissociation of native oligomers can be obtained because of pressurization, leading to changes of enzyme activity [48]. In agreemen<sup>t</sup> with literature, it can be said that complete enzyme inactivation can be di fficult to reach by single pass homogenization, even at the highest homogenization pressures during UHPH. The complete inactivation can be achieved only by increasing temperature and/or the number of recycling passes [24,56].

#### **4. Potential Use of HPH in Winemaking**

The first attempts of using high pressure technologies for must and wine processing date back to the 1990s. Hyperbaric treatment initially consisted of applying high pressure processing in hydrostatic conditions [57]. Nowadays, the e ffects of high hydrostatic pressure on the extraction of color and phenolic compounds from grapes [1,2,58,59], as well as on the inactivation of wild microorganisms in grape, juice, and wine [2,3,57,59–61], are well known.

Nevertheless, compared with high hydrostatic pressure (HHP), high pressure homogenization (HPH) is based on di fferent principles. In fact, HHP is a static batch processing technique, in which the products are pre-packaged and introduced into a pressurized chamber for a given time [62]. In contrast, HPH is a dynamic high pressure technology and the modifications it induces on plant tissues and microbial cells do not depend on the application of pressurization alone, since they are also affected by other physical phenomena such as cavitation, turbulence, and shear, into the homogenizing valve (Sections 2 and 3). For this reason, HPH is not suitable for the processing of grape mash after crushing–destemming, because of the presence of skins and seeds, which may clog the valves of the homogenizer. However, compared to HHP, it allows continuous in-flow processing and, after a preliminary preparation of the fluid (must or wine) by the elimination of skin fragments, seeds, crystals, and other solid particles, it may be more suitable for the treatment of musts and wines in the volumes normally found on a winery scale.

In the following paragraphs, the most interesting applications of HPH technology for winemaking use will be discussed. They are the inactivation of spoilage microorganisms in grape juice and wine, the acceleration of the yeas<sup>t</sup> autolytic process and the production of yeas<sup>t</sup> derivative preparations to be used as processing aids.

#### *4.1. Control of Microbial Populations in Grape Juice and Wine*

The e ffect of high (hydrostatic) pressures on wine microbial populations is well known and it has been reported since 1995 [57]; thirteen wine yeasts species including *Lactobacillus* spp., *Oenococcus oeni*, *Acetobacter* spp., and *Botrytis cinerea*, added at 10<sup>6</sup> CFU/mL to a Moscato wine, were inactivated in 2 min by a HHP treatment at 400 MPa. The positive e ffect of the application of high pressure in static conditions on the inactivation of wine microorganisms was further confirmed in the following years [63]. Mok and co-workers [61] found that total yeasts (2.9 × 10<sup>5</sup> CFU/mL) were completely eliminated from red wine in 30 min at 300 MPa and in 10 min at 350 MPa; at the latter pressure value, lactic acid bacteria (LAB, 2.9 × 10<sup>5</sup> CFU/mL) were destroyed in 5 min. More recently, Morata and colleagues [2] reported significant reduction of yeasts (up to 4 Log units), total aerobic bacteria, and LAB (up to approx. 1 Log unit) in crushed Tempranillo grapes treated by HHP (200, 400, and 550 MPa).

Despite these positive results and the advantage (compared with HHP) of the possibility of using homogenization for continuous in-flow processing, HPH has been poorly exploited for must and wine treatment. Recently, Loira et al. [64] tested UHPH on white must, in comparison with sulfiting (SO2 35 mg/L) and untreated juice. UHPH (300 MPa) determined the complete elimination of wild yeasts (initial load 1 × 10<sup>6</sup> CFU/mL) from the treated samples, while the microbial load of the sulfited must was not significantly changed with respect to the untreated control juice. In addition, UHPH-processed must, stored at 18 ◦C without yeas<sup>t</sup> inoculation, showed the absence of fermentation for eight days. The same trend observed for yeas<sup>t</sup> was also found for wild bacteria (LAB and aerobic bacteria): UHPH treatment (300 MPa) reduced below the limit of detection (LOD 1 CFU/mL) the initial load of 7 × 10<sup>3</sup> CFU/mL [64]. It is interesting to observe that the extent of heating connected with UHPH treatment reported in this study is extremely low (inlet homogenizer temperature: 20 ◦C; outlet temperature: 25 ◦C).

Puig et al. [65] tested HPH for reducing indigenous flora in Parellada and Trepat musts. Results showed that HPH treatment (200 MPa) was able to completely eliminate LAB (3 and 5 Log units reduction, respectively, in Parellada and Trepat), fungi and yeasts (3 and 6 Log units reduction, respectively, in Parellada and Trepat) in both musts, and only a limited residual population of total bacteria (other than LAB) were detected.

Using lower pressure values, Comuzzo and colleagues [66] found less evident results applying HPH on *Saccharomyces bayanus*; an active dry yeas<sup>t</sup> preparation (ADY) was rehydrated, processed by 1–10 passes at 150 MPa and freeze-dried. HPH was carried out in conditions of uncontrolled and controlled temperature regimes, by positioning a heat exchanger at the homogenizer outlet. Results showed that the initial load of the ADY preparation (approximately 10 Log CFU/g) decreased as the number of passes increased, but if the temperature was controlled (Tout, at homogenizer outlet 32 ◦C, in conditions of the controlled temperature regime), the maximum decrease of total yeas<sup>t</sup> population was lower than 4 Log CFU/mL. In contrast, a temperature increase, in the conditions of uncontrolled heating (without heat exchanger), gave an important contribution: Yeast viability was reduced at 1.9 Log units after the 6th pass (Tout 70 ◦C) and below LOD (10 CFU/mL) after the 10th pass (Tout 74 ◦C).

The intensity of the pressure applied may also influence the e fficiency of HPH in eliminating wine yeasts. The same authors [67] reported that the inactivation of a rehydrated *S. bayanus* commercial active yeas<sup>t</sup> strain increased linearly by increasing the pressure, despite that the maximum pressure applied (150 MPa) provoked only a diminution of approximately 2 Log units (Table 2).


**Table 2.** Application of HPH (50–150 MPa) to a water suspension of *S. bayanus* active dry yeas<sup>t</sup> (ADY); effect on yeas<sup>t</sup> viability. Extracted from Comuzzo et al., 2015—Modified [67].

> 1 ADY: Active Dry Yeast; 2 analyzed after HPH and freeze-drying.

The e ffects of HPH on microorganisms of enological interest are evident also in other papers related to fruit juices or other fermented beverages: *S. bayanus* inactivation in apple juice [37]; *S. cerevisiae* and *Lactobacillus plantarum* in orange juice [68]; *S. cerevisiae* and *Lactobacillus delbrueckii* in orange, apple, and pineapple juice [40]; different strains of *Lactobacillus* spp., *Pediococcus* spp., *Acetobacter aceti*, and *S. ludwigii* in beer [69]; and total yeasts in rice wine [70].

Generally speaking, pressures higher than 250 MPa allow a complete inactivation of microorganisms [68]; contrarily, lower pressure values generally require multi-pass processing [40,69], or the combination of mechanical forces and heating [69].

#### *4.2. Acceleration of Yeast Autolysis and Ageing on Lees*

Autolysis is the self-degradation of yeas<sup>t</sup> cell constituents that begins after cell death, promoted by the lytic activity of cellular enzymes [71]. Autolytic phenomena are fundamental during ageing on the lees (*élevage sur lies*), an important technological tool for the production of certain wine typologies, such as white wines from Burgundy or sparkling wines produced by the traditional method (e.g., Champagne, Cava or Franciacorta).

Despite different papers highlighted the possibility of using homogenization for promoting the extraction of intracellular components from *Saccharomyces* cells [22,72–74], very few publications report original data concerning the ability of HPH to accelerate yeas<sup>t</sup> autolysis in wine-like media.

The first evidence about the possibility to use HPH for this purpose was reported by Patrignani et al.[75]. The authors applied HPH treatment (90 MPa) on different yeas<sup>t</sup> strains (*S. cerevisiae* and *S. bayanus*) prior to their use for the preparation of *tirage* solutions, for sparkling wine refermentation (traditional method). The treatment poorly affected yeas<sup>t</sup> viability and refermentation behavior (all the strains allowed to reach a final overpressure of approximately 6 bars), but scanning electron microscopy highlighted that HPH provoked an acceleration of autolysis over a 40 day ageing period. The authors hypothesized that HPH might presumably activate the enzymatic pool involved in autolytic process; it is interesting to observe that temperature was controlled during the experiment: Inlet temperature was 25 ◦C and the samples were immediately cooled to 3 ◦C after the treatment. In such a way, the influence of temperature can be excluded.

The effect of pressure and number of passes on the extraction of intracellular components from *Saccharomyces* cells was described also in other publications [22,76]: The release of ionic compounds, proteins, and other bio-active compounds significantly increased by increasing these two operating parameters.

The possibility to accelerate yeas<sup>t</sup> autolysis is an interesting perspective in winemaking practice. In fact, it is well known that prolonged ageing on the lees may increase the risk of microbial spoilage and production of unwanted metabolites, such as biogenic amines [77]. This risk might be further reduced by HPH treatment, due also to the ability of such technology to reduce wild microorganisms and LAB (Section 4.1).

An interesting approach for managing ageing on lees through HPH technology was described by Carrano [78]; fresh lees were treated at 60 and 150 MPa (single pass) and added to a white wine for ageing on lees. HPH increased the ability of the treated lees to release glucidic colloids in model wine, also determining a significant reduction of viable yeasts and LAB (Table 3). This approach may potentially allow the reduction of the use of sulfur dioxide during ageing on lees, when HPH-processed lees are re-incorporated into the wine.

**Table 3.** Application of HPH (60 and 150 MPa) on fresh lees; effect on yeas<sup>t</sup> and LAB populations. Extracted from Carrano, 2016—Modified [78].


1 *n.d.*: not detected (<10 CFU/mL); 2 measured at homogenizer outlet.

#### *4.3. Production of Yeast Derivative Preparations for Enological Use*

Yeast derivatives (YDs, inactive dry yeasts, and yeas<sup>t</sup> autolysates) are processing aids largely used in the wineries for several purposes, such as nutrients for yeasts or LAB starter cultures or colloidal supplements during wine ageing [79]. They are produced from *Saccharomyces* spp. by natural autolysis (i.e., through the action of endogenous enzymes), combined with heat treatment and/or modification of the pH [80]. Given their mode of preparation, the production of YDs may be considered as a special case of managemen<sup>t</sup> of the autolysis process; for this reason, HPH might also be exploited for the industrial manufacture of these products.

Comuzzo and colleagues [66,67] studied the potential use of HPH for the production of yeas<sup>t</sup> autolysates for winemaking. HPH was able to increase the ability of an ADY preparation of *S. bayanus* to release glucidic colloids, proteins, and amino acids in wine-like solution. This effect was proportional to the pressure applied (0–150 MPa) [67] (Table 4).

**Table 4.** Application of HPH (50–150 MPa) to a water suspension of *S. bayanus* active dry yeas<sup>t</sup> (ADY); effect on the release of proteins and glucidic colloids in wine-like solution. Extracted from Comuzzo et al., 2015—Modified [67].


1 ADY: Active Dry Yeast; 2 Amount released in wine-like solution (pH 3.2, ethanol 12% *v*/*v*) by one gram of ADY or HPH-processed yeas<sup>t</sup> after freeze-drying.

Moreover, the number of passes (1–10) and processing temperature affected the composition of the autolysates and the release of soluble compounds in model wine [66]: lower temperatures, led to higher concentrations of soluble amino acids and proteins, while heating (in conditions of uncontrolled temperature regime—Tout 74 ◦C) provoked a decrease of the amounts of these two groups of molecules. The authors have suggested that HPH processing conditions might be differently set up for tailoring the characteristics of the autolysate, making them suitable for different applications during wine production. For instance, for obtaining a nitrogen supplement for alcoholic fermentation, processing temperature must be controlled to maximize the content of free amino acids useful as nutrient for yeas<sup>t</sup> growth. In contrast, if a colloidal supplement is needed during wine ageing (e.g., for improving wine mouthfeel characteristics), processing temperature could be kept higher, with the advantage of reducing amino acid content and improving microbiological stability during wine storage and ageing [66].

Finally, concerning the possible advantage of using HPH for the production of YDs, it is well known that such kind of products may sometimes negatively affect wine volatile composition, due to the release of exogenous aroma compounds in wine [81,82]. The aroma composition of HPH-processed yeas<sup>t</sup> autolysates differs to that of the products obtained by conventional thermolysis methods, because of a higher concentration of ethyl esters and lower amounts of short-chain fatty acids and carbonyl compounds [67]. The latter are probably connected with lipid fraction and its oxidation, and previous experiments have highlighted that they can be released into the wine, affecting its sensory characters [81].

#### *4.4. Modifications Induced on Wine*

Few data are available about the modifications induced by high pressures on wine characteristics. Such modifications are mainly connected with the thermal stress suffered by the product during hyperbaric treatment. For this reason, high pressure treatments, both static processing (HHP) and

homogenization (HPH), were mainly tested on grapes [2,60], musts [57,64,65], grape by-products [1], or lees [78].

The opportunity to control processing temperature by placing a heat exchanger at the homogenizer outlet might reduce the thermal damage to the product. Keeping temperature below 25 ◦C, the few papers available report that pressurization had a minor impact on wine color and sensory characteristics, both in static [61] and homogenization conditions [65]. At the same maximum processing temperature, Loira et al. [64] found that the white wines obtained from the fermentation of UHPH-processed musts were more fruity and with better aroma than the control (obtained by spontaneous fermentation) and sulfited samples (inoculated with the same *S. cerevisiae* strain as UHPH-treated must). However, UHPH processing of must led to wines with a higher color intensity in comparison with sulfiting (35 mg/<sup>L</sup> SO2).

Apart from the effects of temperature, pressurization alone may also influence wine characteristics. Santos and co-workers [83] analyzed the composition of HHP-processed red wines (500 MPa, 5 min, 20 ◦C), in comparison with a control (stored in stainless steel vats) and with the same wine aged in oak barrels, in stainless steel vats with oak chips, and in stainless steel vats with oak chips plus microoxygenation. After a storage period of 5 months, pressurized wines showed a lower content of monomeric anthocyanins, phenolic acids, and flavonols in comparison with the other wine treatments; in contrast, HHP promoted wine evolution leading to a higher degree of tannin polymerization and pyranoanthocyanins concentration, similar to those found in the samples obtained with microoxygenation and wood contact.

Talcott and co-workers investigated the effects of thermal treatment and HHP (600 MPa for 15 min) on the color and phytochemical stability of Muscadine grape juice, in combination with ascorbic acid and rosemary extract. HHP determined a slight loss of juice color and antioxidant activity with respect to control and thermally-treated samples, and this effect was proportional to the concentrations of added ascorbic acid and rosemary extract [84]. The authors report that the greater loss obtained after HHP processing was likely due to residual activity of polyphenoloxidase enzymes.

Other papers have investigated the effects of high pressure processing (HHP) in comparison with sulfiting and a control wine produced with no preservation treatment (SO2-free) [85,86]. Pressurized wines developed a more brownish color and a slightly lower antioxidant activity during one year of storage [86]. In addition, high pressure processing determined a decreased content of free amino acids and a higher concentration of volatile furans, ketones, and aldehydes [85,86], symptoms of a greater extent of Maillard reaction in HHP processed wines.

Based on the evidence, it is clear that most of the data available about the effects of high pressure treatments on wine composition is related to HHP, while, to the best of the authors' knowledge, very few scientific publications have regarded HPH. Although the possibility of controlling processing temperature may allow the application of HPH to the wines, minimizing thermal damage, there are still too few data concerning the modifications induced by the treatment on the compositional and sensory characteristics of wine itself. For this reason, further experiments need to be carried out, considering a major number of processing conditions, analytical parameters, and wine varieties.

#### *4.5. Other Potential Applications and Perspectives of HPH in Wine*

High pressure treatments, and HPH in particular, are interesting techniques which might be tested for different winemaking applications, not only for those discussed above.

For instance, the ability of HPH to induce protein unfolding and enzyme inactivation (Sections 3.4 and 3.5) might be exploited for must and wine protein stabilization, as well as for polyphenoloxidase inactivation in grape juice.

Furthermore, when applied on must, HPH has demonstrated to be a suitable technique to increase the dominance of commercial non-*Saccharomyces* yeas<sup>t</sup> strains, when such microorganisms are used in sequential inoculation with *S. cerevisiae* [60,64,65]. This represents an innovative approach in managing wine alcoholic fermentation, reducing the competition by wild microorganisms and allowing the

reduction of sulfur dioxide addition. Moreover, this opportunity may be particularly interesting in some specific enological sectors, such as organic winemaking, as well as for the production of SO2-free wines. Concerning fermentations, HPH and UHPH might also be tested for controlling malolactic fermentation during wine storage, as well as for the inactivation of *Brettanomyces* before barrel ageing.

Finally, it is interesting to mention the work of Serrazanetti et al. [87], who found that HPH a ffects membrane fatty acid composition by increasing the percentage of unsaturated fatty acids (UFA) when compared with saturated fatty acids (SFA). The role of UFA as a survival factor for yeas<sup>t</sup> metabolism is well known [4] and this observation might open new perspectives in managing yeas<sup>t</sup> nutrition and pre-fermentative operations.
