**2. High isostatic pressure and high pressure homogenization technologies**

The high isostatic pressure (HIP)—also known as high hydrostatic pressure or high pressure processing—and high pressure homogenization (HPH)—also called as ultra-high pressure homogenization or dynamic high pressure—are emerging process initially developed for food preservation by inactivation of microorganisms, with lower sensory and nutritional changes compared with the thermal process [12]. However, the studies of the consequences of these processes on food matrix highlighted that they were also able to induce changes on the food constituents, allowing the development of new applications and products/ingredients [13].

### **2.1. High isostatic pressure—principle and operation**

**1. Introduction**

50 Enzyme Inhibitors and Activators

mal energy content [1].

of enzymes in many processes.

for changes on the processed product.

for HIP and HPH.

Enzymes are globular proteins that catalyze biochemical reactions. This occurs due to the spatial configuration of the enzymes and catalytic site, which is determined by the quantity and sequence of amino acids and the organization of these chains, with folds and twists induced by attractions and repulsions among the near amino acids, resulting in a structure with mini-

The enzyme reaction occurs due to the interaction of the catalytic site and substrate, forming a complex enzyme-substrate following by the product formation [2]. The maximum velocity of this reaction occurs at specific pH, temperature and salt concentration and, at non-optimum conditions, the enzyme reaction is highly affected [3]. However, in many times, the desirable industrial process conditions are different of the optimum enzyme activity, making difficult the industrial application. Additionally, high costs and low stability limits the extensive use

Several chemical and physical methods were studied aiming to improve the performance of enzymes, withdrawing limitations and consequently increasing the range of application. Among the physical methods proposed, high isostatic pressure (HIP) and high pressure homogenization (HPH) are considered an important way to induce interesting changes on enzymes [4, 5]. HIP and HPH are emerging food processing technologies that involve the use of pressures up to 1000 MPa for HIP and up to 400 MPa for HPH to cause desirable changes in food and other products. The HIP process is based on the principle that the maintenance of a product inside vessels at high pressures induces changes in the molecules. To the contrary, for HPH process, the high shear and sudden pressure drop are the responsible phenomena

The overall evaluation of the results obtained for many authors shows that mild conditions of both processes were able to improve the activity and the stability of several enzymes, whereas extreme process conditions (pressure, time and temperature) induce enzyme denaturation with consequent reduction of biological activity [4–9]. Considering the complexity and diversity involved in the enzyme structure and its ability to react, it is not possible to determine specific conditions that each process is able to promote increase and reduction of enzyme

In terms of molecular structure, the effect of HIP and HPH on enzymes can be explained by the alterations in the quaternary, tertiary and secondary of enzymes, which directly affects the enzymes active site configuration, inducing exposure of hydrophobic amino acids, exposure of SH groups due to unfolding of the protein, a reduction in the total SH content due to new disulfide bonds formation and changes in the α-helix, β-sheet and β-turn ratio composition due to alterations of the secondary structure [4–6, 10, 11]. However, the occurrence of these phenomena—sequence of occurrence, intensity and required pressure—might be different

The impact of each process on enzymes was evaluated by few published revisions [4, 6, 12], however, no one dedicated to compare the effect of HIP and HPH on the main enzymes used

activity, being necessary to evaluate HIP and HPH on each enzyme.

The industrial application of HIP technology started in Japan in the early 1990s and has been gaining popularity and acceptance worldwide. Combining the interests of industry and consumers for these products, the development of new equipment with higher capacities helped to reduce costs and expand both the purchase intent of consumers (due to the perception of high quality products) as the search for new industries by new products, favoring the expansion of this technology [14].

The HIP is usually applied by subjecting the food, commonly vacuum sealed in flexible packaging, at a pressure up to 1000 MPa (10,000 bar), for a pre-defined time and at determined temperature to obtain the desired goal for each product. This process can be used to process liquid, semi-solid, or solid food. Equipment of higher volumes (687 L or 525 L) reaches pressures up to <310 MPa [15] or 600 MPa [16], respectively and temperatures up to <50°C, whereas equipment of lower volumes (<150 L) reaches temperatures up to 95°C and pressures up to 700 MPa [15]. The lab scale equipments reaching extreme pressures and temperature (900 MPa/110°C/chamber of 5L or 1400 MPa/110°C/chamber of 35 mL) [17]. During pressurization, the pressure is transferred instantaneously and evenly throughout the food (isostatic principle), regardless of the size and geometry of the product [18]. This is considered the main advantage of HIP processing, since there is no an equivalent to the so-called point of lower heating rate or "cold point" as in the case of heat conductive processes.

A typical system of HIP consists of a pressure chamber with closure and pressure generating system. Generally, it also have an apparatus coupled to the temperature control of the chamber. The batch process has three stages: the indirect pressurization using a liquid of low compressibility (e.g. water), the retention time at the desired pressure and depressurization. Semi-continuous processing can be obtained using multiple sequential chambers connected in series; while some cameras are under pressurization, others are being pressurized, unloaded, or loaded [14].

The pressurization is accompanied by a uniform temperature rise as consequence of the adiabatic heat of compression, being this specific to each compound [18]. For example, at 25°C, the water increases 3–5°C to every 100 MPa [18]. The adiabatic heating is completely reversed after the release of pressure. Although this temperature increase is relatively small, it can substantially contribute to the lethality of microorganisms in the overall process, resulting in significant implications when pressure is applied at elevated temperature [19]. On the other hand, this temperature increase can impact food structure, changing polysaccharides and proteins do to thermal effects. Therefore, when undesirable effects are observed due to adiabatic heating, the processes need to be carried out at lower temperature.

In molecular terms, the HIP breaks noncovalent bonds, such as ionic and hydrophobic bonds, but has little effect on covalent bonds. As a result, large biomolecules such as proteins and polysaccharides are affected by changes in its secondary, tertiary and quaternary structures (depending on the applied pressure), but small molecules are usually unaffected [6]. As the color components, flavor and vitamins are small molecules, the HIP process has little effect on these molecules in the food [6]. Furthermore, the process of pressurization followed the principle of "Le Chatelier," inducing a reduction in molecular volume and, consequently exponentially accelerating the occurrence of reactions favored by these conditions [20]. Thus, the rates of the chemical or physical reactions resulting in lower volume products are accelerated by the HIP, whereas the reactions that result in an increase in the total volume are retarded.

#### **2.2. High pressure homogenization–Principle and operation**

High pressure homogenization (HPH) is a nonthermal physical process applied for fluid foods [21]. This technology was introduced in the food field in the 1980s to improve the homogenization efficiency and emulsification of dairy products and emulsions, showing the same principle of operation of conventional homogenizers, however, using pressures around 10–15 times higher than usually applied, i.e., pressures up to 350 MPa [12].

In equipment, fluid is forced to pass through a homogenizing valve at high pressures [21]. The passage through the narrow gap (of micrometer order) and the abrupt decompression of the fluid generate an increase in speed (between 150 and 300 ms−1) [22] and an increase in temperature (about 1.5–2.5°C every 10 MPa pressure increase) due to the intense friction in the homogenizing valve region [12]. In addition to shear effects, the fluid undergoes an intense drop of pressure, turbulence and cavitation, what leads to microbiological inactivation and modification of the constituents of the food [12, 13, 21, 22].

The main changes on food constituents are related to disruption of lipids globules, reduction of molecular weight of linear polysaccharides and modification of the quaternary and tertiary structure of proteins [4, 5, 13]. For some applications, these effects are positive and, therefore, the HPH emerged as a suitable operation to improve the versatility of biomolecules (such as polysaccharides and proteins) as food ingredients [13].

An important drawback of HPH technology is the difficult to be industrially implemented to do the small flow capacity of the available equipment at high pressures. Nowadays, the industrial equipment operates at pressures up to 150 MPa with maximum flow rates of 5.000 L/h [23], while equipment that reaches higher pressures (up to 400 MPa) work at maximum flow of 240 L/h [17]. However, due to the high industrial interest, new equipment with higher capacity has been developed, allowing industrial application of this technology.
