Effect of High-Pressure Technologies on Enzymes: Science and Applications

Effect of High-Pressure Technologies on Enzyme: Science and Applications provides a deep, practical discussion of high-pressure processing (HPP) and high-pressure homogenization (HPH) technologies and biochemical approaches, applied across research and industry, with applications ranging from food to pharmaceuticals and commercial enzyme production. Early chapters discuss foundational aspects of HPP and HPH approaches; the science of enzyme modification; and basic aspects of enzyme activity, stability, and structure as studied in biochemical processes. Later chapters consider the effect of HPP and HPH technologies and their mechanisms of controlling enzyme modification to improve enzyme performance for chosen applications. Special attention is paid to the application of HPP and HPH technologies and enzyme modifications in food processing, microbial enzyme modification, drug discovery, and production of other commercial enzymes, as well as the challenges of undesirable enzyme inactivation. The final chapter discusses future directions of the field and technologies, and expanded applications.

• Offers a broad overview of HPP and HPH approaches and technologies applied in enzyme modification
• Introduces fundamental aspects of enzyme activity, stability, and structure as studied in biochemical processes and applications
• Discusses applications of HPP- and HPH-based enzyme modifications in food processing, microbial enzyme modification, drug discovery, and production of other commercial enzymes
• Includes chapter contributions from international leaders in the field, across research and industry

• Language: English
• Publisher: Academic Press
• Release date: Feb 6, 2023
• ISBN: 9780323985826

Book preview

Preface

High-pressure technologies, including high-pressure processing (HPP)—also called high hydrostatic pressure—and high-pressure homogenization (HPH), are nonconventional technologies developed for food processing with better nutrient retention and sensory characteristics of the raw material. However, in the last 15 years, these technologies began to be explored to cover new issues, such as effects on enzymes and improvements in food structure. Some book chapters and review articles (published up to 2017) partially covered innovations regarding the effects of high-pressure technologies on enzymes, but a complete and up-to-date collection is needed to guide researchers and industry in the use of these technologies with a focus on enzyme modifications.

To fill this gap, our book will provide a detailed overview of high pressure technologies on enzymes, describing the effect of these technologies on enzyme activity, stability, and structure.

The book will be divided into five parts, which will address:

Part 1: Introduction: It will cover general aspects of the technologies and more superficial information about their effects on enzymes. This part will help readers understand the organization of the collection and will be useful for new researchers on this subject.

Part 2: Effect of high-pressure technologies on the structure, activity, stability, and kinetic parameters of enzymes: This part will describe in detail the effect of both technologies on the activity, stability, and structure of the enzymes as well as the use of high pressure as a tool to improve enzyme performance and the combination of high pressure with other technologies for modifying enzymes. Thus, this part is important to elucidate the effect of the processes on the characteristics of enzymes, providing a knowledge base for understanding the next chapters.

Part 3: Effects of high-pressure technologies on enzyme activity in different food matrices: This part will discuss the effect of technologies on enzyme activity in different food matrices, showing how the food matrix impacts the effect of HPP or HPH on enzymes and that it must be assessed on a case-by-case basis. This part will allow the audience to find specific information about the effect of each process according to their interest.

Part 4: Applications of high-pressure technologies on enzymes used in nonfood processing applications: This part will show the applications of high-pressure technologies to promote the modification of microbial or commercial enzymes. This part will help readers expand their knowledge about the possibilities of using these technologies in enzymes of commercial interest in nonfood processes.

Part 5: Future challenges of using high-pressure technologies on enzymes: This part will discuss the future of high-pressure technologies on enzymes, describing to the readers the main challenges and perspectives to guide them in future research.

Chapter 1: Use of high-pressure technologies on enzymes

Isabela Soares Magalhãesa; Alline Artigiani Lima Tribstb; Bruno Ricardo de Castro Leite Júniora    a Department of Food Technology (DTA), Federal University of Viçosa (UFV), University Campus, Viçosa, MG, Brazil

b Center for Food Studies and Research (NEPA), University of Campinas (UNICAMP), Campinas, SP, Brazil

Abstract

The first studies of nonconventional technologies for food processing were commonly focused on microbial and enzymatic inactivation. However, with the development of technology and advances in research, new possibilities have emerged. In the enzymatic field, the application of high-pressure processing (HPP) and high-pressure homogenization (HPH) began with a focus on the inactivation of deleterious enzymes (such as pectinases and polyphenol oxidase). In recent decades, new research has proposed the application of both technologies to activate or inactivate different enzymes with diverse commercial interests, including the application of HPP to assist enzyme reactions. In this context, this chapter presents a description of HPH and HPP principles and equipment. In sequence, it presents the general aspects of enzymes and the involved mechanisms of both technologies that promote the activation, inactivation, and stabilization of enzymes. In addition, the impacts of process parameters, food matrix, and/or enzymatic dilution were addressed. Finally, the chapter describes the industrial prospects and future challenges of these technologies for enzyme alterations.

Keywords

Emerging technologies; High-pressure technologies; Food processing; Enzymes; Enzymatic reactions; Enzyme activation

1.1: Introduction

Currently, the consumer demand for processed food products is changing. Although microbiological and chemical safety remains the main important characteristics¹ for processed food, consumers are also looking for products with better preservation of nutrients and sensory profiles, such as fresh ones, clean labels, eco-friendly, and reasonable costs.¹–³

For the food industry, the development of innovative processing is the best way to address these complex demands.⁴ Among these technologies, those involving pressure application (high-pressure processing and high-pressure homogenization) are highlighted by their inability to cleave covalent bonds, achieving microbial inactivation with preservation of most flavor compounds and micronutrients.¹,⁴–⁷ In addition, the process is considered more sustainable than traditional thermal processing, as it does not rely on the use of nonrenewable fuel.²

The first studies of innovative technologies for food processing were commonly focused on microbial inactivation. However, with technology development, processing targets are diversified. Among them, it is possible to highlight the use of technology to inactivate undesirable enzymes and antinutritional factors⁸–¹⁰ and to intentionally change food components or food ingredients to improve texture, color, water-holding capacity, and product yield, minimizing the use of additives and allowing product development.⁴,⁷,⁸,¹¹

Regarding the application of high-pressure processing (HPP) and high-pressure homogenization (HPH) on enzymes, the first studies focused on the inactivation of deleterious enzymes, such as pectinases and polyphenol oxidase, which are able to reduce the stability in processed vegetable products.¹²,¹³ In the last 15 years, new research has proposed the application of both technologies to activate or inactivate different enzymes with diverse commercial interests, including the application of HPP to assist enzyme reactions.¹⁴–¹⁸

Concerning both technologies, the HPP is in the final step of development, with an exponential availability of industrial equipment and food processed by this technology worldwide.¹⁹ On the other hand, HPH can be considered an underdeveloped technology due to the low production capacity (volume/hour) and relatively low operating pressure (insufficient to guarantee microbial inactivation) of most commercial equipment.²⁰ Therefore, current industrial application of HPH focuses on the improvement of the characteristics of some food ingredients and the extraction of compounds of interest, mainly in biotechnological production.²¹

The present chapter starts with a description of HPH and HPP principles and equipment. General aspects of enzymes and the mechanisms of both technologies involved in the activation, inactivation, and stabilization of enzymes are also discussed. Furthermore, the effects of processing parameters, food matrix, and/or enzyme dilution are addressed, and with this information, strategies are proposed to use these technologies to activate/inactivate enzymes of interest. Finally, the chapter describes the industrial perspectives and future challenges of these technologies for enzyme changes.

1.2: High-pressure processing

High-pressure processing (HPP), also known as high isostatic pressure or high hydrostatic pressure processing, is a nonthermal processing firstly developed to guarantee microbiological food safety with minimum impact on heat-sensitive compounds.⁷,⁸

In HPP, the processing variables are pressure, temperature, and holding time. The choice of these parameters is dependent on the processing goal, which can be microbial inactivation, enzyme inactivation/activation, or changes in macromolecule structures and interactions, including fat, protein, carbohydrates, and fibers.²²

The available equipment allows the application of pressure up to 1000 MPa, with time varying from seconds to hours and temperatures between 0 and 120°C.²³ Despite this, processes using nonextreme pressures (< 800 MPa), mild temperatures (20–60°C), and a few minutes have been most investigated. With respect to commercially available products, HPP is mainly used for pasteurization-equivalent food applications, with a holding time ≤ 5 min, pressures ranging from 200 to 600 MPa, and under refrigeration or at room temperature.²³

HPP can process semisolid (but not rigid), viscous, or fluid food, mainly prepackaged.²² The packaging material plays an important role in HPP; it needs to be resistant and tightly sealed to support the volume change caused by pressure (able to withstand a reversible 15% reduction in volume while maintaining its initial characteristics of barrier and integrity)⁷,²⁴; consequently, the HPP efficiency can be affected by the mechanical and physical properties of the packaging materials. Thus, the evaluation of the impact of the pressure on the package material becomes important, such as the migration characteristics of the packaging polymer,²⁵ integrity of mechanical and barrier properties under pressure (water and oxygen permeabilities), sealing strength, heat transfer, and vacuum (to reduce the effect of oxygen).²²

The packaged product is inserted into the chamber vessel flooded with the indirect low-compressible fluid used for pressurization (e.g., water or propylene in processes under freezing temperatures). When the process starts, a pump system or a piston pressurizes the indirect fluid that transmits the pressure instantly to food, regardless of their size and geometry, since the pressure is transferred evenly throughout the sample due to the isostatic principle.⁷ Therefore, there is not a pressure gradient, i.e., there is no low-pressure spot, preventing the product from over-treatment.⁷ After reaching the desired pressure, the holding time count starts, and at the end, the pressure is relieved, finalizing the processing cycle.²⁶ A general scheme exemplifying an HPP process is represented in Fig. 1.1.

Fig. 1.1

Fig. 1.1 Scheme of operation for the high isostatic pressure device. Credit: Authors.

Pressurization is accompanied by adiabatic compression heating. The adiabatic heating rate is specific to each compound (e.g., foods with high water content increase 3–5°C/100 MPa, while fatty foods increase 6–10°C/100 MPa). Therefore, the final temperature of food processing is dependent on the kind of product, inlet temperature of product and of pressurizing fluid, and the final pressure applied.²⁷ In addition, adiabatic heating is completely reversed upon pressure release.

The changes induced by HPP are mainly related to the Le Chatelier principle, which postulates that when a system in equilibrium is disturbed, it responds by minimizing the disturbance, i.e., the high-pressure system favors reactions that result in volume reduction and, conversely, counteracts those involved in an increase in volume.²²

Raw milk was the first food subjected to HPP by Hite in 1899, who observed a shelf-life extension of 4 days after pressurization at 600 MPa/1 h/room temperature.¹ After this, a long time was necessary for engineering development to obtain equipment able to process food using high pressure.⁷ In the decades of 1990 and the beginning of 2000, most of the tests carried out were focused on microbial inactivation and supported the United States Food and Drug Administration²⁸ decision to recognize HPP as a pasteurization process that satisfied the 5-log reduction rule of pertinent pathogens. However, in the last 15 years, several authors have reported the impact of HPP application on food constituents, showing that the technology could be applied strategically to change the technofunctionality of macronutrients.⁶

1.3: High-pressure homogenization

High-pressure homogenization (HPH), also known as ultra-high-pressure homogenization (UHPH) or dynamic high pressure (DHP), is an emerging technology used to process fluids, such as milk, soy beverages, juices and purees, as well as for solutions, such as teas or enzymatic ones.²⁹

HPH technology is based on the same principles of common homogenization applied especially in the dairy industry, but using much higher pressures.²¹ At the beginning of the 2000s, homogenization at high pressures began to be studied as an alternative nonthermal treatment, and its applications were extended to industries other than dairy, with different aims.²¹ In this technology, the operating parameters are pressure, inlet temperature, number of passes, valve design, and flow rate, the last two being dependent on the equipment configuration.⁴

The technology is based on pumping the fluid to a restriction system using high pressures, where the pressurized fluid passes quickly through a convergent section and is then depressurized.³⁰ This process results in high mechanical stress and several physical phenomena associated with kinetics, pressure, and thermal energy. HPH systems have different configurations regarding valve geometries, pumps, and heat exchangers. A general scheme exemplifying an HPH process is represented in Fig. 1.2 and further described.

Fig. 1.2

Fig. 1.2 High-pressure homogenization scheme. Credit: Authors.

In the HPH process, the fluid is pressurized by one or more piston pumps (pressure intensifiers) at pressures up to 450 MPa.³¹ High pressure is the driving force that makes the fluid flow through the homogenization valve, forcing it to pass through a gap with dimensions of some micrometers in the homogenization valve, which is ∼ 1000 times smaller than the other dimensions inside the valve,²⁹ and after this, pressure reduces instantaneously to 0.1 MPa (atmospheric pressure). The flow area reduction leads to a fluid velocity increase (up to 120 m s− 1)³²; consequently, the energy corresponding to the pressure is converted into kinetic energy in the processed fluid. The magnitudes of velocity and operating pressure depend on the gap size, which can be adjusted by moving the valve. To illustrate, a gap size of approximately 2–5 μm is reached for pressures up to 350 MPa.³⁰

With respect to the consequences of abrupt depressurization, two important phenomena occur: velocity profile and cavitation. The velocity profile is governed by the fluid friction against the valve wall, and the difference in the fluid velocity leads to high shear stresses (i.e., tangential force applied into the fluid), as described by Pinho et al.³² Further, the pressure continuously decreases across the valve gap, and it can reach the fluid vapor pressure value at the processing temperature. Thus, part of the liquid can evaporate at the low-pressure regions, and the bubbles collapse in the zones at higher pressures.³³ This phenomenon is called cavitation, liberates punctually a high amount of energy, and results in high values of shear stress and heating. The intensity of cavitation effects depends on the pressure applied, increasing proportionally with the pressure.³⁰

During homogenization, part of the pressure and kinetic energy are also converted to thermal energy as a result of friction. Therefore, the fluid temperature increases due to the heat of compression, homogenization effort, and cavitation,³⁴ rising 15–25°C per 100 MPa increment in homogenization pressure, depending mainly on the physicochemical properties, especially the compressibility of the fluid.³⁴,³⁵ This temperature increment can be desirable or not, depending on the process target (microbial inactivation, enzyme modification, physicochemical or structural changes, and preservation of bioactive compounds). If it is undesired, the reached temperature can be reduced by implementing a cooling system placed immediately after the homogenization valve, restricting the residence time at high temperature to less than 1 s,³⁶ and by using a low inlet temperature of product.⁵ The first option is more effective than the second one due to the high thermal inertia of the equipment.²⁰,³⁶

Furthermore, it is important to highlight that the residence time at high pressures in the homogenizers is a few seconds²⁰; therefore, the processed samples are not subjected to the effect of high pressures observed in the HPP. The shear stress distribution across the product is the main responsible for the effects in HPH processing.³² This effect increases the structure/molecule entropy,³⁷ inducing changes such as cell and tissue disruption, protein unfolding, and polysaccharide cleavage.³²

With respect to microbial inactivation, HPH is effective as a nonthermal pasteurization only when processes are carried out at pressures higher than 250–300 MPa.³¹,³⁸ In addition, pressures near 200 MPa can be used when associated with mild or high temperature, strategically using the heating generated by the shear stress to reach the desired temperature of processing without come up time.³⁹ Despite this, most HPH applications are focused on improving the versatility of biomolecules (such as polysaccharides and proteins) as food ingredients.³⁷ This is explained considering that lower pressures (i.e., up to 200 MPa, which is the pressure usually achieved for most industrial-scale equipment) are effective in promoting these changes.

1.4: Enzymes

Enzymes are globular proteins able to catalyze specific biochemical reactions. They are formed by many amino acids linked by peptide bounds (primary structure) forming a long chain with a specific spatial arrangement (secondary, tertiary, and, sometimes, quaternary structure) determined by inter- and intramolecular noncovalent bindings,⁴⁰ such as hydrogen bonding, disulfide bridges, and van der Waals, electrostatic, and hydrophobic interactions.⁴¹

Thus, enzyme molecules have folds and twists generated by the repulsions and attractions between nearby amino acids, leading to a final structure with a lower energy state that has specific active sites.²⁹,⁴² The enzyme reaction involves: (i) an interaction between the catalytic site and substrate, (ii) enzyme-substrate complex formation, and (iii) complex release and product formation, with no changes in enzyme structure.⁴³

Enzymes are essential in all organisms’ lives.⁴⁴ In food, enzymes can be endogenous to tissues (such as milk, meat, and vegetable products) or added with technological interest, including hydrolysis of compounds of interest (proteins, fat, and polysaccharides), isomerization, O2 consumption/removal, and protein crosslinking.²⁹,⁴²

For endogenous enzymes, the reaction products can be desired or not.²⁹,⁴¹,⁴⁵ For example, browning compounds formed due to the activity of polyphenol oxidase can be desirable in some teas but not in most juices.⁴¹,⁴⁶ Therefore, enzyme inactivation is one of the most common aims of food processing, focusing on improving product appearance and/or taste, and increasing product shelf life.¹³,⁴¹,⁴⁷ Interestingly, for pasteurized products (especially those from fruits and vegetables), enzyme inactivation is often the target of processing due to its higher resistance to thermal and nonthermal processing compared with pathogen resistances.²⁹,⁴⁴

On the other hand, the application of enzymes to catalyze reactions of industrial interest is continuously growing.⁴⁰,⁴⁸ In this case, enzymes can be from animals, plants, or microorganisms and can occur naturally or be produced using genetic engineering. However, for most uses, microbial enzymes are preferred due to their biochemical diversity, genetic manipulation, high productivity, and simplicity to be obtained and purified.²⁹

In general, the replacement of chemical reactions by industrial biocatalysis is desirable. This is explained considering that enzyme reactions are more specific (guaranteeing product purity and less waste generation) and consume less energy due to the mild reaction conditions.⁴⁰ Nevertheless, the substitution of chemicals by enzyme reactions can be limited by biocatalysis costs, the relatively low stability of enzymes in their native state time, and their lower efficacy/yield.⁴⁵ Thus, the competitiveness of enzyme conversion for industrial application depends on improving enzyme activity and stability and reducing costs.²⁹ In this way, the use of different technologies to change enzyme configuration to achieve specific improvements is an interesting strategy, favoring the growth of the biocatalyst industry.⁴⁵

Enzymes have a fragile structure, which can be perturbed by chemical, physical, or physicochemical processes, leading to modification in intramolecular and solvent-molecule interactions that alter the spatial configuration of enzymes.²⁹,⁴²

These changes are called denaturation and can be reversible or not, depending on the intensity and extension (Fig. 1.3). In the first step of denaturation (reversible), the rupture of some interactions that maintain the native structure occurs, but if the denaturing influence is removed, the enzyme can refold to the initial configuration.⁴⁰ However, above a limit of alterations, kinetically irreversible denaturation occurs, with new covalent bonds and aggregation to reach a thermodynamically stable accommodation.⁴⁰ In general, enzyme denaturation is associated with loss of biological activity; however, some studies have shown that some degree of denaturation can improve the stability of enzymes or change its optimum conditions for activity.²⁹,⁴²,⁴⁵,⁴⁹

Fig. 1.3

Fig. 1.3 Representation of enzymes in native and unfolded state. Credit: Authors.

1.4.1: Changes induced in enzymes by high-pressure technologies

Enzyme activity is dependent on its spatial configuration, which is maintained by the linkages and interactions between amino acids.⁴¹,⁴² Most enzymes have maximum activity in the native state, with the parameters of the reaction (such as pH, temperature, and salt concentration) set to maximize the interaction between the enzyme and substrate.²⁹ However, enzyme functionality is affected under nonoptimum conditions, with biological activity loss and changes in substrate specificity.⁵⁰ This occurs because the spatial configuration of the active sites of enzymes is affected by the molecular energy (given by the temperature of the reaction media) and net charge of the system (pH and ionic strength), according to Rao.⁵¹

In this context, slightly denatured enzymes (especially those subjected to reactions able to denature only weak interactions, such as hydrophobic, electrostatic, and van der Waals interactions) can have activity higher than enzymes in the native state under nonoptimal reaction conditions,²⁹,⁴¹,⁴² considering that new active site configurations can become more adapted to react. For example, enzyme resistance to high temperatures can increase with new disulfide bonds in the structure, whereas reduction in the stabilization bounds increases the molecular flexibility, favoring activity at suboptimum temperatures.⁴⁰,⁵¹

Moreover, in some cases, higher activity of denatured enzymes is observed even under conditions determined to be optimum for the native enzyme, which is explained by the exposure of the active site entrapped in the hydrophobic core of the native enzyme configuration.²⁹ On the other hand, when the enzyme is severely denatured (with cleavage of most hydrogen and disulfide bonds), loss of enzyme activity usually occurs. This phenomenon is due to the residues interacting with other exposed groups to reach a new low energy state, resulting in the association and aggregation of protein fragments without catalytic activity.⁴⁰

These consequences of enzyme denaturing can be strategically managed to guarantee enzyme activation/inactivation or stabilization, depending on the industrial interest. Processes such as high-pressure technologies,⁶,⁴⁵,⁴⁹ ultrasound,⁵²,⁵³ microwaves,⁵⁴ and pulsed electric fields⁵⁵ were recently highlighted in the literature as able to promote slight structural modifications in enzymes processed under specific conditions, which were positive regarding their final activity and/or stability.

With respect to specific high-pressure technologies, both HPP and HPH are recognized as able to modify enzymes due to an increase in molecular energy (Fig. 1.4),⁶,²⁹,⁴⁵,⁴⁹ depending on many factors, such as processing conditions, kind of enzyme, and presence/absence of substrates.

Fig. 1.4

Fig. 1.4 Effect of high-pressure technologies on enzyme activity. Credit: Authors.

HPP and HPH alter mainly quaternary and tertiary structures³⁷,⁵⁶ and, under more severe conditions (such as > 700 MPa for HPP and > 150 MPa for HPH associated with high outlet temperature), secondary structures of enzymes.³⁷,⁴¹ These alterations involve exposure of hydrophobic amino acids, exposure of SH groups due to unfolding of the protein followed by reduction in the total SH content due to new disulfide bond formation, and changes in the α-helix, β-sheet, and β-turn ratio composition due to alterations of the secondary structure.³⁷,⁴⁵ On the other hand, the inability of both processes to break covalent linkages guarantees the maintenance of the primary structure of processed enzymes, unless for processes at high temperatures.²⁹,³⁷

Enzyme activation by HPP is usually attributed to exposure or entrapment of the active site and/or split or activation of latent isoenzymes due to pressurization.⁴¹,⁴⁵,⁵⁰ Higher molecular flexibility caused by polar groups hydration can increase the reaction rate of enzymes processed by HPP.⁴⁵ Moreover, when the process is carried out in the presence of a substrate, as occurs in a food matrix or in pressure-assisted catalysis, the velocity of the reaction will be governed by the Le Chatelier principle, which is favored if the volume of the product is lower than that of the substrate.⁴¹

On the other hand, most published research showed that above a specific limit (specific for each enzyme and process condition), changes induced by HPP on molecular structure are sufficient to negatively affect the activity sites and result in enzyme inactivation, which can be reversible or not, depending on the process (Figs. 1.3 and 1.4).⁵⁷

Regarding HPH, most studies have focused on the inactivation of endogenous enzymes in food. In general, changes in enzyme activity are associated with the physical effects of the process, such as high shear, cavitation, and turbulence.²⁹,⁵⁸ These effects cause unfolding of the quaternary structure at a relatively low pressure and modification of the tertiary and secondary structures at higher pressures.³⁷

However, enzymatic inactivation is also dependent on other factors, such as pH, temperature, and the presence of ions,²⁹,⁴² as well as enzymatic activation and stabilization.¹⁵,⁵⁷ The main structural effects described during HPH are the exposure increase of hydrophobic sites and of SH groups due to molecular splitting. In the same way discussed for HPP, enzyme unfolds induced by HPH can result in activation/maintenance/inactivation of its catalytic performance, as the conformational change might expose the active site or prevent its contact with the substrate (Fig. 1.4).⁵⁹

In addition to enzyme configuration, the impact of high-pressure technologies depends on the process parameters: pressure, time (only for HPP), temperature, and number of cycles; the food matrix (integrity, pH, and ionic strength); and the characteristics of the enzyme dilution system (enzyme concentration and dilution media, when technologies are applied to pre-extracted and purified enzymes). These items are deeply discussed in the following sections.

1.4.2: Effects of process parameters on enzyme activity

For HPP, the results of different studies showed that pressures lower than 200 MPa are commonly able to activate or no change enzymes, while pressures > 500 MPa lead to partial or complete inactivation (Fig. 1.4).¹⁷,⁴¹,⁵⁷ Despite this, there are some baro-resistant enzymes that are not inactivated by HPP unless the process is carried out at high temperatures.⁶⁰ The time and temperature of the processes have a positive effect on enzyme inactivation, with the effect of temperature being more effective than the time.⁴¹ Moreover, the use of short pressurization cycles (pulses) can be an alternative to increase the rate of inactivation of enzymes compared with continuous HPP.⁶¹

In relation to HPH, the impact on enzyme activity may vary according to the level of applied pressure, temperature, and number of homogenization cycles. Normally, the increase in the number of passes generates minimal impacts on most of the evaluated enzymes, demonstrating a greater change in activity in the first pressurization cycle. The thermal effect, on the other hand, has a significant influence, and the increase in the inlet temperature reduces the pressure level necessary to initiate the inactivation of the enzyme.⁵⁹

1.4.3: Effects of the food matrix/dilution system on enzyme activity

The food matrix also modulates the consequences of high-pressure technologies on enzymes.¹⁷,⁶²,⁶³ Parameters such as pH and ionic strength of the food (in natura or prepared) can change the configuration of the enzyme and, consequently, the initial exposure of active sites, leading to modification of the process consequences in enzymes.⁴¹

Furthermore, the presence of other food constituents and physical structure integrity (responsible for physical separation between enzyme and substrate, such as observed in fruits) can also affect the enzyme unfolding during pressure processing and the intensity of its consequences during the product shelf life.⁶²,⁶³ This is especially important for food with preserved cellular structure before processing, such as whole or sliced fruits, as even low pressure can disrupt these structures, favoring catalysis by increasing the contact between substrates and enzymes if the processes are not sufficient to cause enzyme inactivation, which can limit the shelf life of the processed product.⁶² Therefore, for products in which enzyme reactions are undesired, changes in product formulation and strategies for improving structural integrity can minimize enzyme reaction occurrence.⁶²,⁶⁴ On the other hand, for HPP-assisted reactions, these parameters must be studied and optimized to maximize and/or accelerate the obtaining of products of industrial interest, such as hydrolyzed starch.⁶⁵

Finally, for commercial enzymes, such as milk coagulants, high-pressure technologies can be strategically used to improve enzyme activity and stability.¹⁵,⁵⁷,⁵⁸,⁶⁶–⁷⁰ In this case, processing (time, temperature, and pressure) and media conditions (pH, ionic strength, and enzyme concentration) must be optimized to maximize the expected effects on the enzymes.

1.5: Enzyme reactions assisted by HPP

The HPP-assisted reaction (Fig. 1.5) is an alternative to improve the catalysis of products of interest. This type of process is interesting because: (i) it induces enzyme activation (through the effects already mentioned), (ii) it promotes structural changes in the substrate, which can facilitate enzyme access and, consequently, contribute to an increase in the hydrolysis rate,⁶² (iii) it favors catalysis if the product has a smaller volume than the substrate, following Le Chatelier’s principle,²² and (iv) an acceleration of mass and energy transfer can occur, favoring the reaction.⁷¹–⁷³ Thus, the HPP-assisted reaction potentializes substrate conversion due to the sum of these effects.

Fig. 1.5

Fig. 1.5 Strategies to enhance enzyme performance. Credit: Authors.

To maximize the performance of HPP-assisted enzyme reactions, process optimization should be carried out considering processing conditions (pressure, time, and temperature); enzyme and subtract concentrations, through the evaluation of inhibition occurrence and limiting factors⁷⁴,⁷⁵; and characteristics of the reaction environment, especially pH and ionic strength, due to its direct effect on enzyme configuration. The best way to optimize it is through evaluation of kinetic parameters of hydrolysis, mainly hydrolysis rate and yield.⁴⁷,⁵²,⁵³

HPP has been used during the enzymatic-assisted hydrolysis of proteins to obtain bioactive peptides. A study showed that the application of 300 MPa induced the unfolding of flaxseed protein structures and increased the degree of hydrolysis by 1.7 times compared with the control, as well as potentiated the antioxidant activity of the hydrolysates.⁷³ In another study, HPP-assisted enzymatic hydrolysis at 400–500 MPa potentiated the technical-functional properties (foaming capacity, oil retention capacity, and solubility) of soy hydrolysates, in addition to reducing the immunoreactivity of proteins without generating a bitter taste.⁷¹ Several other studies also report a significant increase in: (i) the hydrolysis degree of whey proteins,⁷⁶ (ii) the rate of hydrolysis reaction of bovine serum albumin,⁷² and (iii) the antioxidant activity of protein hydrolysates from fish.⁷⁷

1.6: Challenges, future perspectives, and final remarks

HPP and HPH can be considered promising technologies to be applied in enzyme processing. These technologies can promote the activation, inactivation, and stabilization of different enzymes according to the type of enzyme and desired purpose.

Although food processed by HPP has already been sold worldwide, the specific use of this technology to improve enzyme performance to obtain biocatalytic products of commercial interest has only been evaluated on a laboratory scale. Similarly, HPH is considered a developing technology. Thus, to use these technologies on an industrial scale focusing on enzyme activation/stabilization, future studies need to be carried out in a comparative way to determine the differences and equivalences between the processes and the costs involved. Other challenges are the cost of implementing and maintaining these technologies, as well as the scalability of HPH, which makes it difficult to adopt the technology industrially. Currently, industrial equipment operating at higher pressures has reduced flow.

On the other hand, the food industry’s interest in these technologies is growing as they are environmentally friendly, innovative, and sustainable options. In addition, they have advantages when compared to heat treatment, such as the preservation of sensory and nutritional characteristics of foods submitted to these new methods. This great potential already drives the development of new equipment with greater capacity and higher pressures.

Furthermore, growing social pressure and food policy against the use of additives in food have stimulated the development of biocatalytic processes in recent years, aiming to produce replacers for conventional stabilizers, emulsifiers, and preservatives. In addition, many products of enzyme reactions have high value-added nutraceutical/pharmaceutical applications. Thus, the use of emerging technologies to improve the rate and yield of this catalysis is a research field with great interest and application.

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