High-Pressure Homogenization On Food Enzymes: © 2020 Elsevier Inc. All Rights Reserved

High-Pressure Homogenization on Food Enzymes
Bruno RC Leite Júniora, Mirian TK Kubob, Pedro ED Augustoc, and Alline AL Tribstd, a Department of Food Technology (DTA),
Federal University of Viçosa, Viçosa, MG, Brazil; b Department of Chemical Engineering, Escola Politécnica, University of São Paulo,
São Paulo, SP, Brazil; c Department of Agri-food Industry, Food and Nutrition (LAN), Luiz de Queiroz College of Agriculture (ESALQ),
University of São Paulo, Piracicaba, Brazil; and d Center for Food Research, University of Campinas (UNICAMP), Campinas, Brazil
© 2020 Elsevier Inc. All rights reserved.
Introduction 1
High-Pressure Homogenization 2
Enzymes 3
General Aspects 3
Mechanisms Involved in Enzyme Activation, Inactivation, and Stabilization 5
General Effects of High-Pressure Homogenization on Enzymes 6
Effects of High-Pressure Homogenization on Specific Food Enzymes 10
a-Amylase 10
Alkaline Phosphatase 10
Amyloglucosidase 11
b-Galactosidase 11
Glucose Oxidase 11
Lactoperoxidase 12
Lipases 12
Lysozyme 12
Pectate Lyase 13
Pectin Methylesterase 13
Peroxidase 14
Polyphenol oxidase 14
Proteases 15
Alkaline Protease from Bacillus licheniformis 15
Fungal Protease Obtained from Rhizomucor miehei 15
Neutral Protease from Bacillus subtilis 15
Papain 15
Pepsin 16
Plasmin 16
Rennet 16
Trypsin 16
High-Pressure Homogenization of Food Products: The Challenge for Inactivating Endogenous Enzymes 17
Fruit- and Vegetable-Based Products 17
Milk and Dairy Products 17
Economic Perspectives, Future Challenges, and Final Remarks 18
Acknowledgment 19
References 19
Further Reading 22
Introduction
Consumer demand for processed food products similar to fresh ones, with high quality and microbiological and chemical safety,
clean label, sustainable, and reasonable costs is growing. As a consequence, there is a need for the development of innovative food
processes, with an increasing interest for nonconventional technologies. The nonconventional technologies, mainly the nonthermal
ones, are able to guarantee food safety and shelf life stability while retaining nutritional and sensory quality.
High-pressure homogenization (HPH) is a nonthermal, nonconventional, emerging technology for food processing. HPH is also
referred as dynamic high-pressure processing (by being a continuous process) and as ultra-high-pressure homogenization (for pres-
sures higher than 200 MPa). Basically, HPH consists of pressurizing a fluid to flow through a narrow gap valve, which greatly
increases its velocity, resulting in depressurization with consequent cavitation and high shear stresses. During the process, several
physical phenomena successively and/or simultaneously are involved. Among them, the most important contributive factor is the
shear stress distribution across the product. This leads to changes in biopolymers (as protein and carbohydrate structures) resulting
Reference Module in Food Sciences https://doi.org/10.1016/B978-0-08-100596-5.22999-5 1

2 High-Pressure Homogenization on Food Enzymes
in microbial inactivation and in alterations of various properties and quality parameters of the product, including color, stability,
consistency, rheology, and bioaccessibility of bioactive compounds.
Homogenization technology has been typically employed in the processing of dairy beverages and cosmetics with the aim of
promoting stability of emulsions and avoiding creaming (Patrignani and Lanciotti, 2016; Hayes et al., 2005). The application of
HPH in food processing initially focused on microbial inactivation, with the aim of achieving reduction of microorganisms similar
to thermal pasteurization with better preservation of sensory and nutritional quality attributes (Patrignani and Lanciotti, 2016).
The impact of HPH technology on enzymes begun to be studied only after 2005 (Lacroix et al., 2005), around 15 years later than
studies on microbial inactivation using HPH. In addition, only in the last 5 years the studies about changes on enzymes caused by
HPH gained visibility. This is a problem because, for several products, especially fruit- and vegetables-based ones, enzymes have
higher resistance than nonspore-forming microorganisms, therefore must be chosen as the processing target (Terefe et al., 2014).
Thus, if HPH processing parameters are only established considering microbial safety, they can fail in the maintenance of product
stability resulting in serum separation, browning, and protein agglomeration during the product shelf life (Terefe et al., 2014).
More recently, HPH has also being studied as a strategy to promote desirable structural changes on enzymes of commercial
interest, being able to improve enzyme activity (especially at non-optimum conditions) and stability, which is very important to
improve the enzyme’s performance, reduce costs, increase the range of application, and, consequently, increase the feasibility of
industrial enzyme processes (Rao et al., 1998; Iyer and Ananthanarayan, 2008).
The present article starts with a description of the principles and equipment of HPH. Then, it presents a review of enzymes,
including its general aspects and the mechanisms involved in the process of activation, inactivation, and stabilization. Furthermore,
specific effects of HPH on studied enzymes are discussed, as well as the consequences on food quality with endogenous enzymes.
Finally, the article describes the industrial perspectives and future challenges of HPH use for enzymes’ modulation.
High-Pressure Homogenization
High-pressure homogenization (HPH), also called ultra-high-pressure homogenization (UHPH), is a technology used for process-
ing of fluids continuously. Consequently, it is also called dynamic high-pressure (DHP) processingdalthough the desired mecha-
nisms are not strictly related with pressure. 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, being then depressurized (Floury et al.,
2004a). It results in high mechanical stress and several physical phenomena associated to kinetic, pressure, and thermal energy.
Different configurations of HPH systems are found in industrial and laboratory scales, with different types of high-pressure
homogenizers, homogenization valve geometries, pumps, heat exchangers, accessories, and others. A general scheme exemplifying
an HPH process is represented in Fig. 1 and further described. Due to the equipment particular features (related with its dimen-
sions), HPH cannot be used for particulate food. In contrast, it can be applied for dispersions, such as milk, soy beverage, juices,
and purees, as well as for solutions, such as teas or enzyme solutions.
Figure 1 Schematic representation of a high-pressure homogenization system, indicating the product temperatures: T1 (initial temperature), T2
(HPH valve inlet temperature), T3 (HPH valve outlet temperature), and T4 (final temperature).
High-Pressure Homogenization on Food Enzymes 3
During HPH process, the nonhomogenized fluid, depending upon processing objective or desired effect (microbial inactivation,
enzyme modification, physicochemical or structural change, and bioactive compounds preservation), is either pre-chilled or heated
to a certain temperature using a heat exchanger. Subsequently, the fluid is pressurized by one or more piston pumps (pressure inten-
sifiers), at pressures up to 400 MPa (4000 bar, 4000 atm, 58,000 psi).
High pressure is the driving force that makes the fluid flow through the homogenization valve and further. The fluid is forced to
pass through a narrow passage (called valve gap), which has dimensions in the order of some micrometers (i.e., 1000 times
smaller than the other dimensions inside the valve). According to the Mass Conservation Law, as the area for flowing is reduced,
the velocity is increased. Consequently, based on the Energy Conservation Law, as the velocity is increased, the product pressure
is reduced. As a result, the energy corresponding to the pressure is converted into kinetic energy. The magnitudes of velocity and
operating pressure depend on the gap size, which can be adjusted by moving the valve. For instance, with a typical gap size of
around 2–5 mm, the homogenization pressure can reach up to 350 MPa (Floury et al., 2004a).
By flowing through the valve gap, the product passes from a pressure in the order of 100 MPa to atmospheric pressure (in the
order of 0.1 MPa). Therefore, two important phenomena take place: huge increase in velocity, and change in flow profile and cavi-
tation. Considering the Momentum Conservation Equation, fluid friction against the walls results in a velocity profile. The differ-
ence in the fluid velocity leads to high shear stresses (i.e., tangential force applied into the fluid), resulting in important changes in
the product properties and constituents (Pinho et al., 2011)dsuch as cell and tissue disruption, protein unfolding, enzyme struc-
tural changes, and polysaccharides cleavage.
Furthermore, pressure continuously decreases across the valve gap and it can reach the fluid vapor pressure value at that temper-
ature. At the gap exit, the area of flowing is greatly increased, reducing the fluid velocity and then increasing the pressure until the
outlet pressure (atmospheric pressure). As a consequence, part of the liquid evaporates at the low-pressure regions, and the bubbles
collapse in the zones where pressure increases (Floury et al., 2004b). This phenomenon is called cavitation, which liberates a high
amount of energy and results in high values of shear stress and heating. The intensity of cavitation effects increases with increasing
homogenization pressure (Floury et al., 2004a).
During the passage of the fluid through the gap, part of the pressure and kinetic energy is also converted to thermal energy, as
a result of friction. Therefore, the fluid temperature increases due to heat of compression, homogenization effort, and cavitation
(Martínez-Monteagudo et al., 2016)dwhich can be undesirable when an enzyme solution of commercial interest is processed.
The temperature can rise about 1.5–2.5 C per 10 MPa increment in homogenization pressure, depending on the equipment geom-
etry and the physicochemical properties of the fluid (Martínez-Monteagudo et al., 2016; Harte et al., 2016). In cases where thermal
effects and overheating must be avoided, the temperature rise may be controlled by implementing a cooling system (e.g., by placing
a heat exchanger immediately after the homogenization valve, as illustrated in Fig. 1) and by using low inlet temperature of product
(Dumay et al., 2013; Rojas et al., 2019).
It is noteworthy that instead of the pressure before the gap, the shear stress distribution across the product is the main factor
responsible for the effects of HPH processing. The fluid product is subjected to the high-pressure condition for a very small period
of time, which is much lower than the required process time to observe the effects of pressure in a processed sample, as in high
hydrostatic pressure processes. Intense shear stress distribution across the fluid physically affects product constituents (microorgan-
isms, enzymes, cells, polysaccharides, proteins, etc.).
Therefore, in view of the several physical phenomena successively and/or simultaneously involved during HPH, the structure of
the product and its constituents are modifieddwhich can change the enzyme activity and stability in different ways.
Enzymes
General Aspects
Enzymes are defined as proteins that have a biocatalyst’s role (Terefe et al., 2014; Iyer and Ananthanarayan, 2008). Proteins are
formed by long chains of amino acids with peptide bonds (primary structure) and that have a specific spatial arrangement deter-
mined by inter- and intramolecular noncovalent bindings (Iyer and Ananthanarayan, 2008). Hydrogen bonds, formed between
hydrogen donor groups (especially –OH and ¼ NH) and hydrogen acceptor groups (especially ¼ O, –O–, and ¼ N–), are the
main forces responsible for the secondary structure of a protein. Depending on the amino acid distribution in the polypeptide
chain, secondary structure is organized mainly as a-helices or b-sheet forms (Iyer and Ananthanarayan, 2008; Walstra et al., 2006).
Further folding in the protein chain leads to a tertiary structure, which is stabilized essentially by hydrophobic, electrostatic, and
van der Waals interactions, intramolecular –S–S– bridges, and internal salt bridges (Walstra et al., 2006). The most common
arrangement for tertiary structure is the globular one (chain folded as a spherical shape). Fibrous protein configuration is more
specific and occurs for proteins that contain exclusively b-sheet in the secondary structure and form regularly elongated structures.
Such proteins function as a building material. Other proteins can have a disordered structure, since they have not standard folding,
although containing some secondary and tertiary structure (Walstra et al., 2006). Globular structure is preferentially formed for
proteins that have sufficient proportion of hydrophobic amino acid residues. In these structures, hydrophobic side groups tend
to interact in the core of the folded molecule, and hydrophilic groups at the outside of the molecule, which means, in contact
with water and probably forming hydrogen bonds. At this condition, protein is considered hydrated (Iyer and Ananthanarayan,
2008; Walstra et al., 2006).
The segregation between hydrophilic (external face of the protein) and hydrophobic groups (core of protein) normally is incom-
plete. Depending on the sequence of residues, some hydrophilic residues become entrapped in the core while some hydrophobic
ones are forced to face the external surface of protein. When the protein structural arrangement results in large hydrophobic patches
outside, association of polypeptide subunits into larger units can occur, forming the quaternary structure. Therefore, this last orga-
nized structure is found only in some proteins (Iyer and Ananthanarayan, 2008; Walstra et al., 2006).
Enzymes are globular proteins that catalyze biochemical reactions (Leite Júnior et al., 2017) and are essential in the physiology
and metabolism of living things, including bacteria, yeasts, fungi, plants, and animals (Terefe et al., 2014). This catalytic activity
arises from an active site, which is determined by the three-dimensional configuration of the molecule (Iyer and Ananthanarayan,
2008; Hendrickx et al., 1998) formed from a specific sequence of amino acids arranged in the configuration of the lowest energy
state, called the native state (Leite Júnior et al., 2017; Aguilar et al., 2018a). The biochemical reaction involving enzymes occurs
through the interaction of the catalytic site and the substrate, forming a complex enzyme substrate followed by product formation
(Leite Júnior et al., 2017).
The enzymes have a delicate structure, easily perturbed by chemical, physical, or physicochemical processes that can alter the
balance of intermolecular and solvent–protein interactions (Hendrickx et al., 1998; Yaldagard et al., 2008). Enzyme denaturation
is any change in the enzyme spatial structure, which can results in alterations in its biological activity (Iyer and Ananthanarayan,
2008; Aguilar et al., 2018a). Denaturation is the separation of quaternary structure subunits and/or the unfolding of the enzyme
tertiary structure to a disordered polypeptide chain. In the denatured conformation, key amino acid residues of the active site
are no longer aligned closely enough to guarantee the expected functional activity (Iyer and Ananthanarayan, 2008). Denaturation
occurs as a two-step phenomenon: firstly a reversible unfolding of the original enzyme caused by the rupture of the interactions that
maintain the native structure occurs, followed by the kinetically irreversible step, in which the unfolded structure forms aggregates
and new covalent bonds, reaching a thermodynamically stable accommodation (Iyer and Ananthanarayan, 2008; Aguilar et al.,
2018a). Therefore, if the denaturing influence is removed, the denaturation can be reversible up to a limit of structural changes.
After this limit, the changes are irreversible and commonly associated with loss of enzyme activity and stability (Iyer and Anantha-
narayan, 2008). On the other hand, for several enzymes, the maximal catalytic activity and stability does not occur in native confor-
mation (Leite Júnior et al., 2017; Aguilar et al., 2018a; Liu et al., 2010a; Tribst et al., 2018). Fig. 2 shows a schematic representation
of enzymes in native and denatured conformation.
Enzymes are primarily classified into six different classes: oxidoreductases, transferases, hydrolases, lyases, isomerases, and
ligases. In food, enzymes can be endogenous to the tissues (fruits, milk, meat, and vegetable products) or intentionally added as
a processing aid to promote hydrolysis of compounds of interest (proteins and polysaccharides), reduction of the media (for O2
removal), isomerization, and protein cross-linking. Commonly, endogenous enzymes promote undesirable changes in processed
food products (such as browning, phase separation, and oxidation), consequently reducing their shelf life. Therefore, in several
cases, enzyme inactivation is the target of industrial food processing, especially for fruit- and vegetables-based products (Terefe
et al., 2014; Leite Júnior et al., 2017).
The development of industrial biocatalysis started in 1960s (Iyer and Ananthanarayan, 2008) and is continuously growing due
to improved production technologies, engineered enzyme properties, and expansion of new applications (Iyer and Ananthanar-
ayan, 2008). Most of the enzymes used in industry are obtained from microorganisms, due to their broad biochemical diversity,
susceptibility to genetic manipulation, high productivity, and relatively simple production and purification steps (Rao et al., 1998).
The main advantages of using industrial biocatalysis are the high specificity of enzymes (molecular and chiral), activity under
mild conditions, high turnover number, and biodegradability (Iyer and Ananthanarayan, 2008). Even considering the advantages
of enzyme catalysis compared to chemical catalysis, the intensive replacement of chemical catalysts is limited due to the relatively
low stability on enzymes in their native state and also their prohibitive cost (Eisenmenger and Reyes-De-Corcuera, 2009). Therefore,
the development of competitive biocatalysts to be used in industrial processing depends on the improvement of enzyme activity
and stability at conditions compatible with diverse industrial processes (such as temperature, pH, presence of ions, and organic
substances) (Eisenmenger and Reyes-De-Corcuera, 2009). This challenge can be overcome through physical and chemical modifi-
cations in the enzymes, and also through genetic engineering applied to organisms that produce the enzymes. Examples of better
performance of enzymes after physical changes include the use of dense or supercritical gases such as supercritical carbon dioxide
(Senyay-Oncel et al., 2014), high-pressure processing (Leite Júnior et al., 2016, 2017; Tribst et al., 2017; Martínez-Monteagudo
et al., 2017), and HPH (Aguilar et al., 2018a; Tribst et al., 2018; Leite Júnior et al., 2014, 2017).
Figure 2 Schematic representation of enzymes in native and unfolded state.

Mechanisms Involved in Enzyme Activation, Inactivation, and Stabilization

Enzymes have sensitive structure that can be easily denatured by changing their quaternary or tertiary configuration, as previously
explained. These conformational changes can alter the functionality of the enzyme resulting in biological activity loss or increase
and changes in substrate specificity (Hendrickx et al., 1998).
The most common observed effect of enzyme denaturation is the loss of activity. A majority of enzymes have their maximum
activity at native arrangement, for which the other parameters of catalysis (temperature, pH, and salt concentration) are set on to
guarantee optimized conditions for enzyme–substrate interaction and consequently maximum yield of catalysis products. Fig. 3
shows a typical curve for enzyme activity measured at different temperature, pH, or salt concentration. The huge differences on
the enzyme activity at distinct temperatures are explained considering that final enzyme arrangement during the catalysis depends
on the energy input into the molecule (high temperature increases agitation at the molecular level, leading to an open molecular
structure). Other catalysis parameters, such as pH and ionic strength, may cause alteration in enzyme net charges, favoring or
hampering inter- and intramolecular ionic repulsion. Therefore, these parameters directly affect the volume of the native enzyme,
its conformation, and consequently its ability to interact with the substrate.
These changes in enzyme activity depending on different catalytic parameters explain why several slightly denatured enzymes
(especially using denaturing processes that mainly affect the hydrophobic, electrostatic, and van der Waals interactions, but preserve
covalent bindings) can have higher activity at non-optimum conditions. This occurs because modifications in the overall surface
charge of the proteins are known to alter the optimal pH/ionic strength of the enzyme (Rao et al., 1998) and the introduction
of new disulfide bonds can increase the enzyme activity at high temperatures (Rao et al., 1998), while the disruption of stabilization
bonds can increase the enzyme flexibility and, consequently, activity at lower temperatures (Iyer and Ananthanarayan, 2008). There-
fore, at partially unfolded arrangement, it is possible that the final configuration of the activity site becomes more feasible to interact
with the substrate at adverse processing parameters (e.g., non-optimum temperature, and pH) than the native state.
Some studies showed higher activity for unfolded enzymes at conditions optimized for the native form. This phenomenon is
commonly attributed to enzymes that have some active sites entrapped in the hydrophobic core of the native structure, which
can be exposed after a partial denaturation, increasing the number of active sites available for catalysis.
Different to activation processes, inactivation is associated with the occurrence of severe alterations in the enzyme structures,
which can be induced by physical (pressure, temperature, and radiation) or chemical modifications. In this case, the intense changes
in the native structure due to the rupture of interactions (hydrophobic, electrostatic, and van der Waals) and disulfide and hydrogen
bonds lead to unmasking of buried reactive residues that became available for interaction with other exposed groups. This occur-
rence is natural to molecules, allowing them to reach a new low state of energy and commonly results in protein fragment associ-
ation and aggregation, consequently without activity as biocatalyst (Iyer and Ananthanarayan, 2008).
In addition to high activity, enzyme stability is another important factor, especially from industrial perspective (Iyer and Anan-
thanarayan, 2008; Leite Júnior et al., 2017). Enzymes that have intrinsic natural stability (from extremophilic organisms) generally
have different amino acid sequences, with high content of arginine and proline and lower content of lysine, asparagines, and gluta-
mine. The enzymes from extremophilic organisms have lesser tendency to unfold, when compared to their mesophilic counterparts
(Iyer and Ananthanarayan, 2008). These stable enzymes have more interactions (i.e., hydrogen bonds, electrostatic interactions,
hydrophobic interactions, disulfide bonds, and metal binding) and superior conformational structure, i.e., rigid, higher packing
efficiency, reduced entropy of unfolding, stability of a-helix structure, and conformational strain release (Mozhaev and
Martinek, 1984).
Stable enzymes can be obtained through genetic manipulation of mesophilic organisms, protein engineering, chemical/physical
modifications, immobilization, and medium engineering by using additives (Iyer and Ananthanarayan, 2008). Generally, changes
on enzymes that favor hydration of charged groups and nonpolar groups, disruption of bound water, stabilization of hydrogen
bonds, increase of hydrogen and disulfide bonds, and increase in the hydrophobicity of the enzymes can be related to enzyme stabi-
lization (Eisenmenger and Reyes-De-Corcuera, 2009; Mei et al., 1999; Mozhaev et al., 1996).
110 120 120

Residual activity (%)
100 100 100
90 80 80
80 60 60
70 40 40
60 20 20
50 0 0
4 5 6 7 8 0 20 40 60 80 100 0 0.2 0.4 0.6 0.8 1
pH Temperature (°C) NaCl (M)
Figure 3 Typical curves of enzyme activity measured at different pH, temperature, and salt concentration.
General Effects of High-Pressure Homogenization on Enzymes
The impact of HPH in enzymes is a relatively new study field. First studies occurred only in 2005, around 15 years later than studies
on microbial inactivation using HPH. These works focused on the inactivation of food endogenous enzymes, mainly on fruit- and
vegetables-based products (Lacroix et al., 2005; Welti-Chanes et al., 2009; Liu et al., 2009a). Only in the last 5 years the studies
about changes on enzymes caused by HPH gained adequate visibility.
Most studies about the effect of HPH processing on enzymes focused on the inactivation of endogenous enzymes, such as pectin
methylesterase (PME) in orange (Lacroix et al., 2005; Welti-Chanes et al., 2009; Velázquez-Estrada et al., 2012), clementine juice
(Navarro et al., 2014), peach juice (Yildiz, 2019), and mix of apple and kiwi juices (Yi et al., 2018); polyphenol oxidase (PPO)
in apple juice (McKay et al., 2011; Bot et al., 2018), chinese pear (using a microfluidizer, equipment that produces similar effects
than high-pressure homogenizer) (Liu et al., 2009a), mushroom extract (using a microfluidizer) (Liu et al., 2009b), peach juice
(Yildiz, 2019), and apple mixed with kiwi juice (Yi et al., 2018); and pectate lyase (PL) in banana juice (Calligaris et al., 2012)
and plasmin (Hayes and Kelly, 2003; Datta et al., 2005; Huppertz et al., 2004; Iucci et al., 2008). More recent studies evaluated
the impact of HPH on isolated enzymes of commercial interest, such as amyloglucosidase (Tribst and Cristianini, 2012a),
a-amylase (Tribst and Cristianini, 2012b), glucose oxidase (Tribst et al., 2014; Tribst and Cristianini, 2012c), neutral protease
(Tribst et al., 2012a), trypsin (using a microfluidizer) (Liu et al., 2010a), lysozyme (Tribst et al., 2017, 2018), calf rennet (Leite
Júnior et al., 2014), and porcine pepsin (Leite Júnior et al., 2015a).
HPH processing can change enzyme activity due to the physical effects of the process, which includes intense shear in the
homogenization valve region, cavitation, and turbulence (Leite Júnior et al., 2017; Martínez-Monteagudo et al., 2017). For enzymes
with quaternary structure, dissociation occurs at relatively low pressure. Changes in tertiary structure normally involve higher expo-
sure of hydrophobic sites, which can result in rearrangement and aggregation after the homogenization process (Porto et al., 2018).
The effect of HPH on secondary structure (that commonly requires pressures > 150 MPa and high outlet temperature) is mainly
related to changes on hydrogen and disulfide bonds, with consequent changes on percentages of a-helix, b-turn, b-sheet, and unor-
dered coil content (Porto et al., 2018). Finally, no changes in the primary structure were reported, since HPH is not able to break
covalent linkage (Porto et al., 2018). Despite these general statements about the effects of HPH on enzyme structures, it is not
possible to predict the effects of HPH processing on individual enzymes (Leite Júnior et al., 2017).
Results obtained so far show that HPH induces changes on enzymes differently than thermal processing, HPH being able to
cause activation of some enzymes, commonly at low pressure (Leite Júnior et al., 2014, 2015a; Liu et al., 2009a,b; Tribst and Cris-
tianini, 2012a; Tribst et al., 2013). Other authors observed few or no changes on the activity of enzymes considered as thermal resis-
tant, such as a-amylase (Tribst and Cristianini, 2012b), and proteases from Rhizomucor miehei (Leite Júnior et al., 2015b) and from
Bacillus licheniformis (Aguilar et al., 2018b), being also baroresistant. Finally, enzyme inactivation was also found to be dependent
on many factors such as pH of the sample, presence of ions, and processing temperature (Leite Júnior et al., 2014; Welti-Chanes
et al., 2009; Velázquez-Estrada et al., 2012; Navarro et al., 2014; Calligaris et al., 2012).
The same behavior is observed for enzyme stabilization with HPH able to increase (Liu et al., 2010a; Tribst et al., 2014, 2018;
Tribst and Cristianini, 2012c), decrease, or not alter the stability of enzymes (Aguilar et al., 2018b), depending on the enzyme and
the processing condition (Martínez-Monteagudo et al., 2017).
The difficulty to predict the impact of HPH in enzymes can be explained by the multifactorial parameters involved in the changes
of enzyme by this process (Leite Júnior et al., 2017; Martínez-Monteagudo et al., 2017), including:
(i) Operational parameters of the equipment and process characteristics, such as pressure applied, inlet temperature of the
samples, temperature during homogenization, holding time at high temperature after homogenization, and number of
process cycles applied (Velázquez-Estrada et al., 2012; Calligaris et al., 2012; Tribst et al., 2012a; Zamora and Guamis, 2015;
Marszałek et al., 2017);
(ii) Sample pretreatments, such as prewarming/blanching (Plazzotta and Manzocco, 2019);
(iii) Homogenization valve design that directly affects the mechanical energy on the molecules, changing its conformation (Dumay
et al., 2013);
(iv) Product characteristics, mainly pH and ions (salt) content, which are able to change the initial enzyme configuration (Navarro
et al., 2014; Tribst et al., 2012a) and also enzyme content and pulp concentration;
(v) Characteristics of the enzymes, especially structure (tertiary or quaternary), individual sensitivity to mechanical process, and
existence of isoenzymes (Lacroix et al., 2005; Velázquez-Estrada et al., 2012; Tribst et al., 2013);
(vi) Presence of substrate during the HPH process, which can also have structure altered by homogenization (Lacroix et al., 2005;
Marszałek et al., 2017; Oliveira et al., 2018);
(vii) Conditions used to measure the residual activity (pH, temperature, and salt concentration) (Tribst et al., 2012a,b, 2014,
2018).
In general, reduction of enzyme residual activity is caused by a combination of high temperature (high inlet temperature and/or
holding time after homogenization valve) and high pressure (Dumay et al., 2013; Welti-Chanes et al., 2009; Velázquez-Estrada
et al., 2012), with the thermal history being the main responsible for enzyme inactivation (Leite Júnior et al., 2017; Martínez-
Monteagudo et al., 2017). For example, the effect of the HPH processing on PME residual activity in orange juice was studied
by Welti-Chanes et al. (Welti-Chanes et al., 2009) that observed a linear decreasing trend of residual activity when pressure
increased, but achieved an adequate inactivation level (70%) only after processing the juice at 45 C (inlet temperature) and
110
PME Residual activity (%)

90
70
50
30
0 50 100 150 200 250 300
Pressure (MPa)
inlet at 22ºC inlet at 35ºC inlet at 45ºC
Figure 4 Effect of high-pressure homogenization on pectin methylesterase in orange juice at different inlet temperatures. Image based on data from
Welti-Chanes et al. Welti-Chanes et al. (2009).
250 MPa. At room temperature, the inactivation at 250 MPa was about 40%. Fig. 4 illustrates this observation. Enzyme inactivation
occurs when the physical processes are sufficient to cause unfolding followed by aggregation of enzymes, changing their tertiary
structure and, consequently, the active site configuration (Marszałek et al., 2017).
The application of prewarming (up to 50 C/10 min) also enhances the inactivation of endogenous enzymes, as observed for
PME in orange juice (Lacroix et al., 2005; Velázquez-Estrada et al., 2012). Moreover, combining HPH with other technologies
may improve the level of inactivation. For example, combining HPH and ultrasound improved PPO inactivation in apple juice
compared to HPH applied alone. However, probably the energy consumption necessary for the combined process and the high
levels of enzyme inactivation by ultrasound alone does not justify the application of HPH and ultrasound together (Bot et al.,
2018).
Commonly, pressures up to 200 MPa together with inlet temperature lower than 30 C results in low or no inactivation of
enzymes, especially for endogenous enzymes of fruit- and vegetable-based products (Lacroix et al., 2005; Welti-Chanes et al.,
2009). Moreover, at these mild conditions, an increase of enzyme activity/stability was also reported. For endogenous enzymes,
Calligaris et al. (Calligaris et al., 2012) observed 3.5 times increase of PL activity in banana juice processed at 100 MPa using
lab-scale high-pressure homogenizer of two stages, and Liu et al. (Liu et al., 2009a,b) observed up to 70% of PPO activation in
pear and mushroom enzyme extracts after processing the samples at 160 MPa using a high-pressure microfluidization treatment
at different inlet temperatures.
For enzymes of commercial interest, this activation is desirable and was observed for neutral protease (Tribst et al., 2012a),
glucose oxidase (Tribst and Cristianini, 2012c), amyloglucosidase (Tribst and Cristianini, 2012a), lysozyme (Tribst et al., 2017),
calf rennet (Leite Júnior et al., 2014), and porcine pepsin (Leite Júnior et al., 2015a) depending on the conditions (pH and temper-
ature). Moreover, compared to native arrangement, higher thermal stability was observed for trypsin (Liu et al., 2010a) and glucose
oxidase (Tribst et al., 2014) processed by HPH under mild conditions, but not for lysozyme (Tribst et al., 2018). Additionally, after
HPH processing, glucose oxidase (Tribst and Cristianini, 2012c) and lysozyme (Tribst et al., 2018) showed high stability to storage
in aqueous solution and lysozyme also showed stability increase at high NaCl concentration and very low pH (Tribst et al., 2018).
The main hypothesis to explain the enzyme activation observed at low pressure and temperature considers that process at these
conditions was able to cause some changes in enzyme structure, increasing their hydrophobicity and consequently exposing active
sites that were entrapped in the enzyme hydrophobic core in the native conformation (Leite Júnior et al., 2017; Liu et al., 2009a,
2010a; Tribst et al., 2014). Moreover, it is possible that the pressurization process activates latent isoenzymes and consequently
increases the observed activity (Leite Júnior et al., 2017). Results from Liu et al. (Liu et al., 2009a) also suggest some degree of alter-
ations in secondary structure can also occur in PPO while no change in it was observed for glucose oxidase processed at 150 MPa
(Tribst et al., 2014). The increase of thermal stability is attributed to increase of the number of disulfide bonds, which results from
increase of SH groups exposure due to unfolding, followed by reduction of total SH content, due to new S-S formation (Liu et al.,
2009a, 2010a). Although these results are reported in literature, the available data is scarce and not sufficient to clearly explain how
the degree of such structural modifications affects the activity or stability of enzymes (Martínez-Monteagudo et al., 2017).
A gradual increase on enzyme inactivation was observed at pressures higher than 200 MPa, even with sample inlet at room
temperature (Velázquez-Estrada et al., 2012). However, it is important to consider that processes carried out at 200 MPa results
in temperature increment of > 30 C (Harte et al., 2016; Martínez-Monteagudo et al., 2017). Therefore, enzyme inactivation by
HPH at this condition is mainly related to the inherent increase of temperature in the homogenization valve. In contrast, for nonre-
sistant enzymes, a significant inactivation can occur even at pressures, as low as 50 MPa. Examples include neutral protease in phos-
phate buffer (Tribst et al., 2012a) and calf rennet in acetate buffer (Leite Júnior et al., 2014). The differences in the individual
resistance of enzymes can be attributed to the existence of isoenzymes and also to the rigidity/flexibility of each enzyme, determined
by the kind and number of hydrogen and disulfide bonds and electrostatic, hydrophobic, and van der Waals interactions (Iyer and
Ananthanarayan, 2008).
180

150
120
90
60
30
0
0 50 100 150 200 250 300 350
Pressure (MPa)
sensitive enzyme activated enzyme resistant enzyme
Figure 5 Generic effect of high-pressure homogenization processing on enzymes with different resistance to inactivation.
Fig. 5 shows a generic model that illustrates enzyme activation/inactivation induced by HPH.
The application of multiple subsequent cycles is considered as an alternative to achieving a lower residual activity of enzymes.
For some enzymes, cycles have an additive effect on the inactivation of enzymes, although an asymptotic behavior is observed
(Welti-Chanes et al., 2009). Lacroix et al. (Lacroix et al., 2005) observed only 20% of reduction of PME activity in orange juice pro-
cessed at 170 MPa/5 cycles and Bot et al. (Bot et al., 2018) reached 50% of inactivation of PPO in apple juice processed at 150 MPa/
10 cycles, while higher inactivation of PME (around 70%) was observed when 5 cycles were carried out at 250 MPa (Welti-Chanes
et al., 2009). In contrast, at low pressures (100 MPa), no additive effect on inactivation of PME was observed (Welti-Chanes et al.,
2009). Therefore, the effectiveness of the subsequent cycles of HPH to inactivate resistant endogenous enzymes is questionable,
especially at low pressures.
In addition, subsequent cycles can be useful when HPH is applied to intentionally increase the activity of some commercial
enzymes, as observed for glucose oxidase processed at 150 MPa (3 cycles) that showed an activity at 75 C three times higher
than native sample (Tribst et al., 2013). Fig. 6 illustrates the impact of HPH cycles in different enzymes and products. Finally, it
is important to consider that, although results of recirculation of processed products have been observed as able to increase the
desirable effect on enzymes (activation or inactivation), this approach may not be realistic from an industrial perspective (Martí-
nez-Monteagudo et al., 2017), considering the product volume required to be processed.
240
180
120
60
0
1 2 3 4 5 8 10
Cycles of HPH
PPO (apple juice at 150 MPa) PME (orange juice at 100MPa)
PME (orange juice at 250 MPa) Neutral protease (buffer at 200 MPa)
Glucose-oxidase (buffer at 150 MPa) Amyloglucosidase (buffer at 150 MPa)
Figure 6 Effect of high-pressure homogenization (HPH) cycles on different enzymes in different matrices. The activity of neutral protease measured
at 20 C, glucose oxidase measured at 75 C, and amyloglucosidase at 80 C. Image based on data from Welti-Chanes et al. Welti-Chanes et al.
(2009), Lacroix et al. Lacroix et al. (2005), Tribst, Augusto & Cristianini Tribst et al. (2013), and Bot et al. Bot et al. (2018).
The effect of products/processing media characteristics, especially salt content and pH, can alter the impact of HPH on each
enzyme. This occurs because the spatial configuration of an enzyme and, consequently, the exposure of its active sites can signifi-
cantly change depending on the ionic balance between the media and the enzyme (Tribst et al., 2012a). Therefore, the enzyme can
be processed at different initial configuration (major or less exposure of structure and active sites), impacting the final effect of
process (Tribst and Cristianini, 2012a).
In this way, it is interesting to observe that few changes on the pH of the products can increase the impact of HPH, as observed by
Lacroix et al. (Lacroix et al., 2005), who observed a reduction of residual activity of PME in orange juice from 80% to 30% for
samples processed at 170 MPa/3 passes when the pH was reduced from 4.0 to 3.5. Similarly, reduction on PME in orange and clem-
entine juices processed at 150 MPa was up to 50% and 90% higher when the pH of juices was reduced from 3.9 to 3.5 and 3.1,
respectively (Navarro et al., 2014). According to these authors, the impact of pH can be as important as processing temperature
for PME inactivation in citrus juices (Fig. 7 illustrates these results).
In contrast, other characteristics of fruit-based products, as ripeness stage (and consequently fruit Brix/acidity ratio), seem to
have only minor effects (Navarro et al., 2014). These characteristics, added to individual differences of enzymes/isoenzymes found
in each product, possibly explain the differences in the process parameters required to reach an adequate level of inactivation of the
same enzyme in different samples (Lacroix et al., 2005; Navarro et al., 2014).
The pH was also observed as an important parameter when enzyme activation is desirable, as observed for amyloglucosidase
(AMG), for which different levels of activation were observed at pH 2.9, 4.3 (optimum for AMG at native arrangement), and 6.5
(Tribst and Cristianini, 2012a). For b-galactosidase, significant inactivation occurred only when the enzyme was processed
(150 MPa) at non-optimum pH (6.4 or 8.0), according to Tribst et al. (Tribst et al., 2012b). The calcium requirement for a-amylase
stability was evaluated, but results showed that this enzyme was very stable to HPH, even in the absence of calcium (Tribst and
Cristianini, 2012b). For illustrating, Fig. 8 shows the residual activity of glucose oxidase processed at different pH and activity
measured at 80 C.
For enzymes of commercial interest that are processed/diluted in water or buffer, the solution concentration can alter the effect of
HPH processing, since shear stress occurrence increases at high concentrations (Leite Júnior et al., 2015b). In addition, different
valve geometries can deliver different intensity of mechanical forces to molecular structure (Dumay et al., 2013; Calligaris et al.,
2012), which also helps to explain the inconsistency of the results obtained in many studies that investigated the inactivation of
the same enzymes.
Another important aspect to consider in the use of HPH for processing food products is that the process causes changes not only
in the enzymes but also in the exposure of the substrates. When a fruit juice is processed by HPH, the remaining intact cells and
tissues are disrupted, releasing the intracellular content. As a consequence, the enzymes and substrates are also released to the
serum. This can change the enzyme activity and commonly accelerates the undesirable reactions caused by endogenous enzymes,
resulting in the reduction of the suspension stability and product browning (McKay et al., 2011). In contrast, despite the higher
residual PME activity, Lacroix et al. (Lacroix et al., 2005) observed a relatively stable suspension of the orange juice processed by
HPH. This phenomenon can be explained by breakdown of the pectin structure as a result of the mechanical forces during homog-
enization, reducing the particle size and consequently delaying the clarification by decreasing the particle’s Stokes radius and
thereby slowing down sedimentation.
In milk, the main changes occur in fat globule, which has a significant particle size reduction after homogenization. According to
Dumay et al. (Dumay et al., 2013), the lipolysis tends to be favored by this process, since homogenization causes an increase of fat
globule/water interfacial area (site for lipase activity). Results from Smoczy nski (Smoczynski, 2016) corroborates this fact, showing
significantly higher activity of porcine pancreatic lipase (added lipase) in milk previously homogenized at 20 or 100 MPa than
observed for unprocessed milk. On the other hand, protease activity tends to be limited because the increase of protein adsorbed
onto the fat globule fragments hinders the access of proteases (Oliveira et al., 2018).
100
PME residual activity (%)
80
60
40
20
0
Clementine / inlet at 58ºC Orange/ inlet at 58ºC Clementine / inlet at 68ºC Orange/ inlet at 68ºC
pH 3.9 pH 3.5 pH 3.1

Figure 7 Effect of high-pressure homogenization (HPH) on the activity of pectin methylesterase (PME) in different citrus juices and at different pH
and inlet temperature. Image based on data Navarro et al. Navarro et al. (2014).
250

200
150
100
50
0
5 5.7 6.5
pH of homogenization
50 MPa 100 MPa 150 MPa

Figure 8 Residual activity of glucose oxidase processed by high-pressure homogenization at different pH (activity measured at 80 C). Image based
on data from Tribst, & Cristianini Tribst and Cristianini (2012c).
The overall evaluation of the results on the effect of HPH on enzymes suggests that the process cannot be considered effective
against endogenous enzymes (especially heat-resistant enzymes/isoenzymes found in fruit- and vegetables-based products),
although there are some individual successes. Therefore, when HPH is used to extend shelf life of this kind of products, blanching
processes or heating is mandatory in order to guarantee an adequate level of inactivation of undesirable endogenous enzymes. On
the other hand, the ineffectiveness of HPH for enzyme inactivation can be useful to preserve/improve the performance of enzymes
with antimicrobial activity, such as the lactoperoxidase system and lysozyme. Moreover, for some commercial enzymes, low pres-
sures can be used to improve their performance, enhancing their activity, stability, and specificity. Thus, in terms of enzyme modi-
fications, HPH needs to be used with caution and the impact on each enzyme/characteristics of the food matrix needs to be
evaluated individually, to better choose the strategy to improve activity or minimize enzyme inactivation, based on the desired goal.
Effects of High-Pressure Homogenization on Specific Food Enzymes

a-Amylase
a-Amylase (EC 3.2.1.1) is an enzyme that catalyzes the cleavage of the internal a-(1,4) glycosidic linkage of starch and polysaccha-
rides into dextrins and oligosaccharides (Tribst and Cristianini, 2012b; Wong et al., 2003; Michelin et al., 2010). This enzyme is
found in fruits and vegetables and acts in fruit ripening process, hydrolyzing starch with consequent tissues softening and with
the development of sweet taste (Tribst and Cristianini, 2012b).
The main commercial application of a-amylase is in starch hydrolysis, followed by application in bakery, brewery, corn syrup
and alcohol production, detergents, and in the textile and paper industry (Tribst and Cristianini, 2012b; Wong et al., 2003;
Gupta et al., 2003; Haki and Rakshit, 2003; Souza and Magalhães, 2010).
Tribst and Cristianini (Tribst and Cristianini, 2012b) evaluated the effect of the HPH process on activity and storage stability of
a-amylase from Aspergillus niger. Results showed no changes in the enzyme activity at 15, 45, and 75 C after homogenization pro-
cessing up to 150 MPa. In addition, the HPH enzyme stability was also unaltered for 4 days. The combination of 150 MPa and high
inlet temperatures (65 C) was also ineffective to change the a-amylase activity (Tribst and Cristianini, 2012b). Therefore,
a-amylase was highly resistant to the HPH processing, remaining active even in products that are subjected to combined processes
of high temperature and high-pressure homogenization.
Alkaline Phosphatase
Alkaline phosphatase (EC 3.1.3.1) is a hydrolase that catalyzes the hydrolysis of phosphate groups (dephosphorylation) of several
molecules including nucleotides and proteins. In the food industry, especially in the dairy industry, alkaline phosphatase activity in
milk has a great technological importance, due to its thermal resistance being similar to the pathogen with the highest thermal resis-
tance found in milk. So, this enzyme has been used as an efficiency index for the pasteurization of milk (Datta et al., 2005).
Some authors have studied the effect of HPH on milk alkaline phosphatase activity (Hayes et al., 2005; Datta et al., 2005; Picart
et al., 2006). Picart et al. (Picart et al., 2006) observed that HPH process using inlet temperature of 24 C was able to activate the
enzyme (up to 150 MPa), caused no changes at 175 MPa, and 94% inactivation at 300 MPa. In another study, Datta et al. (Datta
et al., 2005) observed no reduction of enzyme activity when the process was carried out at 200 MPa and inlet temperature was lower
than 15 C. On the other hand, these authors observed that the increase of the inlet temperature (up to 45 C) led to a reduction in
the enzyme activity. A complete inactivation of this enzyme was reached at 200 MPa, using inlet temperature higher than 50 C
(Datta et al., 2005). Similar results were obtained by Hayes et al. (Hayes et al., 2005), who verified the inactivation of this enzyme in
processes carried out at 250 MPa with an inlet temperature of 45 C. In both studies, the authors considered that the outlet
temperature (>75 C) was the determining factor for the observed inactivation (Hayes et al., 2005; Datta et al., 2005).
Amyloglucosidase
Amyloglucosidase (AMG) (EC 3.2.1.3) is an enzyme able to produce glucose from starch, mainly by hydrolysis of the L-1,4 gluco-
sidic bound (Tribst and Cristianini, 2012a; Svensson et al., 1982; Adeniran et al., 2010). The main application of AMG is the starch
saccharification (Mamo and Gessesse, 1999), which is used as ingredient in the food industry, or converted to high-quality ethanol
to be used in several areas, such as the cosmetic and pharmaceutical industries (Tribst and Cristianini, 2012a; Kumar and Satyanar-
ayana, 2009; Zanin and Moraes, 1998; Rani et al., 2000). In addition, AMG is used in the juice industry for the hydrolysis of the
starch naturally found in vegetables and some unripe fruits (Tribst and Cristianini, 2012a; Ribeiro et al., 2010).
HPH effects on AMG from Aspergillus niger were investigated by Tribst and Cristianini (Tribst and Cristianini, 2012a) who did not
observe changes in the enzyme activity for almost all samples processed up to 200 MPa at pH 2.9, 4.3, and 6.5, when the activity was
measured at 35 and 65 C (optimum temperature). However, when AMG was processed at pH 4.3 and its activity was evaluated at
optimum temperature, a slight increase in activity (5%–8%) was observed after processes > 100 MPa. Moreover, when activity was
measured at 80 C, a significant increase in the residual activity was observed after HPH processing at different pH. Sample homog-
enized at pH 2.9 showed a gradual activity increase, with maximum increment (100%) after processing at 200 MPa, while samples
processed at pH 4.3 and 6.5 showed, respectively, a maximum residual increase of 20% and 30% after homogenization at 100 MPa
(Tribst and Cristianini, 2012a). Therefore, the impact of HPH on AMG activity depends on the pressure and the pH of the enzyme
solution subjected to the processing. It is interesting to highlight that the main gain of residual activity occurred at high temperature,
which is particularly important from an industrial perspective, considering the huge application of AMG in the saccharification
process of the starch (to convert dextrins into glucose) and that this saccharification step occurs immediately after initial starch
hydrolysis by a-amylase at temperatures near 100 C (Tribst and Cristianini, 2012a).
b-Galactosidase
b-Galactosidase (EC 3.2.1.23) catalyzes the hydrolysis of lactose into glucose and galactose (Jurado et al., 2002; Mahoney et al.,
2002) and has been used in the production of lactose-free or low-lactose milk and dairy products (Tribst et al., 2012b). Considering
that lactose intolerance reaches 3%–70% of people from different origin, reduction of lactose content in dairy products can substan-
tially increase the consumption of these products (Tribst et al., 2012b; Jurado et al., 2002; Mahoney et al., 2002).
As a promising method for changing the enzyme activity and stability, Tribst et al. (Tribst et al., 2012b) evaluated the effect of
HPH (up to 150 MPa) on b-galactosidase from Kluyveromyces lactis at different pH. The results showed that the enzyme (at pH 7.0)
remained active at pressures up to 150 MPa and that enzyme stability under refrigerated storage was not affected. On the other
hand, a gradual loss of activity was observed when this enzyme was homogenized at non-optimum pH (6.4 and 8.0), with
minimum activity (30% residual activity) after processing at 150 MPa/pH 8.0. Thus at milk pH (6.8), b-galactosidase will be prob-
ably preserved by HPH, allowing the application of this technology in milk (aiming microbial reduction or functional properties
improvement) without inactivating b-galactosidase intentionally added to produce low-lactose or lactose-free products.
Glucose Oxidase
Glucose oxidase (GO) (EC 1.1.3.4) is a dimeric glycoprotein that catalyzes the oxidation of b-D-glucose to gluconic acid using
molecular oxygen as an electron acceptor with simultaneous hydrogen peroxide production (Tribst and Cristianini, 2012c; Bankar
et al., 2009; Fiedurek and Gromada, 1997).
GO has been used to decrease the oxygen content in packaged food, reducing the oxidation effects and, therefore, its undesirable
consequences on sensory and nutritional quality of these products (Tribst and Cristianini, 2012c; Bankar et al., 2009). Additionally,
GO is used as a biosensor for the determination of D-glucose content in body fluids, foodstuffs, beverages, and during fermentation
processes (Tribst and Cristianini, 2012c; Bankar et al., 2009; Rauf et al., 2006). GO is an unstable enzyme at pH extremes, high
temperatures, and aqueous solutions (Tribst and Cristianini, 2012c; Bankar et al., 2009). Therefore, GO stabilization against these
factors is required to improve its commercial applications.
In this context, the results obtained by Tribst and Cristianini (Tribst and Cristianini, 2012c) showed that, although the applica-
tion of low pressures (50 MPa in pH 5.0) has reduced the activity of commercial GO from Aspergillus niger, the HPH at 150 MPa
increased the enzyme activity by 25% at 75 C. Moreover, the storage stability of GO increased between 100% and 400% after
homogenization at different pressures.
The results confirmed that HPH is able to increase or decrease GO activity, depending on the processing parameters. This activity
change may be associated to continuous modifications in enzyme structure due to homogenization pressure and pH of solution,
responsible for different enzyme arrangement during processing (Tribst et al., 2014). The results found, including higher activity at
high temperature and greater stability in aqueous solution, indicate that HPH can increase the range of commercial GO
applications.
In another study, Tribst et al. (Tribst et al., 2014) evaluated the effect of HPH on GO activity at different pH values, thermal
stability, and structural conformation. HPH applied at 75 and 150 MPa promoted improvements in activity, especially at non-
optimal pH values (4.5 and 6.5) or low temperatures (15 C). In addition, the process at 75 MPa maintained the thermal resistance
of GO while the process at 150 MPa resulted in a decrease in the enzyme thermal resistance. These results are correlated with the
permanent changes in the GO secondary and tertiary structures after the process of HPH (Tribst et al., 2014).
Lactoperoxidase
Lactoperoxidase (EC 1.11.1.7) is an oxidoreductase that uses hydrogen peroxide to oxidize the thiocyanate ion to hypothiocyanate.
This enzyme is naturally found in raw milk, colostrum, and saliva and plays a role in the protection of animals against infections,
with bactericidal effect against Gram-negative bacteria and bacteriostatic against Gram-positive bacteria (Hayes et al., 2005; Vannini
et al., 2004; Naidu and Naidu, 2000).
Although raw milk contains lactoperoxidase and thiocyanate, there is not enough hydrogen peroxide to activate the enzyme
system (Naidu and Naidu, 2000; Harper, 2000). Some studies evaluated the effect of HPH on the antimicrobial activity of this
enzyme (Vannini et al., 2004). The results showed an increase in the lactoperoxidase antimicrobial activity at 75, 100, and
130 MPa with a drastic and intense reduction in the viability of Escherichia coli, Pseudomonas fragi, and Salmonella enteritidis in
skim milk (Vannini et al., 2004). On the other hand, milk processing at 250 MPa using 45 C (inlet temperature) inactivated
the lactoperoxidase in milk (Hayes et al., 2005).
Lipases
Lipases (EC 3.1.1.3; triacylglycerol hydrolases) are enzymes that hydrolyze the ester linkages of triacylglycerol molecules, releasing
free fatty acids, diacylglycerols, monoacylglycerols, and glycerol. In addition, these enzymes also catalyze reactions of esterification,
transesterification, and interesterification of lipids. Lipases may be from animal, vegetable, and microbial source. Commercial
lipases can be used in the formulation of detergents, in the modification of oils and fats, in the food and pharmaceutical industry,
in the production of biodiesel, in tanneries, and in the treatment of effluents, among other applications (Gandhi, 1997; Jaeger &
Eggert, 2002; Lanciotti et al., 2006; Vannini et al., 2008)
In food technology, lipases are used to improve aroma and texture in various products and to hydrolyze milk fat with release of
short-chain fatty acids, intensifying the development of aromas and flavors in cheeses, accelerating the ripening process (Lanciotti
et al., 2006; Vannini et al., 2008). In bakery, the addition of lipases to breads and cakes increases bread volume, improves texture
and aroma, and delays syneresis, extending the products’ shelf life. Lipases are also used for fat removal in meat and fish and for
improving the aroma of meat products. In addition, the enzymes can be employed in the production of emulsifiers, such as mono-
acylglycerols containing linoleic acid (Gandhi, 1997; Jaeger and Eggert, 2002). In contrast to desirable activity, lipases naturally
present in the food or produced by microorganisms may shorten the shelf life of fresh products due to the development of undesir-
able flavors.
Some studies have evaluated the effect of HPH on lipase activity (Datta et al., 2005; Iucci et al., 2008; Picart et al., 2006; Vannini
et al., 2008; Lanciotti et al., 2004). In milk, Datta et al. (Datta et al., 2005) verified an increase of 140% in activity of this enzyme at
200 MPa and inlet temperature of 10 C, with inactivation at 200 MPa if the temperature reached in the outlet of homogenization
valve was higher than 71 C (Datta et al., 2005). In addition, some authors evaluated cheeses made from milk processed by HPH at
100 MPa and reported an increase in lipolytic enzymes activity, from endogenous milk enzymes or those produced by microorgan-
isms (Pinho et al., 2011; Hayes and Kelly, 2003; Vannini et al., 2008; Lanciotti et al., 2004).
Thus, milk homogenized at these conditions can be used for cheese production, accelerating its ripening by improving flavor,
texture (Vannini et al., 2008), color, and aroma (Lanciotti et al., 2006). On the other hand, if HPH is used for milk processing, aim-
ing to extend the product’s shelf life (Oliveira et al., 2018), it must be associated with heat treatment to guarantee adequate level of
lipase inactivation, avoiding milk lipolysis during storage. From the observed results, it is clear that the HPH process parameters
(pressure and inlet temperature) can be strategically chosen to induce lipase activation or inactivation.
Lysozyme
Lysozyme is an N-acetyl-muramylhydrolase (EC 3.2.1.17) which hydrolyzes the b-glycosidic bond between the C1 of N-acetylmur-
amic acid and C4 of N-acetylglucosamine present in the peptidoglycan of the microbial cell wall, leading to wall lysis and cell death
(Tribst et al., 2008). Lysozyme is a low-cost, natural, and safe antimicrobial enzyme. This enzyme is widely distributed in nature,
being found in tears and mucus of animals. The chicken egg white is the main commercial source of lysozyme (Tribst et al., 2017).
Lysozyme is used in the food industry with the main purpose of avoiding the late blowing of hard and semihard cheeses such as
Gouda, Danbo, Grana Padano, and Emmental. Several patents show the efficacy of lysozyme, alone or in combination with other
hurdles, as a preservative for fruits, vegetables, meats, and beverages. Commercially, chewing gums, candies, and mouthwashes
already use lysozyme as a natural preservative (Losso et al., 2000).
Some studies have evaluated the effect of HPH on the enzyme (muramidase) and antimicrobial activities of lysozyme
(Vannini et al., 2004; Tribst et al., 2008; Iucci et al., 2007). Tribst et al. (Tribst et al., 2008) observed that lysozyme was resistant
to HPH (up to 200 MPa), without loss of muramidase and antimicrobial activity. On the other hand, an intense reduction on mur-
amidase activity was observed at pressures above 250 MPa.
In addition, other studies showed increased antimicrobial activity of lysozyme processed at 75 MPa (Vannini et al., 2004) or
100 MPa (Iucci et al., 2007). For example, combining lysozyme and pressure homogenization at 150–170 MPa, a greater reduction
in Lactobacillus brevis count was observed compared to the HPH processing applied alone (Tribst et al., 2008). These results highlight
that combining mild processing conditions of HPH with lysozyme can guarantee adequate levels of microbial inactivation, safety by
processing at lower pressures, and, consequently, involving equipment of lower cost.
In another study, Tribst et al. (Tribst et al., 2017) evaluated the effect of HPH on muramidase and antimicrobial activities of hen
egg white lysozyme. The results showed an increase on muramidase activity by up to 29% under mild process conditions
(<120 MPa at 20 C), especially at non-optimum conditions of pH and temperature for the enzyme. When processes were carried
out at 50 C, lower activation (<18%) or inactivation (up to 20% at 180 MPa) was observed depending on the pH and temper-
ature evaluated (Tribst et al., 2017). These results indicate that HPH processing under severe conditions (pressure, inlet tempera-
ture) delivered enough energy to result in undesirable unfolding of lysozyme. In addition, although an increase in muramidase
activity was observed, no improvement of lysozyme of inhibitory effect against Bacillus cereus and Geobacillus stearothermophilus
was observed (Tribst et al., 2017).
Finally, Tribst et al. (Tribst et al., 2018) using X-ray crystallography showed discrete and transient alterations in lysozyme struc-
ture after HPH. In addition, these authors verified a higher activity in the presence of NaCl (increase up to 6 times at pH 4.5) and
a higher resistance to low pH (with activity increase up to 110% at pH 4.0). Therefore, the results highlighted that HPH reversibly
changed lysozyme functionality in different ways and that it can be an interesting alternative for improving lysozyme performance.
Pectate Lyase
Pectate lyase (EC 4.2.2.2) is an enzyme that hydrolyzes pectic fraction of fruit and vegetable products, altering viscosity and consis-
tency of juices, resulting in undesirable phase separation (Calligaris et al., 2012; Payasi and Sanwal, 2003).
In this context, Calligaris et al. (Calligaris et al., 2012) used HPH processing to promote the inactivation of this enzyme in
banana juice. These authors verified that the processes carried out above 150 MPa (4 C inlet temperature) using a prototype
unit homogenizer equipped with a single homogenization R design valve (flow rate of 200 L/h and maximum pressure of
400 MPa) caused an irreversible inactivation of this enzyme. According to these authors, it is assumed that the mechanical stress
applied by the homogenization valve promoted irreversible changes in the structural conformation of this enzyme leading to inac-
tivation (Calligaris et al., 2012). On the other hand, these authors verified an activation of up to 262% in PL activity after the process
of 100 MPa (4 C inlet temperature) using lab-scale high-pressure homogenizer (flow rate of 10 L/h) of two stages, with ceramic
ball valves (Calligaris et al., 2012). These results show that activation/inactivation of PL is dependent on the applied pressure, valve
design, and the flow conditions of the equipment. Thus, in addition to the process conditions (homogenization pressure and inlet
temperature), other parameters (such as valve design) should be considered to determine the HPH effects on each enzyme.
Pectin Methylesterase
The pectinolytic enzymes hydrolyze pectins of fruit and vegetable tissue. These enzymes are found naturally in plants and are group-
ed into three types of enzymes: de-esterifying or demethoxylating agents (e.g., pectin methylesterase (PME), EC 3.1.1.11dwhich
remove methyl ester groups), depolymerizing agents, e.g., polygalacturonase (PG, which catalyze the cleavage of the glycosidic
bonds of the pectic substances), and protopectinases (which solubilize protopectin to form pectin).
Pectinolytic enzymes can be applied in many technological operations in the food industry. They are used in the fruit juice indus-
tries to reduce viscosity (improving filtration and clarification efficiency), in the preliminary treatment of grapes in wineries, in
maceration, in liquefaction and extraction of juices from plant tissue, in tea fermentation, in coffee and cocoa (improving the extrac-
tion of vegetable oils), and in the extraction of tomato pulp. Pectinases are also used to reduce excessive bitterness in citrus peels,
restore the aroma lost during drying, and increase the amount of antioxidant agents in extra virgin olive oil (Uenojo and Pastore,
2007). On the other hand, these enzymes, especially PME, may cause phase-separation in cloudy juices, negatively affecting their
appearance. In this way, many authors studied juice processing by HPH aiming at the inactivation of PME (Lacroix et al., 2005;
Welti-Chanes et al., 2009; Velázquez-Estrada et al., 2012; Navarro et al., 2014; Suárez-Jacobo et al., 2012; Carbonell et al., 2013).
Lacroix et al. (Lacroix et al., 2005) used HPH alone or combined with preheating to inactivate PME in orange juice. These authors
observed that the HPH at 170 MPa (1–5 passes) without heating reduced only 20% of PME activity. Moreover, the same study re-
ported that the thermal process at 50 C/10 min and the juice pH reduction to 3.0 were more efficient than the HPH processing,
being able to reduce up to 90% of the PME activity (Lacroix et al., 2005).
In another study, Welti-Chanes et al. (Welti-Chanes et al., 2009) evaluated the effect of HPH on PME activity in Valencia Late
orange juice, which was processed at pressures up to 250 MPa for five passes and three inlet temperatures (22, 35, and 45 C). A
reduction of up to 50.4% of PME activity was observed in juice homogenized after one pass at 250 MPa/45 C. In addition, the
increase in the number of passes increased the effectiveness of PME inactivation, reaching reduction of up to 80% after processing
at 250 MPa/5 passes (Welti-Chanes et al., 2009). These authors noted that HPH processing could be an alternative to reduce PME
activity, and consequently avoid sensory and physiochemical changes in the juice (Welti-Chanes et al., 2009).
Velázquez-Estrada et al. (Velázquez-Estrada et al., 2012) also reported the effectiveness of the HPH process for PME inactivation
in orange juice, with a maximum reduction of 96% after processing of 300 MPa at 20 C. Similar results were obtained by Suárez-
Jacobo et al. (Suárez-Jacobo et al., 2012), who observed absence of PME activity in apple juice processed at 300 MPa and using 4 C
of inlet temperature. On the other hand, Carbonell et al. (Carbonell et al., 2013), using lower pressures (up to 150 MPa), observed
a maximum reduction of 75% in PME activity in Lane Late orange juice processed at inlet temperature of 31 C. Using similar pres-
sure and temperature conditions (up to 150 MPa/31 C inlet temperature), Navarro et al. (Navarro et al., 2014) obtained greater
reductions in the activity of PME (above 90%) in Lane Late oranges and clementine juices due to the samples’ pH reduction to
3.1. Thus, these results show that the effectiveness of HPH on PME inactivation depends on the applied process conditions (pressure
and inlet temperature), fruit type, enzyme characteristics (probably concentration and isoenzymes), pulp concentration, and
juice pH.
Peroxidase
Peroxidase (POD) (EC 1.11.1.x) is an oxidoreductase that catalyzes the peroxidation of several molecules, including phenolic
compounds, using hydrogen peroxide in the initial stage of the reaction as an electron acceptor. This enzyme is known for its
high thermal resistance attributed to the numerous isoenzymes found in diverse food/matrix. The products of the POD reaction
imply the modification of the sensory characteristics of fruits and vegetables, especially when PPO is active, due to a synergistic effect
between the enzymes. Therefore, several strategies are adopted to inhibit POD activity.
Yi et al. (Yi et al., 2018) evaluated the effect of HPH processing (60 MPa and 4 C of inlet temperature) on this enzyme and
observed that no reduction in POD activity occurred in apple juice and a mixture of apple juice and kiwi fruit puree. This was ex-
pected considering the low pressure and inlet temperature used in the test and that heat-resistant enzymes are normally resistant to
HPH processing.
Despite the importance of POD inactivation, there are not many studies that describe the effect of HPH on these enzymes. It is
possible that the relative low efficiency of HPH processing in inactivation of enzymes discouraged research with the objective of
inactivating this heat-resistant enzyme.
Polyphenol oxidase
Polyphenol oxidase (PPO) (EC 1.10.3.2 or EC 1.14.18.1) is a metalloprotein that catalyzes hydroxylation reactions of monophe-
nols present on plants to diphenols and, sequentially, the oxidation of the diphenols to quinones. Quinones are unstable and form
melanins by polymerization, which are compounds of high molecular weight and brown color. Therefore, PPO is considered an
important enzyme in processing of vegetables, due its ability to cause browning of products including potato, apple, pear,
plum, and peach (Liu et al., 2009a; Espin et al., 1998; Alfred, 2006; Marusek et al., 2006; Yasuyuki et al., 2006; Suárez-Jacobo
et al., 2012).
Enzymatic browning is responsible for 50% of the rejection of fruits and vegetables by consumers (Espin et al., 1998; Yasuyuki
et al., 2006; Marusek et al., 2006; Alfred, 2006). Thus, strategies such as cooling, vacuum packaging, pH reduction, and uses of addi-
tives that react with the enzyme or with the intermediate compounds formed are applied to minimize the oxidative reaction and the
deterioration of in natura fruits and vegetables. In addition, heat treatment can be used to inactivate PPO in the processed products.
However, time and temperature normally required for PPO inactivation results in undesirable sensory and nutritional changes in
several vegetables.
As an alternative to heat treatment, some authors studied the effect of HPH on PPO activity in fruits and vegetables (Liu et al.,
2009; Bot et al., 2018; Liu et al., 2009; Suárez-Jacobo et al., 2012). From the results, it was suggested that pressures of 300 MPa or
higher can be effective for PPO inactivation, as observed by Suárez-Jacobo et al. (Suárez-Jacobo et al., 2012) in apple juice. On the
other hand, results from other authors (that carried out processes at 150 MPa) showed that HPH was almost ineffective and, in
some cases, induced enzyme activation.
Liu et al. (Liu et al., 2009b) observed activation of mushroom PPO at pressures up to 150 MPa for three passes (12% increase).
These authors verified that this increase is correlated with conformational changes of the enzyme. The circular dichroism analysis
demonstrated that the secondary structures such as a-helix were affected, with decrease of a-helix content when PPO activity
increased. In addition, changes were observed in the tertiary structure of the enzyme, in which the tyrosine and tryptophan residues
of mushroom PPO were exposed to solvent and the sulfhydryl group content on the surface of mushroom PPO was increased. Thus,
these results highlight that the HPH process was able to promote measurable structural changes in mushroom PPO, which can be
associated to the increase in activity.
In another study, Liu et al. (Liu et al., 2009a) studied the effect of HPH on PPO from Chinese pear (Pyrus pyrifolia Nakai). These
authors observed an increase of up to 83% in activity after HPH processing at 180 MPa at room temperature. In addition, PPO
activity increased with subsequent processing cycles, independent on the applied pressure (Liu et al., 2009a). This result again high-
lights the high resistance of this enzyme to HPH processing.
Bot et al. (Bot et al., 2018) evaluated the effect of HPH and ultrasound processing (individually or combined) on the activity of
PPO in apple juice. For 50% inactivation of PPO activity, 10 passages were required at 150 MPa using 8 C as inlet temperature.
Moreover, no sufficient gains were observed in the HPH ultrasound processing to justify the combined use of the technologies.
In addition, the results showed that the temperature involved in processing affected the enzyme activity rather than homogenization
pressure applied (Bot et al., 2018), confirming the high resistance to the HPH process of PPO from different sources.
These results of HPH effects on PPO reinforce the notion that different responses on enzyme activity can be observed depending
on the food matrix, enzyme concentration, the type of enzyme, and the applied process conditions. This reinforces the need for the
evaluation of each product individually.
Proteases
Proteases (EC 3.4.x.x) are enzymes that catalyze the hydrolysis of proteins into peptides and amino acids (Tribst et al., 2012a;
Sumantha et al., 2006). They are a large and complex group of enzymes, which differ in properties such as substrate specificity,
the active site and the catalytic mechanism, the optimum pH and temperature, and stability profile (Tribst et al., 2012a; Sumantha
et al., 2006). Proteases are classified as acid, neutral, and alkaline, depending on the pH of maximum activity (Esakkiraj et al., 2009);
moreover, they are also subclassified as carboxyl protease, cysteine protease, metalloprotease, and serine protease, according to their
active sites and sensitivity to inhibitors (Tribst et al., 2012a; Hartley, 1960).
Proteases have 60%–65% of the commercial enzymes market share, being used mainly in detergents, meat, beer, animal feed,
leather, and dairy industries (Tribst et al., 2012a; Esakkiraj et al., 2009).
Alkaline Protease from Bacillus licheniformis

Aguilar et al. (Aguilar et al., 2018b) evaluated the effect of HPH on activity and stability of alkaline protease from B. licheniformis LBA
46. The enzyme was processed at pressures up to 200 MPa at different pH values (pH of 4, 7, and 9) and the activity was measured at
40, 60, and 90 C immediately after processing and after 24h of refrigerated storage. No changes in protease activity and stability
were observed at the studied conditions, highlighting the relatively high resistance of this enzyme to HPH processing.
Fungal Protease Obtained from Rhizomucor miehei

Calf rennet is the traditional milk coagulant that is used in cheese processing; however, its use is limited due to low availability
(Andrén et al., 2011; Chitpinityol and Crabbe, 1998). The protease from Rhizomucor miehei is the most used rennet substitute among
microbial sources, with 35% of the coagulants market share in France (Andrén et al., 2011). It is an aspartic protease (EC 3.4.23.23)
with a single polypeptide chain and highly similar arrangement to chymosin (Chitpinityol and Crabbe, 1998). Moreover, this
fungal protease has a high thermal stability, which is a disadvantage due to the difficulty in stabilizing the whey obtained from
cheese manufacturing (Kumar et al., 2010) without significantly damaging the whey functionality (Leite Júnior et al., 2015b; Har-
boe et al., 2010).
In this context, Leite Junior et al. (Leite Júnior et al., 2015b) evaluated the effect of HPH (up to 190 MPa using three subsequent
passes) on the proteolytic activity (PA) and milk-clotting activity (MCA) of this enzyme at low (2%) and high (20%) concentra-
tions. In addition, the rheological behavior of milk coagulation using the HPH processed enzyme was also evaluated. The results
showed that the energy supplied from HPH to enzyme at low concentration (2%) was not enough to promote positive changes in
the studied protease. The high resistance of protease from R. miehei to HPH might be explained due to the high amount of glyco-
sylated fractions in the enzyme, which is recognized as a resistant structure that protects the protease from physical denaturation
caused by heating (Leite Júnior et al., 2015b; Yang et al., 1997), and, possibly, also due to HPH denaturing effects.
On the other hand, a slight increase in PA (3%) and MCA (10%) was observed for the enzymes processed at 190 MPa at high
concentration (20%), with consequently faster clotting and higher consistency of the milk gel (Leite Júnior et al., 2015b). These
results suggest that high enzyme concentration increases the molecular collision in the homogenization valve and that this increase
in shear was necessary to modify the protease in the observed way.
Neutral Protease from Bacillus subtilis

Neutral proteases are applied in milk and soy protein modification, nitrogen control, cleaning processes, and protein synthesis
(Tribst et al., 2012a; Sumantha et al., 2006; Schallmey et al., 2004).
Tribst et al. (Tribst et al., 2012a) evaluated the effect of HPH on the activity and stability of a neutral protease from Bacillus sub-
tilis. The results showed that this process was able to change the optimum temperature of this enzyme from 55 to 20 C after HPH at
200 MPa with a 30% increase in enzyme activity (Tribst et al., 2012a). The authors noted that these changes can be reversible or
irreversible, depending on the homogenization pressure, inlet temperature, and enzyme storage conditions. For example, the
activity reduction observed after the processing of 200 MPa at 55 C was reversible after storage. In addition, the homogenized
enzyme at 200 MPa/20 C showed a slight activity increase during storage.
Papain
Latex of the green papaya fruit (Carica papaya) contains a mixture of cysteine endopeptidases, known as papain (EC 3.4.22.2).
Papain is used extensively in the pharmaceutical, cosmetic, and detergent industries, as well as in the dental and tannery applica-
tions. In the food industry, papain is applied in the clarification of beers for the hydrolysis of precipitated proteins that promote
turbidity of the beverage. It is also used in the softening of meats without altering their flavor and the aroma. Its application to
cheeses is not feasible due to high nonspecific PA, which can result in undesirable bitterness in cheeses (Moraes et al., 1994; Ayello
and Cuddigan, 2004; Sangeetha and Abraham, 2006; Amri et al., 2012).
The effect of the HPH on papain was studied by Liu et al. (Liu et al., 2010b), who observed a slow and continuous activity reduc-
tion when samples were homogenized between 120 and 180 MPa (10% of reduction at 180 MPa), and a reduction in the enzyme
stability after 24h of storage. This reduction in the stability was attributed to the conformational change in the enzyme structure,
which was confirmed by amino acid residues analysis (Liu et al., 2010b).
Pepsin
Pepsin comprises the major group of acid proteases from the stomach of warm-blooded animals. Pepsin hydrolyzes N- and
C-terminal bonds, but also cleaves synthetic dipeptides such as Glu-Tyr or Phe-Phe with less efficiency (Leite Júnior et al.,
2015a). Porcine pepsin (EC 3.4.23.1) is available as a commercial product and is applied in food industry for hydrolysis of
meat melting, soy protein, and gelatin and as a reagent for the method of detection of Trichinella spiralis. In addition, this enzyme
exhibits milk coagulation activity by having specificity over the Phe105-Met106 binding of k-casein. However, high nonspecific
activity causes undesirable changes in taste and aroma of fresh cheeses during storage (Leite Júnior et al., 2015a; Andrén et al.,
2011; Chitpinityol and Crabbe, 1998; TangPepsin et al., 2013).
Aiming to improve the application of porcine pepsin in the dairy industry, Leite Junior et al. (Leite Júnior et al., 2015a) inves-
tigated the effect of HPH on this enzyme (proteolytic and milk-clotting activities), and the consequences of using the processed
enzyme in milk coagulation and gel formation. These authors observed that the process did not promote modification in the
nonspecific activity after processing; however, during storage (60 days) a slight reduction of 5% was observed for samples processed
at 50, 100, and 150 MPa. In addition, HPH increased the MCA of the enzyme processed at 150 MPa, being 15% higher than the
non-processed samples after 60 days of storage. Due to this increase, the HPH processed enzyme produced faster aggregation
and a more consistent milk gel (a more compact protein network with lower porosity) when compared with the nonprocessed
enzyme. Thus, HPH was able to change the proteolytic characteristics of porcine pepsin, with improvements in the milk coagulation
step and gel characteristics. Despite the observed improvements, it is important to note that HPH processing did not change porcine
pepsin sufficiently to guarantee a proteolytic profile adequate for cheese production. Therefore, HPH processed enzyme should be
used with caution in this application.
Plasmin
Proteases may have undesirable effect on milk and dairy productsdan example is plasmin (EC 3.4.21.7) (endogenous milk
protease) which acts on casein (especially on the b-casein fraction), favoring the formation of short-chain peptides and bitter taste
in cheeses and the phenomenon of age gelation in UHT milk. Such effects may be limited by the reduction of the plasmin concen-
tration in the milk, which can be obtained through strict sanitary control of the cows, since plasmin is produced as the immune
response of the animals in situations of infection.
Another strategy would be the use of HPH to promote the inactivation of this enzyme. Although some studies have described
reductions in plasmin activity by the HPH process (Hayes and Kelly, 2003), Iucci et al. (Iucci et al., 2008) showed that previously
reported reductions in plasmin activity by HPH processing probably was not related to enzyme inactivation by unfolding but to the
association of casein micelles (where plasmin is naturally found in milk) with milk fat globules during homogenization. As a result,
part of the plasmin is removed by centrifugation before the residual activity measurement (Huppertz et al., 2011), causing an
analytical error. Thus, no reduction in activity was observed in the processes carried out up to 200 MPa (Iucci et al., 2008). This
result has highlighted that plasmin is stable to inactivation by HPH in milk. On the other hand, reductions in plasmin activity
caused by HPH appear to be more relevant by combining high pressure and high temperature. For example, milk processing at
200 MPa and inlet temperature at 50 C (outlet at 80 C) resulted in a 90% reduction in plasmin activity (Datta et al., 2005).
Rennet
Rennet is the extract obtained from the abomasum of ruminant animals. This extract is rich in acid proteinases that have coagulant
activity on the milk under appropriate conditions of temperature and pH. Proteinases found in ruminant abomasum are chymosin
(EC 3.4.23.4), pepsin A (EC 3.4.23.1), pepsin B (EC 3.4.23.2), and gastricsin (EC 3.4.23.3) (Andrén et al., 2011; Chitpinityol and
Crabbe, 1998; Fox et al., 2004). Currently, only 20%–30% of the cheeses produced in the world use rennet (Leite Júnior et al., 2014;
Andrén, 2011; Chitpinityol & Crabbe, 1998; Fox et al., 2004).
In this context, Leite Junior et al. (Leite Júnior et al., 2014) used HPH process to induce improvements in the hydrolytic char-
acteristics of rennet. The results obtained for HPH showed that this process was able to increase the MCA of rennet, especially those
that have chymosin in its constituents. Specifically, HPH caused a reduction in PA with increase in pressure (up to 52% loss of
activity at 190 MPa) without affecting the milk coagulation activity. In addition, rheological tests showed higher G0 values for
the milk coagulated with the enzyme processed at 190 MPa (Leite Júnior et al., 2014). The changes caused by HPH in the rennet
may improve the cheese manufacturing process and the quality of the product (higher consistency of the milk gel and lower prote-
olysis during storage).
Trypsin
Trypsin (EC 3.4.21.4) is a serine proteinase and acts by cleaving the ester and peptide bonds involving the carboxyl groups of argi-
nine and lysine (Fuchise et al., 2009). Trypsin is isolated from the mammalian pancreas and also from different fish species
(Klomklao et al., 2009). This enzyme has been widely used in the leather manufacturing process, in the biological area during
proteomic experiments, and in the food industry (Liu et al., 2010a). An application of trypsin in the food industry involves the
production of a natural seafood flavor (Barcia et al., 2008).
The effect of HPH on trypsin was studied by Liu et al. (Liu et al., 2010a), who observed that homogenization at 80 MPa
enhanced the pH stability and thermal stability of the enzyme (increase of 10%), although it did not show any effect on its activity
(Liu et al., 2010a). These results are correlated with modifications promoted by the HPH process on the conformational structure of
this enzyme. In this context, the process was able to increase the maximum emission fluorescence intensity (Liu et al., 2010a), which
indicates a gradual exposure of hydrophobic groups initially buried in the interior of the molecules (Yin et al., 2008). In addition,
the increase in exposed SH contents as well as the decrease in total SH contents and in a-helix intensity suggested that trypsin was
unfolded after HPH (Liu et al., 2010a).
High-Pressure Homogenization of Food Products: The Challenge for Inactivating Endogenous Enzymes
Fruit- and Vegetable-Based Products
In fruit- and vegetable-based products, the activity of native enzymes is almost always undesirable. Endogenous pectinases (PME,
PG, and PL) cause juice stability loss due to pulp hydrolysis (Terefe et al., 2014; Dumay et al., 2013). The oxidase enzymes (mainly
POD and PPO) cause undesirable enzymatic browning, noncharacteristic flavor formation, and nutrient loss (Terefe et al., 2014;
Dumay et al., 2013). Commonly, the pectinase and oxidase enzymes in fruits and vegetables have isoenzymes which are very resis-
tant to inactivation by physical processes (heating and pressure) (Lacroix et al., 2005). In fact, for most of fruit- and vegetables-based
products, processing parameters (pressure, time, and temperature) are designed with the inactivation of the most resistant enzyme
as targets, instead of microbial reduction, since enzyme inactivation needs more intense process than that required to ensure micro-
biological safety of the product in pasteurization (Lacroix et al., 2005).
Among plant endogenous enzymes, inactivation of PME is the most extensively studied, in diverse matrices such as orange
(Lacroix et al., 2005; Welti-Chanes et al., 2009; Velázquez-Estrada et al., 2012), clementine juice (Navarro et al., 2014), peach juice
(Yildiz, 2019), and mixture of apple and kiwi juices (Yi et al., 2018). The effect on PPO is the second most studied, and inactivation
effect was studied in apple juice (McKay et al., 2011; Bot et al., 2018), Chinese pear (Liu et al., 2009a), mushroom extract (Liu et al.,
2009b), peach juice (Yildiz, 2019), and apple mixed with kiwi juice (Yi et al., 2018). PL inactivation was only determined in banana
juice (Calligaris et al., 2012). Moreover, several studies suggested reduction of enzymes that causes juices browning (especially PPO)
due to the limited color changes observed during storage in products such as on strawberry juice (Karacam et al., 2015) and Annurca
apple juice (Maresca et al., 2011).
In most of the studies, HPH failed when applied alone to inactive these endogenous enzymes (Lacroix et al., 2005; Velázque-
z-Estrada et al., 2012; Yildiz, 2019; Liu et al., 2009b) and commonly the inactivation increased with increased thermal effects asso-
ciated with the processes (e.g., initial warming for blanching, high inlet temperature, and high temperature reached in the
homogenization valve). Other strategies, as pH reduction and use of multiple subsequent HPH processing, seem to enhance the
inactivation of endogenous enzymes.
In conclusion, the control of undesirable enzyme activity can be achieved by: (i) processing at 400 MPa associated to mild
temperature (50–80 C), targeting strong reduction of the activity of baroresistant enzymes and (ii) use of mild pressure condition
(up to 300 MPa) and temperatures aiming at inactivation of sensitive enzymes, together with other technological hurdles to control
the residual activity during product shelf life (e.g., package with good barrier to oxygen, use of hydrocolloids/stabilizers to avoid
phase separation and antioxidants). Moreover, it is useful to teach consumers that visible changes induced by enzymes naturally
occur and do not represent safety risk (Leite Júnior et al., 2017).
On the other hand, HPH can be strategically used to improve the activity and stability of commercial enzymes, which are used as
processing aids in the industrial processing of fruits and vegetables, mainly for extraction, clarification, and filtration. In this case,
enzymes are preprocessed to improve their performance. For example, it was observed that HPH process changed the optimum
temperature of a neutral protease from B. subtilis from 55 to 20 C, which is very interesting from an industrial perspective (Tribst
et al., 2012a). Moreover, HPH at 150 MPa increased the activity of GO at 75 C and the enzyme stability during refrigerated storage
in solutions, which facilitates oxygen consumption in products to which the enzyme is added to extend their shelf life. Therefore, the
use of GO processed by HPH tends to improve the sensory and nutritional quality of stored juices by limiting the occurrence of
oxidation (Tribst and Cristianini, 2012c). For this enzyme, an activity increase of 150% was observed after processing for three cycles
of HPH at 150 MPa (Tribst et al., 2013). Finally, HPH at 100 MPa was able to increase the activity of amyloglucosidase at temper-
atures lower than 100 C, facilitating enzyme application for clarification of juices with high starch content (Tribst and Cristianini,
2012a). For more specific information about each enzyme, please read section “Effects of high pressure homogenization on
specific food enzymes”.
Milk and Dairy Products

Enzymes in dairy products include: (i) endogenous enzymes of milk (phosphatase, lactoperoxidase, lysozyme, lactoferrin, and
plasmin), (ii) proteases and lipases produced by microorganisms naturally found in milk (lactic acid bacteria) or from contamina-
tion (especially caused by psychrotrophic bacteria present in milk stored for long time before processing), and (iii) enzymes from
bacterial starters intentionally added in fermented dairy products, such as yogurt, fermented milk, and cheese (Huppertz et al.,
2004; Vannini et al., 2004; Delgado et al., 2012).
The enzymes in milk are not able to cause perceptible changes in raw or pasteurized milk due to the relative short shelf life and
low storage temperature of milk, which decreases enzyme activity. To the contrary, commercial sterile milk (obtained by ultra high-
temperature processing) are degraded by proteases (endogenous and from contaminants), resulting in age gelation phenomena,
responsible for the end of UHT milk shelf life (Huppertz et al., 2004; Oliveira et al., 2018). In dairy products, the activity of
some enzyme is always desirable (e.g., enzymes that act as antimicrobials, preventing dairy product contamination) whereas the
activity of other enzymes is desirable depending on the product (e.g., lipase and protease are important for cheese ripening, but
high activity can result in rancidity and/or bitter flavor) (Delgado et al., 2012).
In general, enzymes are more resistant to HPH than thermal processing for similar degree of microbial inactivation (Leite Júnior
et al., 2017). Therefore, HPH can have a positive effect for the preservation or improvement of the antimicrobial activity of lacto-
peroxidase, lactoferrin, and lysozyme (Vannini et al., 2004; Iucci et al., 2007; Patrignani et al., 2013). On the other hand, insufficient
inactivation of enzymes by HPH may result in defects of flavor and texture that can occur due to intense activity of proteases and
lipases (Dumay et al., 2013; Oliveira et al., 2018). In addition to effects of HPH on individual enzymes, it is important to consider
how the process affects the milk globally. For example, results obtained by Oliveira et al. (Oliveira et al., 2018) showed that
although HPH was not able to change the activity of the protease from Pseudomonas fluorescens, it was able to reduce 50% of prote-
olysis in whole milk because the homogenization caused fat globule fragmentation and the fragments absorbed the protein, which
reduced protease activity. Thus, the use of HPH as an alternative to thermal pasteurization of milk prior to the manufacturing of
dairy products of extended shelf life needs to be evaluated carefully. If necessary, HPH can be combined with mild/high temperature
(high inlet temperature or holding time at high temperature after homogenization valve) to guarantee the process effectiveness to
inactivate undesirable enzymes (Huppertz et al., 2004).
Conversely, several works highlight the use of HPH to induce enzyme production by microorganisms due to baric stress and lysis
of microbial cells, which facilitate the removal of intracellular microbial endoenzymes (Hayes and Kelly, 2003; Vannini et al.,
2008). These phenomena can be strategically used to increase enzyme activity in cheeses during ripening (lower pressures) for
reducing the time to reach the desirable flavor and texture (Vannini et al., 2008; Lanciotti et al., 2004). Moreover, some works
showed significant gains of activity and specificity of milk coagulants processed in buffer, highlighting HPH as a technology that
can be used to improve the performance of this kind of enzymes (Leite Júnior et al., 2014, 2015a, 2015b). For more specific infor-
mation about each enzyme, please read section “Effects of high pressure homogenization on specific food enzymes”.
Economic Perspectives, Future Challenges, and Final Remarks
HPH is considered as a promising technology for fluid food processing, especially for heat-sensitive products, such as fruit- and
vegetable-based products. However, in general, HPH processing alone is not enough to obtain microbiologically safe food and/
or to adequately inactivate undesirable endogenous enzymes. Therefore, for food preservation, HPH process can be combined
with other complementary technologies (hurdle technology), such as use of packaging with specific barrier properties, refrigerated
storage, and application of mild heating. For example, an initial warming of the fluid product can be performed prior to the passage
through the homogenizer, increasing the inlet temperature and consequently, the temperature in the homogenization valve, which
results in a greater inactivation degree of endogenous enzymes.
Depending on the processing parameters, medium characteristics, and type of enzyme, HPH can not only inactivate enzymes,
but also activate or stabilize them. Such effects of activation and stabilization are particularly important for enzymes of commercial
interest. Unlike detrimental endogenous enzymes, which need to be inactivated, the maintenance of enzymes of commercial
interest is desirable. HPH technology can be used as a procedure to improve enzyme activity, specificity, and stability, especially
at non-optimum conditions, which is very interesting from an industrial perspective. These improvements can lead to reduced
enzymes costs and better performance, resulting in higher products yield, novel enzymes application, faster enzymatic processes,
and products with better sensory and chemical characteristics.
In addition to enzyme modification, the application of HPH technology has also been found to promote desirable changes on
food structure with positive impact on its properties, such as improving physical stability, reducing or even avoiding pulp sedimen-
tation during storage, increasing consistency and viscosity, and improving the bioaccessibility of nutrients. These changes permit
a reduced need of additives, which is particularly interesting to obtain clean-label products.
Therefore, by choosing an appropriate equipment geometry, HPH configuration system, inlet temperature, homogenization
pressure, number of passes, and other processing parameters, HPH can be a valuable technology to potentially improve the sensory
and nutritional quality of liquid foods, extend shelf life, modify product formulation, and increase performance and stability of
some enzymes of commercial interest. HPH is considered as a sustainable process that has potential application in a large number
of liquid foods, including fruit- and vegetable-based products (juice, puree, nectar, and pulp), and milk and dairy products
(yoghurt, cheese).
Nevertheless, an important challenge facing the HPH technology and restraining its broad use in food industry is the lack of
validation at large scale, since the majority of studies have been carried out in lab scale (Misra et al., 2017), which is limiting uptake
by dairy, juice, and puree industries. In fact, the availability of HPH equipment at the industrial scale is still limited. More efforts are
needed to develop reliable systems able to perform at industrially applicable and relevant capacity for food processing. However,
this issue of scale may not be a limitation when HPH technology is used to promote desirable changes on enzyme solutions.
Overall, the main drawbacks of HPH technology are the costs of equipment, and the high energy and equipment maintenance
cost due to wear. Equipment erosion can cause deleterious reactions in the product, and requires high costs and frequency of main-
tenance. Research in the area is increasing and engineering development is expected to increase energy efficiency and reduce mate-
rials’ abrasion.
In conclusion, the future of HPH technology will depend upon the research toward the development of industrial systems and
improvement of HPH equipment in terms of functionality, flexibility, and capacity to work in a food processing line and wear life-
time. Continuous development of new products differentiated from traditional ones by sensory and structural features or nutri-
tional and functional properties is also important to expand the use of HPH technology.
Acknowledgment
The authors are also grateful to the National Council for Scientific and Technological Development (CNPq, Brazil) for funding the Postdoctoral grant
of MTK Kubo (155725/2018-1) and the productivity grant of PED Augusto (306557/2017-7).
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Further Reading
Iyer, P.V., Ananthanarayan, L., 2008. Enzyme stability and stabilization – aqueous and non-aqueous environment. Process Biochem. 43, 1019–1032 [this review provides deep
explanation about enzymes structure and its relationship with enzyme activity and stability].
Zamora, A., Guamis, B., 2015. Opportunities for ultra-high-pressure homogenisation (UHPH) for the food industry. Food Eng. Rev. 7 (2), 130–142 [this review provides more details
concerning the evolution of HPH and shows general effects on other food constituents and microbial contamination].

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High-Pressure Homogenization On Food Enzymes: © 2020 Elsevier Inc. All Rights Reserved

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High-Pressure Homogenization on Food Enzymes

Reference Module in Food Sciences https://doi.org/10.1016/B978-0-08-100596-5.22999-5 1

Figure 2 Schematic representation of enzymes in native and unfolded state.

Mechanisms Involved in Enzyme Activation, Inactivation, and Stabilization

110 120 120

Residual activity (%)

100 100 100

General Effects of High-Pressure Homogenization on Enzymes

PME Residual activity (%)

Residual activity (%)

sensitive enzyme activated enzyme resistant enzyme

pH 3.9 pH 3.5 pH 3.1

Residual activity (%)

50 MPa 100 MPa 150 MPa

Effects of High-Pressure Homogenization on Specific Food Enzymes

Alkaline Protease from Bacillus licheniformis

Fungal Protease Obtained from Rhizomucor miehei

Neutral Protease from Bacillus subtilis

Milk and Dairy Products

Economic Perspectives, Future Challenges, and Final Remarks

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