doi: 10.1111/1471-0307.

12622

REVIEW
Impact of nonthermal processing on different milk
enzymes
TALHA AHMAD,1 MUHAMMAD ZUBAIR BUTT,1 RANA MUHAMMAD
AADIL*1 MUHAMMAD INAM-UR-RAHEEM,1 ABDULLAH,2
~ S,4 CELSO F
A L A A E L - D I N B E K H I T , 3 J O N A S T G U I M A R AE
4 4,5
BALTHAZAR, RAMOM S ROCHA, ERICK A ESMERINO,4
^ I C A Q F R E I T A S , 4 M AR
M ON  C I A C S I L V A , 5 A Y S H A S A M E E N 1 and
5
ADRIANO G CRUZ
1
National Institute of Food Science and Technology, University of Agriculture, Faisalabad 38000 Pakistan,
2
Department of Food Science and Human Nutrition, University of Veterinary and Animal Sciences, Lahore, 54000
Pakistan, 3Department of Food Science, University of Otago, Dunedin 9054 New Zealand, 4Faculdade de Medicina
Veterin

aria, Universidade Federal Fluminense (UFF), Niter
oi, Rio de Janeiro, 24230-340 Brazil, and 5Instituto
Federal de Educacß~ ao, Ci^encia e Tecnologia do Rio de Janeiro (IFRJ), Mestrado Profissional em Ci^encia e
Tecnologia de Alimentos (PCTA), Rua Senador Furtado 121, Rio de Janeiro, 20270-021 Brazil

Milk is highly perishable and deteriorates rapidly during storage. Although the thermal processing
technologies successfully inactivate many enzymes and microorganisms up to a required level, they
can negatively affect the natural flavour of dairy foods and decrease their nutritional value. Alter-
native nonthermal technologies have been established as an interesting approach to produce safe
and healthy dairy products, without compromising their nutritional quality. These techniques have
the ability to inactivate milk enzymes without affecting the milk quality. In addition, the combination
of two different nonthermal techniques and mild heating has proven to be more effective to provide
safety to milk when compared to the treatments alone. This review aims to evaluate the impact of
nonthermal technologies, in particular, ultrasound, pulsed electric field, high-pressure processing
and ultraviolet irradiation on milk enzymes.
Keywords Enzyme inactivation, Novel techniques, Ultrasound, Pulsed electric field, High-pressure
processing, Irradiation.

proteins and lipids, allowing the development of

INTRODUCTION
Modern food processing has focused on the pro-
duction of a safe, high-quality product with a
long shelf life. Milk is a perishable food; thus,
the preservation of long shelf life milk is a chal-
lenge for the dairy industry as exogenous
microbes and enzymes can cause rapid deterio-
ration. Undesirable microbial and enzymatic
activities must be prevented to extend the shelf
life of foods and maintain quality (Aadil et al.
*Author for 2015a). Up to date, approximately 70 indige-
correspondence. E-mail: nous milk enzymes have been discovered and
dilrana89@gmail.com many native milk enzymes have their signifi-
(RMA)
cance in dairy products (Fox and Kelly 2006).
© 2019 Society of Milk enzymes also play an important role in
Dairy Technology microbial growth, once they break down
microorganisms (Indumathi et al. 2018).
Although the thermal treatments are used to
confer stability and safety of milk and dairy
products (Amaral et al. 2017), the conventional
heating can lead to changes in the organoleptic
(texture, aroma and flavour) and nutritional (vi-
tamin losses) properties of the processed prod-
ucts (Barba et al. 2016). Moreover, thermal
processing requires high amounts of energy,
which affects the costs of the final product and
thus the profitability of the industry (Coutinho
et al. 2018). Most of the negative effects of
thermal treatment can be reduced by the use of
nonthermal technologies, thus resulting in a food
product with improved quality. Several nonther-
mal technologies are often referred to as cold

Vol 70 International Journal of Dairy Technology 1

Vol 70
pasteurisation, which ensures the product safety, retains the
nutritional attributes of food while saving time, and reduces
the processing costs (Ghosh 2017). Various nonthermal
techniques for milk preservation have been studied, includ-
ing ultrasound (sonication), irradiation or ultraviolet (UV)
irradiation processing, high-pressure processing (HPP) and
pulsed electric field (PEF). When applied alone or in combi-
nation with heat, these techniques can reduce the microbial
load and inactivate enzymes. The effects of these novel
technologies on microorganisms have been extensively
investigated due to their role in improving safety and delay-
ing food spoilage (Aadil et al. 2018; Roobab et al. 2018;
Balthazar et al. 2019). However, the impact of these tech-
nologies on the enzymes involved in dairy processing has
not been reviewed extensively; thus, the inactivation of
enzymes from milk using nonthermal techniques should be
investigated. Furthermore, given the interest and the great
potential of novel nonthermal technologies in the dairy
industry, it is essential to examine their suitability for milk
processing and to evaluate their potential as an alternative
to heating techniques. This review aimed to provide a com-
prehensive overview of the application of different novel
nonthermal technologies for the inactivation of milk
enzymes, focused on the advantages and disadvantages of
each technique.
ULTRASOUND TECHNOLOGY
The Ultrasound (US) is an emerging nonthermal technology,
which is developed to minimise processing and to improve
quality and ensure the safety of food products (Aadil et al.
2013; Huang et al. 2017; Manzoor et al. 2019; Zia et al.
2019). As a replacement for the conventional treatment, US
has been used to control the microstructure and modify the
texture characteristics of fat-containing products, and the
functional properties of food proteins (Arvanitoyannis et al.
2017). The US can also be used in different processes,
including the inactivation of enzymes and microbes (Islam
et al. 2014; Aadil et al. 2015b). Acoustic cavitation plays
an important role in the US process, once it involves a sud-
den expansion and contraction of nano- or micro-bubbles of
gas present in the liquid subjected to sonication. During the
process, a net inflow of gas into the bubbles leads to the
growth of bubbles up to a resonance size range. Further
expansion of the size of the bubbles can cause a quick and
immediate collapse resulting in hot spots, with temperatures
up to 5000 K and pressure values higher than 1000 atm.
Within a liquid, such collapse occurs at different sites and
increases the physical shear forces and the generation of
free radicals (Bhaskaracharya et al. 2009). In addition, the
macromolecular chains can also be broken by sonication
(Islam et al. 2014).
Ultrasonic waves have high sensitivity tos enzymes, lead-
ing to enzyme activation or inactivation, as a function of
2 © 2019 Society of Dairy Technology
the physiological properties of the enzymes and the treat-
ment conditions (Nadar and Rathod 2017). Protein denatura-
tion is the main mechanism for enzyme inactivation, which
can occur either by the production of a shear force resulting
from the collapse of cavitation bubbles or the formation of
free radical by sonolysis of water molecules (O’donnell
et al. 2010). The enzymatic activity can increase due to the
conformational changes of the enzymes, which allows the
access of substrates and improves the surface area for inter-
actions (Nadar and Rathod 2017).
ENZYME INACTIVATION BY US
The effects of US on milk enzymes are presented in Table 1.
Different researchers have studied the effects of different
ultrasonic treatments on the inactivation of milk enzymes
(Khanal et al. 2014). Generally, the enzyme activity
decreased with the increase in the enzyme concentration,
while higher solid content (i.e. high protein and fat) can
improve enzyme inactivation (Villamiel and de Jong 2000).
Villamiel and de Jong (2000) studied the impact of US
treatment on the enzymes of whole milk in a continuous
flow system. The enzymes alkaline phosphatase (AP), c-glu-
tamyltranspeptidase (c-GTP) and lactoperoxidase (LPO)
from whole milk are found to be quite resistant to sonica-
tion treatment. No enzyme inhibition was observed for AP
and LPO from whole milk subjected to the US treatment
(120 µm, 20 kHz and residence time of 56.3 sec) with a
3.5% reduction in c-GTP activity. When increasing the resi-
dence time to 70.3 sec, no impact was observed in AP
activity, with a reduction of 14.5% and 12.8% in c-GTP
and LPO enzymes activity, respectively (Villamiel and de
Jong 2000). No significant reductions were observed for the
AP activity with the increase in the residence time to
102.3 sec, while the c-GTP and LPO activities decreased by
22.1% and 14.4%, respectively (Villamiel and de Jong
2000). Cameron et al. (2009) reported no changes in the AP
and LPO activities of raw milk under similar US power for
longer treatment times (124 µm and exposure time of 5 and
10 min), which confirms the findings reported by Villamiel
and de Jong (2000) about the enzyme resistance to the US
treatment.
In skimmed milk, only c-GTP slightly decreased (5.8%
reduction) after the US treatment (120 µm, 20 kHz and resi-
dence time of 70 sec. The LPO and AP activities were not
affected with the increase in the residence time to
102.3 sec, while a reduction of 17.2% was observed in the
c-GTP activity (Villamiel and de Jong 2000). Khanal et al.
(2014) reported similar results for the AP activity, with no
significant reduction after 1 and 5 min of treatment time
(amplitude 80%, 20 kHz), with 5.8% reduction after 10 min
of process. The activity of bile salt-stimulated lipase (BSSL)
in human milk was investigated at a constant US energy
(1000 J) and US power of 3.62 W (Christen et al. 2012).
Vol 70

Table 1 Effect of US on milk enzymes

Percent Reduction
Enzyme Sample Treatment Plan (%) Reference
c-GTP Whole milk Amplitude: 120 µm, frequency: 20 kHz, residence time: 3.5 Villamiel and de Jong
AP 56.3 sec 0 (2000)
LPO 0
AP Amplitude: 120 µm, frequency: 20 kHz, residence time: 0
c-GTP 70.3 sec 14.5
LPO 12.8
AP Amplitude: 120 µm, frequency: 20 kHz, residence time: 1.8
c-GTP 102.3 sec 22.1
LPO 14.4
c-GTP Skimmed milk 17.2
AP Amplitude: 120 µm, frequency: 20 kHz, residence time: 1.4
c-GTP 70.3 sec 5.8
AP Raw milk Amplitude: 124 µm, exposure time: 5 and 10 min AP = 0 Cameron et al. (2009)
BSSL Untreated Human Power: 3.62 W, exposure time: 276 sec 9 Christen et al. (2012)
milk
AP Milk Amplitude: 91.2 µm, exposure time: 1 min 0 Khanal et al. (2014)
Amplitude: 91.2 µm, exposure time: 5 min 0
Amplitude: 91.2 µm, exposure time: 10 min 5.2
US, ultrasound; AP, alkaline phosphatase; LPO, lactoperoxidase; c-GTP, c-glutamyl transpeptidase; BSSL, bile salt-stimulated lipase; SMUF,
simulated milk ultrafiltrate.

After 276 sec of treatment time, the enzyme activity
reduced to 9% in human milk, which suggested that the US
technology alone is not effective to inactivate milk enzymes,
with a reduction only for the c-GTP activity upon long
exposure time. A combination of mild thermal treatment
and the US, such as thermosonication (TS), was suggested
to be effective for inactivating the milk enzymes (Villamiel
and de Jong 2000).
ADVANTAGES AND DISADVANTAGES
Several advantages of the use of the US to control the
microbial activity have been reported. It has been suggested
that large cells such as yeasts are more susceptible to the
cavitation effects due to the larger surface area as compared
to small sized cells. Generally, bacterial spores (i.e. Bacillus
and Clostridium spp.) are more resistant to the US when
compared to vegetative bacteria, which are in the growth
phase. Commonly, vegetative microbes are less resistant
than fungi, while anaerobes are less resistant than aerobes
(Chandrapala et al. 2012). Low cost, simplicity and energy
saving are the main characteristics of US techniques. More-
over, a better homogenisation of milk can be achieved by
using the US as compared to the conventional method
(Berm udez-Aguirre et al. 2011). In milk, the ultrasonic
waves can remove gases and enhance oxidative stability due
to the removal of air (Cameron et al. 2009). Despite the
advantages of US, its disadvantages include the generation
of free radicals during treatment, which can damage the pro-
tein, amino acids and fat, by catalysing nondesirable reac-
tions (Arvanitoyannis et al. 2017).
HIGH-PRESSURE PROCESSING
High-pressure processing is a nonthermal technology used
in the food industry, which consists of treatment at a pres-
sure above 100 MPa within the range of >100–700 MPa. In
HPP, the pressure of the product is generated as a result of
mechanical pressure exerted on a fluid, normally water, sur-
rounding the treated product (Lerasle et al. 2012). High-
pressure processing seems to be a better alternative for food
preservation when compared to conventional techniques
such as thermal processing (Aadil et al. 2017). As enzyme
molecules are protein subsets, the secondary, tertiary and
quaternary structures of enzymes can be changed by apply-
ing pressure, which can expose the amino acid and sulfhy-
dryl groups, resulting in an increased number of
hydrophobic sites and inducing the modification of enzyme
functionality (Santos et al. 2018).
The two principles responsible for the effects of HPP
include Le Chatelier’s and isostatic principles. Le Chate-
lier’s principle states that a pressure is exerted to the system
on its equilibrium position, and then, the system will react
to prevent damage caused by the pressure applied, while the
change in volume will be assisted under the high pressure,
which may result in the inactivation of enzymes or

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microorganisms. The isostatic principle states that uniform
pressure from every direction compresses food products and
then recovers their original shape when the pressure is
released. The product is compressed regardless of the pro-
duct size and geometry because the transmission of pressure
to the core is not mass/time-dependent (Naik et al. 2013).
Three parameters characterise the HPP, including pressure,
temperature and time, which contributes to the process
design (Naik et al. 2013). High-pressure processing can lead
to the disintegration of casein micelles into casein particles
with smaller diameter, along with a decrease in milk light-
ness and turbidity, and increased viscosity (Mandal and
Kant 2017). Moreover, the pressure-based dissociation of
colloidal calcium phosphate and the denaturation of whey
proteins may lead to changes and/or improvement of milk
characteristics (Trujillo et al. 2002). Recent studies on the
use of pressure-assisted thermal processing (PATP) and
pressure-assisted thermal sterilisation (PATS) reported heat-
induced changes by using heat and pressure. According to
Ludikhuyze and Hendrickx (2001), the mechanism of
enzyme inactivation by HPP is similar to the protein denatu-
ration. The application of pressure on the enzyme structure
may cause partial or complete and reversible or irreversible
unfolding, which can lead to changes in the enzyme activity
due to the structural changes of its active site.
According to Cheftel (1992), high-pressure processing can
lead to several changes in the enzymes, including:
• Reversible or irreversible, partial or complete enzyme
inactivation may cause conformational changes in the pro-
tein structure due to the HPP at room temperature. The
extent of the changes depends on several factors, includ-
ing pressure, temperature, time, type of enzyme, and
micro-environmental conditions (e.g. pH and presence of
solutes or other biological compounds).
• Macromolecules such as protein are denatured by pres-
surisation and may become more reactive to the enzy-
matic reaction.
• The changes in the cell membrane of intracellular orga-
nelles or the removal of intracellular enzymes from the
cell cytoplasm can improve the enzyme–substrate interac-
tions.
EFFECT OF HPP ON ENZYME INACTIVATION
The effect of HPP on milk enzymes is presented in Table 2.
There are few studies on enzyme inactivation by HPP in
milk, probably due to the resistance of enzymes to this tech-
nology. Sakharam et al. (2011) reported that lipase, LPO
and xanthine oxidase of milk are resistant up to 400 MPa,
while AP, phosphohexose isomerase and c-glutamyltrans-
ferase (GGT) were partially inactivated at pressures of 600,
350 and 400 MPa, respectively. Complete inhibition of
these enzymes was found at pressures of 800, 550 and
630 MPa, respectively. Milk AP is moderately pressure
4 © 2019 Society of Dairy Technology
resistant, and no inactivation was found at moderate HPP
treatment (400 MPa, 60 min at 20 °C) (Lopez-Fandino
et al. 1996). In contrast, after a more intense HPP treatment
(500 MPa, 90 min), a 50% AP inactivation was observed,
with 100% inactivation at 800 MPa for 8 min (Rademacher
et al. 1998). The LPO, xanthine oxidase and lipase of milk
subjected to HPP were found to be resistant at 400 MPa for
60 min, with no inactivation of AP (Naik et al. 2013),
which was completely inactivated at 800 MPa for 6 min.
Hence, the total enzyme inactivation in milk requires a pres-
sure of at least 800 MPa, which is regarded as an intense
HPP treatment. The GGT and lipoprotein lipase activity has
been studied in raw milk, and the results revealed that the
HP treatment (300–400 MPa, 0–180 min) improved the
enzymatic activities (Pandey and Ramaswamy 2004), and
lipase was found to be resistant over this entire range.
Plasmin activity reduced by 86.5% in raw milk after
applying the HPP treatment (400 MPa, 60 °C) for 15 min
(Garcıa-Risco et al. 2000). Rademacher and Kessler (1997)
also observed a 15% reduction (400 MPa, 8 min) of plas-
min in milk, indicating that the treatment time is a very
important factor. A much higher (up to 90%) reduction was
observed when the pressure increased to 600 MPa. Another
study concluded that the pressure treatments (400, 600,
800 MPa) for 20 min can reduce the AP activity of raw
bovine milk by 30, 60 and 99.7%, respectively (Johnston
1995).
The combination of heat treatment and HPP appears to be
a very effective strategy to inhibit the milk enzymes. The
effect of HPP at different temperatures on different enzymes
from whole milk was investigated (Datta et al. 2005). The
results revealed that the plasmin activity reduced by 74% at
200 MPa and 64 °C (exposure time 20 sec), whereas a 90%
reduction in enzyme activity was achieved at 80 °C. Com-
plete inactivation of lipase was achieved at >71 °C under
similar treatment conditions, while LPO was inactivated at
80 °C (HPP 200 MPa, exposure time of 20 sec) (Datta
et al. 2005).
Seyderhelm et al. (1996) observed a 20% loss in the LPO
activity of bovine milk when subjected to HPP treatment
(600 MPa, room temperature, 30 min), which reduced to
16% of the initial activity at 45 °C under similar conditions.
In addition, half of the phosphatase activity was lost after
the HPP treatment at 600 MPa and 45 °C for 10 min (Sey-
derhelm et al. 1996), with no substantial reduction of LPO
in bovine milk after the HPP treatment (450–700 MPa)
(Mazri et al. 2012). Phosphohexose isomerase of bovine
milk was found to be inactivated at 500 MPa and 20 °C for
10 min, while GGT can be completely inactivated at
600 MPa and 20 °C for 30 min (Rademacher and Hinrichs
2006). Although milk enzymes are quite resistant to the
HPP treatment, the resistance varies from enzyme to
enzyme, and LPO and AP have been found to be the most
resistant when compared to the other milk enzymes. The
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Table 2 Effect of HPP on milk enzymes

Enzyme Sample Treatment plan Percent Reduction (%) Reference

Phosphohexose isomerase Milk 550 Mpa 100 Sakharam et al. (2011)
c-glutamyltransferase (GGT) 630 Mpa 100
AP 800 MPa, 8 min, 20 °C 100 Rademacher et al. (1998)
600 MPa, 90 min, temp: 20 ° 50
400 MPa, 60 min, temp: 20 °C 0 Lopez-Fandino et al. (1996)
AP Milk 400 Mpa, 60 min 0 Naik et al. (2013)
800 MPa, 6 min 100
Raw milk 800 MPa, 20 min 99.7 Johnston (1995)
c-glutamyltransferase (GGT) 400 MPa, 30 min 15 Pandey and Ramaswamy (2004)
Milk 400 MPa, 8 min 15 Rademacher and Kessler (1997)
Bovine milk 600 MPa, 30 min, 20 °C 100 Rademacher and Hinrichs (2006)
LPO Raw milk 700 MPa, 73 °C 100 Ludikhuyze et al. (2001)
Bovine milk 600 MPa, 25 °C, 30 min 20 Seyderhelm et al. (1996)
600 MPa, 45 °C, 30 min 16
Phosphatase 600 MPa, 45 °C, 10 min 50
Plasmin Milk 200 MPa, 65 °C, 20 sec 74 Datta et al. (2005)
200 MPa, 80 °C, 20 sec 90
Lipoprotein lipase 200 MPa, above 71 °C, 20 sec 100
LPO 200 MPa, 80 °C, 20 sec 100
HPP: high-pressure processing, AP: alkaline phosphatase, LPO: lactoperoxidase, GGT: c-glutamyltransferase.

use of high pressure (e.g. 800 MPa) or moderate HPP and
moderate thermal treatments is required to inactivate all
indigenous milk enzymes.
ADVANTAGES AND DISADVANTAGES OF HPP
HPP has many advantages over the thermal treatments since
the latter results in the loss of vitamins and changes in fla-
vour, while the HPP treatment provides nutritional integrity
to the product, which increases the shelf life of the products
while remaining fresh. HPP can be applied to foods that are
sensitive to thermal processing, and it is known for its abil-
ity to modify the milk structure, especially proteins. HPP
has the ability to maintain shelf life, with a minimal effect
on the nutritional and sensory characteristics of food (Erk-
men and Karatas 1997). In addition, pressures above
300 MPa can raise the chymosin coagulation time of milk
(Buffa et al. 2001).
Most of the important food deteriorating enzymes are
quite resistant to pressure; thus, a complete enzyme inactiva-
tion is difficult (Patterson et al. 2006). However, several
enzymes, like phosphatase, are comparatively pressure sen-
sitive in the range of 400–800 MPa (Palou et al. 2004).
Moderate HHP treatment, for example 400 MPa, cannot
inactivate the enzymes lipase, xanthine, oxidase and LPO in
milk (Naik et al. 2013). Various disadvantages can also
occur when using HPP processing. Liepa et al. (2016)
reported that HPP can decrease the size of casein micelles,
increase the free fatty acids levels and cause denaturation of
whey proteins, which can affect the appearance and the
functionality of milk.
IRRADIATION/ULTRAVIOLET
Irradiation for food preservation consists of the application
of electromagnetic waves to food products to confer a
longer shelf life. In general, electromagnetic wave spectrum
consists of energy and frequencies such as c ray, UV rays,
infrared irradiation and radio waves. Most of the studies
have investigated the effects of c and UV rays on milk
enzymes. UV rays are the short electromagnetic rays with a
wavelength range of 200–400 nm. For a practical purpose,
UV light can be divided into three different subclasses,
namely UV-A (320 to 400 nm), UV-B (280 to 320 nm) and
UV-C (200 to 280 nm) (Bintsis et al. 2000). Ionising radia-
tion involves the use of radioisotopes like Cobalt-60 and
Cesium-137, which are the main sources of c rays produc-
tion (Roobab et al. 2018).
Enzymes are more resistant to irradiation than microor-
ganisms, and complete enzyme destruction requires 5 to
10 times of radiation dose for microbial destruction. Esti-
mated gamma irradiation D values for enzymes should be
higher than 50 kGy, and four times the D-value are
required for complete inactivation (Rahman 2004). The
effect of irradiation on various enzymes from bovine and
human milk has been studied (Glew 1962;
Eitenmiller et al. 1971; Christen et al. 2013a; Christen
et al. 2013b).

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Table 3 Effect of irradiation on milk enzymes

Enzyme Sample Treatment Plan Percent reduction (%) Reference

Lysozyme Bovine milk c-irradiation: 102 500 Gy, dose rate: 3.6 Gy/s 44.3 Eitenmiller et al. (1971)
Human milk c-irradiation: 102 500 Gy, dose rate: 3.6 Gy/s 31.5
Bovine milk c-irradiation: 200 000 Gy, dose rate: 3.6 Gy/s 53.9
Human milk c-irradiation: 200 000 Gy, dose rate: 3.6 Gy/s 45.9
Phosphatase Raw whole milk Irradiation: 0.2 Gy/s, exposure time = 12 h 9.8 Kung et al. (1953)
Irradiation: 0.2 Gy/s, exposure time = 6 h 7.4
AP Whole milk Irradiation 20 000 Gy 23.6 Glew (1959)
Whole cow milk Irradiation 240 000 Gy 100
Irradiation 250 000 Gy 100 Glew (1962)
Lysozyme Human milk UV-C 4683 J/I 25 Christen et al. (2013b)
UV-C 3474 J/I 16
UV-C 2084 J/I 9
AP UV-C 3474 J/I 0
UV-C 4863 J/l 0
UV-C 2084 J/l 0
BSSL UV-C 4863 J/I 0
UV-C 2084 J/I 3.3
AP, alkaline phosphatase; BSSL, bile salt-stimulated lipase.

Irradiation affects biological materials both directly and
indirectly. The direct effect is through energy deposition
that targets molecules and leads to chemical reactions, while
the indirect effect is based on the production of free radicals
such as superoxide anions, hydrogen radicals and hydroxyl
radicals from the radiolysis of water. The strong oxidising
(hydroxyl radical) and reducing (hydrogen radical) agents
have the ability to change the molecular structure of organic
matter (Rahman 2004). Mostly, free radicals in the solvent
phase affect the enzymes indirectly. The presence of
enzymes in their natural environment makes them more
resistance to irradiation effect (Rahman 2004). Thus,
enzymes present in food are more resistant to the radiation
than microorganisms. A 0.1 kGy radiation dose can damage
2.8% of DNA molecules, which can be dangerous for living
cells, while a similar dose only causes 0.14% reduction of
the enzymatic activity (Grandison 2012).
ENZYME INACTIVATION BY I/UV
The effect of irradiation on milk enzymes is presented in
Table 3. It is known that milk enzymes have high resistance
to irradiation. Glew (1959) studied the AP activity in whole
milk under irradiation dose of 20 000 Gray (Gy) and
240 000 Gy and found that a dose of at least 240 000 Gy
was necessary to completely inactivate AP, while
20 000 Gy inactivated only 23.6% of the enzymes, which
was confirmed by Glew (1962), who reported that milk AP
is extremely radio-resistant. The phosphatase activity in
whole milk at irradiation of 0.2 Gy/s has been studied
(Kung et al. 1953), with a decrease in the enzyme activity
of only 7.4% and 9.8% after 6 and 12 h, respectively (Glew
1962). Thus, milk AP has been found to be resistant to irra-
diation. Moreover, irradiation doses for milk sterilisation
cannot inactivate the enzyme phosphatase, which proves the
high resistance of this enzyme to irradiation (Procter and
Goldblith 1951). The effect of c-radiation on bovine milk
lysozyme revealed that a c-radiation dose of 102500 Gy
resulted in 44.3% reduction, while a 200 000 Gy dose
caused 53.9% reduction of the lysozyme activity (Eiten-
miller et al. 1971).
In human milk, c-radiation doses of 102 500 Gy and
200 000 Gy reduced the lysozyme activity to 31.5% and
45.9%, respectively (Eitenmiller et al. 1971), while other
study reported that UV-C irradiation at 2048 J/I, 3474 J/I
and 4683 J/I reduced the lysozyme activity to 9%, 16% and
25%, respectively (Christen et al. 2013b). According to the
authors, the higher total solid content may have exerted a
protective effect to lysozyme. Similarly, the activities of AP
and BSSL from human milk have been studied under differ-
ent UV-C doses (Christen et al. 2013a), with no effects on
AP and BSSL even after the UV-C treatments at 3474 J/I
and 4863 J/I (Christen et al. 2013a). In contrast, the same
researchers reported that an exposure of 23 h at 239,683 J/I
was enough to inactivate 60% of AP activity. The UV-C
dosage required to destroy specific microorganisms (Escher-
ichia coli, Staphylococcus epidermidis, Enterobacter cloa-
cae and Bacillus cereus) from human milk has no
significant effect on the heat-sensitive enzymes (Christen
et al. 2013a). These results showed that most milk enzymes
have high resistance to the irradiation treatment, and higher
doses are required to completely inactivate them.

6 © 2019 Society of Dairy Technology

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ADVANTAGES AND DISADVANTAGES OF I/UV
The irradiation technology is effective to reduce losses in
fresh food and can enhance the shelf life of the products. In
addition, energy can be saved, as it uses lower energy as
compared to other preservation processes, such as refrigera-
tion. The hygienic quality of solid food can also be main-
tained through radiation treatment. Although the radiation is
effective in reducing the microbial load, enzymes have been
proven to be more resistant to this treatment. Low radiation
doses have no significant effect on the milk enzymes, while
higher doses can inactivate the enzymes, despite affecting
the nutritional profile, flavour and protein denaturation.
Moreover, the availability of a proper infrastructure within a
country is the basic requirement for the implementation of
this technology. Flavour changes can occur in milk and
dairy products when subjected to irradiation treatments,
even at low dosages (Grandison 2012). The development of
rancid off-flavour in milk after irradiation treatment has
been reported by different researchers (Matak et al. 2007;
De Oliveira et al. 2015), probably due to the generation of
free radicals, which affects the milk lipid fraction, particu-
larly, the fatty acid composition (De Oliveira et al. 2015).
However, some authors have suggested that the application
of irradiation at low doses or under frozen conditions can
be effective without changing the organoleptic characteris-
tics (Odueke et al. 2016). In milk, sensory and chemical
changes can occur with the use of UV irradiation at 254 nm
(Matak et al. 2007). Also, the major disadvantages of irradi-
ation include the need for a critical minimum capacity along
with the product volume, due to the high costs of operation.
PULSED ELECTRIC FIELD
Pulsed electric field is an emerging nonthermal preservation
technology to inactivate the spoilage organisms and patho-
gens in liquid foods such as milk or juices by using short
high-voltage pulses (Aadil et al. 2015c). It involves the use
of short pulses (µs or ms) of high voltage (kV/cm) to foods
that placed between two and more electrodes (Zhang et al.
2017). The aim of using PEF is to produce safe food with
better nutritional and sensory properties when compared to
food products subjected to heat treatment alone (Bendicho
et al. 2002a; Liu et al. 2015, 2016). PEF technology has
gained popularity by preventing the negative effects of ther-
mal treatments during food processing. It can destroy the
microorganisms and inactivate the enzymes, without having
any major unwanted effects on the organoleptic properties
of foods (Lasekan et al. 2017). The important processing
parameters used for enzyme inactivation by PEF include the
treatment time, electric field strength, pulse width, pulse
form, pulse number and pulse frequency (Bendicho et al.
2003a).
© 2019 Society of Dairy Technology
For the enzyme inactivation, a greater reduction in the
enzyme activity is obtained by using the high electric field
and temperature. It has been reported that the conforma-
tional changes in the structure of the enzyme during PEF
treatment may be responsible for the modification of the
enzyme activity (Vega-Mercado et al. 1995a; Ho et al.
1997; Castro et al. 2001). The enzymes are basically pro-
teins, whose catalytic activity depends on both the confor-
mation of surrounding proteins and the native configuration
of their active site. Amino acid (AA) groups found in
enzymes can lead to a highly asymmetric distribution of
charge, thus forming strongly polar and charged regions in
the protein structure (Laberge 1998), while the intense elec-
tric field causes the denaturation, breakdown of covalent
bonds, and the oxidation–reduction reactions in the protein
structure (Barsotti and Cheftel 1999). Giner et al. (2000)
concluded that the mechanism of enzyme inactivation is not
completely clear for PEF technology.
ENZYME INACTIVATION BY PEF
The effect of PEF on milk enzymes is presented in Table 4.
The impact of PEF on the inactivation of AP has been stud-
ied, with no changes in the enzyme activity (Grahl and
M€arkl 1996; Ho et al. 1997; Van Loey et al. 2001). How-
ever, Grahl and M€arkl (1996) reported a 10% inactivation
of AP in milk subjected to PEF treatment at 20 pulses and
21.5 kV/cm for 25 µs, and 25% and 60% inactivation for
LPO and lipase. The effects of high-voltage pulses on lac-
tate dehydrogenase from milk have been studied, with no
reduction in the enzyme activity after PEF treatment (200
pulses at 31.6 kV/cm for 0.96 µs, 1.1 Hz and temperature
at 30 °C) (Barsotti et al. 2001).
Bendicho et al. (2003a) have studied the effect of PEF on
protease activity from Bacillus subtilis in skim milk and
whole milk and found 81.1% and 57.1% reduction of activ-
ity after the PEF treatment (35.5 kV/cm for 896 µs and
111 Hz), respectively. With respect to SMUF (stimulated
milk ultrafiltrate), Bendicho et al. (2003b) found 62.7%
reduction by just decreasing the temperature to 40 °C. Simi-
larly, the PEF treatment (70 pulses at 18.8 kV/cm for
400 µs and at 22 °C) resulted in a 60% reduction of AP in
raw milk, 2% in full-fat milk, and 65% in skim milk (Bar-
bosa-Canovas et al. 1998). A 96% reduction in the AP
activity was observed after PEF treatment chamber, which
consists of two stainless steel electrodes to generate the
electric field at temperatures up to 44 °C after the PEF treat-
ment (70 pulses at 13.2 kV/cm) (Barbosa-Canovas et al.
1996). In whole milk, the plasmin and xanthine oxidase
activities reduced about 7% and 30%, respectively, immedi-
ately after the PEF treatment (preheating 55 °C, holding
time: 24 sec and 25.7 kV/cm for 34 µs) (Sharma et al.
2018).
7
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Table 4 Effect of PEF on Milk enzymes

Percent
Enzyme Sample Treatment plan Reduction Reference
AP Milk 21.5 kV/cm, number of pulses: 20 pulses About 10% Grahl and M€arkl (1996)
Raw 21.5 kV/cm, total energy in put up to 400 kJ/I 0
milk 18.8 kV/cm, number of pulses: 70, pulse width: 400 µs, 60% Barbosa-Canovas et al. (1998);
temp: 22 °C, Castro et al. (2001)
13.2 kV/cm, number of pulses: 70, temp: 44 °C 96% Barbosa-Canovas et al. (1996)
6.7–20 kV/cm, number of pulses: 200, temp: 25 °C 0 Van Loey et al. (2001)
Batch mode HIPEF,
6.7–20 kV/cm, number of pulses: 200, temp: 70 °C, 74%
Batch mode HIPEF
Milk 6.7–20 kV/cm, No. of pulses: 200, pulses width: 2 us, 100%
batch system
SMUF 18.8–22 kV/cm, No. of pulses: 70, Batch mode HIPEF 65% Castro et al. (2001)
Whole 18.8–22 kV/cm, No. of pulses: 70, Batch mode HIPEF 59%
milk
LPO Milk 21.5 kV/cm, No. of pulses: 20 About 25% (Grahl and M€arkl 1996)
Raw 21.5 kV/cm, total energy in put up to 400 kJ/I 0
LPO milk 19 kV/cm, No. of pulses: 100, total pulsing energy of 0 van Loey et al. (2001)
up to 500 kJ/kg, pulse width: 5 µs
35 kV/cm, temp: 30°C, pulsing energy input of up to <10% Jaeger and Knorr (2007)
220 kJ/kg
37.6 kV/cm, number of pulses: 200, temp: <35°C 0 De Luis et al. (2009).
Protease SMUF 35.5 kV/cm, 111 Hz, pulse width: 866 ls 81% Bendicho et al. (2003b)
Protease (Pseudomonas Skim 15 kV/cm, No. of pulses: 98, f: 2 Hz, temp: 50 °C, 60% Vega-Mercado et al. (1995b)
fluorescens) milk pulse width: 2 µs
25 kV/cm, No. of pulses: 16, continuous mode HIPEF 0 Vega-Mercado et al. (2001)
Protease from B. SMUF 16.4–27.4 kV/cm, No. of pulses: 100, batch mode 10% Bendicho et al. (2001b)
subtilis HIPEF treatment
17.6–25.2 kV/cm, No. of pulses: 100, continuous mode 15%
HIPEF treatment
16.4–27.4 kV/cm, No. of pulses: 60, pH: 5, batch mode 50% Bendicho et al. (2001c)
HIPEF treatment
16.4–27.4 kV/cm, No. of pulses: 60, temp: 63 °C-5 min, 30%
batch mode HIPEF treatment
Protease from B. Skim 35.5 kV/cm, temp: 46 °C, pulses width: 866 µs, 81.1% Bendicho et al. (2003a)
subtilis milk continuous PEF treatment
Whole 35.5 kV/cm, temp: 46 °C, pulses width: 866 µs, 57.1%
milk continuous PEF treatment
SMUF 35.5 kV/cm, temp: 40 °C, pulses width: 866 µs, 62.7% Bendicho et al. (2003b)
continuous PEF treatment
16.4–27.4 kV/cm, pulses width: 320 µs, batch mode 0
PEF treatment
Lipase Milk 21.5 kV/cm, No. of pulses: 20, About 60% Grahl and M€arkl (1996)
Raw 21.5 kV/cm, total energy in put 400 kJ/I 60%
milk
Lipase from SMUF 16.4–27.4 kV/cm, No. of pulses: 100 About 62% Bendicho et al. (2002b)
Pseudomonas 26.1–37.3 kV/cm, No. of pulses: 100, continuous mode 13% Bendicho et al. (2001d)
fluorescens HIPEF treatment
16.4–27.4 kV/cm, No. of pulses: 100, pH: 5 52%
16.4–27.4 kV/cm, No. of pulses: 100, temp: 63 °C- 35% Bendicho et al. (2001e)
15 min., batch mode HIPEF treatment

(continued)

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Table 4 (Continued).

Percent
Enzyme Sample Treatment plan Reduction Reference
Plasmin (bovine milk) 30 kV/cm, No. of pulses: 50, f: 0.1 Hz, temp: 15 °C, 90% Vega-Mercado et al. (1995a)
pulses width: 2 µs
Peroxidase Raw 13–19 kV/cm, No. of pulses: 200, temp: 25 °C, batch ˃3% Van Loey et al. (2001)
milk mode HIPEF
13–19 kV/cm, No. of pulses: 200, temp: 44–52 °C, 2–13%
batch mode HIPEF treatment
AP Raw PEF intensity: 25 kV/cm, temp: 60 °C 29% Shamsi et al. (2008)
skim PEF intensity: 31 kV/cm, temp: 60 °C 56%
milk PEF intensity: 35 kV/cm, temp: 60 °C 67%
PEF, pulsed electric field; AP, alkaline phosphatase; LPO, lactoperoxidase; SMUF, simulated milk ultrafiltrate.

Grahl and M€arkl (1996) have also studied the effect of
high-voltage pulses on AP, LPO and lipase from raw milk
and found no large-scale inactivation under the high electric
field pulses conditions, while up to 60% lipase inactivation
was observed after the treatment with high energy input.
Van Loey et al. (2001) reported no reduction of LPO in
raw milk subjected to the PEF treatment (100 pulses at
19 kV/cm for 5 µs) with a total energy of up to 500 kJ/kg,
while Jaeger and Knorr (2007) observed a <10% inactiva-
tion of LPO under the PEF treatment (35 kV/cm at 30 °C)
and pulsing energy up to 220 kJ/kg. Moreover, De Luis
et al. (2009) have reported no changes in the LPO concen-
tration either in milk or whey after the PEF treatment (37–
6 kV/cm using 200 pulses and temperature < 35 °C). Van
Loey et al. (2001) found a 74% reduction of AP from raw
milk after batch mood (BM) treatment (>200 pulses at 6.7–
20 kV/cm) along with the heat treatment (70 °C), while no
inactivation was found at 25 °C. Moreover, the authors also
found 2–13% peroxidase inactivation after applying the BM
treatment (more than 200 pulses at 13–19 kV/cm) along
with the heat treatment (44–52 °C), with a reduction <3%
at 25 °C.
The effect of the PEF treatment (14 kV/cm at a pulsing
rate of 1 Hz) on the milk protease was studied in sterilised
skim milk, and 40% inactivation was observed, which
increased to 60% when using other PEF conditions (15 kV/
cm with 98 pulses and 2 Hz) (Vega-Mercado et al. 1995b),
with no protease inactivation after reducing the number of
pulses (Vega-Mercado et al. 2001). Bendicho et al. (2001a)
studied the effect the PEF treatment (10 pulses of 20 kV/
cm) on the protease from Pseudomonas fluorescens in skim
milk and found a 25% reduction of the enzyme activity,
with an inactivation level from 37.9 to 81.1% after applying
a continuous flow PEF treatment (35.5 kV/cm for 866 µs
with 67–111 Hz and additional temperature of 46 °C).
Shamsi et al. (2008) studied the AP activity in raw
skimmed milk subjected to different PEF intensities and
temperatures. At 25 kV/cm and 60 °C, the enzyme activity
decreased to 29%, while reductions of 56% and 67% were
achieved under the same temperature after increasing the
PEF intensity to 31 kV/cm and 35 kV/cm, respectively.
In SMUF, the PEF treatment (35.5 kV/cm for 866 µs and
at 111 Hz) reduced the protease activity by 81% (Bendicho
et al. 2003b), while a reduction of 62% lipase from P. fluo-
rescens was observed after using 100 pulses at 16.4–
27.4 kV/cm (Bendicho et al. 2002b). Vega-Mercado et al.
(1995a) studied the effect of PEF by using continuous mood
treatment on plasmin, with a 90% reduction after the PEF
treatment (10–50 pulses at 15–45 kV/cm for 2 µs and at
0.1 Hz, 50 °C). Castro et al. (2001) investigated the effect
of PEF on the inactivation of AP in SMUF, nonfat milk,
full-fat milk and whole milk. After the PEF treatment (70
pulses at 22 kV/cm), a 65% reduction of AP activity was
observed, and a 59% reduction was observed for full-fat
milk and whole milk subjected to 70 pulses at 18–8 kV/cm.
The authors investigated the effect of PEF on the protease
from B. subtilies suspended in SMUF. After applying BM
treatment (>100 pulses at 16.4–27.4 kV/cm), only 10%
inactivation was achieved, which increased to 15% when
PEF was applied in a continuous flow mood treatment
(>100 pulses at 17.6–25.2 kV/cm) (Bendicho et al. 2001b).
The effect of BM treatment (>60 pulses at 16.4–27.4 kV/
cm) along with acidification (pH 5) on the protease from
B. subtilies in SMUF was also studied, with a 50% inactiva-
tion against 30% protease inactivation after using a mild
heat treatment (63 °C for 5 min) (Bendicho et al. 2001c).
Bendicho et al. (2001d) studied the effect of BM treatment
along with acidification (pH 5 and up to 60 pulses at 16.4–
27.4 kV/cm) on lipase from P. fluorescens in SMUF and
reported a 52% inactivation against only 13% achieved by
applying a continuous flow treatment (>100 pulses at 26.1–
37.3 kV/cm), while the mild heat treatment (63 °C for

© 2019 Society of Dairy Technology 9

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15 min) together with the BM treatment led to a 35% inac-
tivation (Bendicho et al. 2001e). By applying the continu-
ous treatment (35.5 kV/cm for 866 µs and frequency from
67–111 Hz) at 40 °C on the protease from B. subtilis in
SMUF, 62.7% inactivation was found, with no inactivation
without the use of mild heat treatment or acidification (Ben-
dicho et al. 2003b). Hence, the sensitivity of milk enzymes
can vary as a function of the PEF treatment, and higher
PEF conditions are required to complete enzyme
inactivation.
ADVANTAGES AND DISADVANTAGES OF PEF
PEF involves the application of high-voltage pulses to liquid
or semiliquid food placed between two electrodes for a few
seconds and is used for the enzymatic inactivation of milk
and dairy products, juices and egg products. This technol-
ogy can also be helpful in extending the shelf life of milk.
The main disadvantage of PEF is the leakage of metal ions
from the electrodes into the food products, once a signifi-
cant increase in the metal contents, including Fe, Zn, Mn
and Cr, has been reported in the treated products (Evrendi-
lek et al. 2004). The high cost of processing is another main
concern that has limited interest in this technique. Although
PEF can destroy vegetative bacteria, it cannot inhibit spores;
thus, the use of thermal processing is required for the spore
inhibition. In addition, it cannot inactivate enzymes in the
products that require low temperatures of storage to control
enzymatic reactions. Food products with high electrical con-
ductivity cannot be treated with PEF (Yang et al. 2016).
THERMOSONICATION ON THE ENZYME
INACTIVATION
The ultimate goal of the modern food industry is to improve
safety and to minimise food degradation. The combination
of different techniques allows effective processing while
minimising the negative effects of using a single treatment
to achieve the process goal (Aadil et al. 2015d). Enzyme
inhibition and destruction of microbes can be achieved more
effectively by combining moderate heat treatments along
with other techniques. The extensively investigated combi-
nations include the US and temperature (TS), pressure and
US (nano sonication), and temperature and HPP. TS can
reduce the thermal resistance of enzymes, thus reducing the
temperature and the time required for enzyme inhibition
(O’donnell et al. 2010). The effect of TS on the milk
enzymes is presented in Table 5.
The application of TS (amplitude 120 µm, intensity
20 kHz) in whole milk at 75.5 °C for 102.3 sec completely
inhibited the AP and c-GTP activities, while a 69.2% reduc-
tion was observed for the LPO activity (Villamiel and de
Jong 2000). Treatments at 119 µm or 133 µm for 2.5 min
and 60 oC caused a 72% reduction of Staphylococcus
10
aureus V8 protease activity in whole milk (Vijayakumar
2012). In skim milk, the TS treatment (amplitude 120 µm,
intensity 20 kHz, temperature 75.5 °C for 102.3 sec) caused
complete inhibition of AP and c-GTP, while LPO activity
reduced to 52.8% (Villamiel and de Jong, 2000). These
results demonstrate that LPO is more resistant to the TS
treatment when compared to AP and c-GTP from milk.
Raw skim milk subjected to two TS conditions (152 µm
and 133 µm for 3 min at 60 oC) exhibited a reduction of
total plasmin activity to 80.9% and 83.24%, respectively
(Vijayakumar 2012). A reduction of 72% of S. aureus V8
protease activity was achieved in skim milk when subjected
to the TS treatment (133 µm, 2.5 min, 60 oC) (Vijayakumar
2012).
Moreover, a study on human milk reported that the TS
treatment (60 W, 45 °C, 10 min) reduced the lysozyme and
BSSL activities by 51% and 91.7%, respectively. At 50 °C
(60 W and treatment time of 10 min), the remaining BSSL
and lysozyme activities were 4.8% and 39% (Czank et al.
2010), which shows that BSSL of human milk is more sen-
sitive to the TS treatment. Thus, the combination of these
techniques has proven to be more effective when compared
to single treatments, despite the high cost of these
technologies.
APPLICATIONS OF NONTHERMAL
TECHNOLOGY AT A LAB AND INDUSTRIAL
SCALE
Nonthermal techniques have been studied extensively, and
the industrial applications have grown rapidly in the last
few years. The basic purpose of these technologies is to
provide consumers with safe and healthy food. The use of
irradiation is usually limited by legal requirements, and the
major hindrance of this technology may be the development
of off-flavours in irradiated dairy products (Odueke et al.
2016).
Although the US alone is not very effective for microbial
inactivation, it can accelerate the rate of thermal sterilisation
of foods. The US process can lead to a more effective
homogenisation of milk, which when applied in milk before
cheesemaking can result in an increased cheese yield due to
protein binding on the fat globule membrane (Soria and Vil-
lamiel 2010). Sonication can reduce the viscosity of skim
milk (Zisu et al. 2013) and the fermentation time and pro-
duction of fermented milk with lower lactose and higher
oligosaccharides contents (Nguyen et al. 2009). In addition
to pasteurising milk, TS can disintegrate large milk fat glob-
ules simultaneously (Bermudez-Aguirre et al. 2008). In
milk, high-power US is generally employed for inactivation
of microorganisms and enzymes, homogenisation, and
extraction of b-galactosidase (lactase) (Evrendilek 2014).
HPP can be extensively used in dairy processing.
Yoghurts made with high-pressure treated milk can ferment
© 2019 Society of Dairy Technology
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at a higher pH, with no changes in the physical properties
when compared to yoghurt made from heat-treated milk
(Evrendilek 2014). The application of HPP to milk may be
an alternative to the use of food additives in yoghurt prepa-
ration, which can affect flavour, aroma and mouth-feel of
yoghurt (Sfakianakis and Tzia 2014). HPP can be used to
improve the microbiological safety and quality of milk to
produce better quality cheeses (Liepa et al. 2016).
The PEF treatment (40 kV/cm, 30 pulses and 2 ls pulse)
extended the shelf life of raw skim milk to 2 weeks at 4 °C
(Fernandez-Molina et al. 2000; Fernandez-Molina et al.
2005). Chymosin coagulation time of PEF-treated milk has
been reported to be increased by 10% (Garcia-Amezquita
et al. 2013). Some authors have reported that the PEF treat-
ment of milk for the manufacture of butter, ice cream and
cheese provided organoleptic characteristics to the processed
products similar to fresh products (Sampedro et al. 2005).
However, the application of these technologies has also
faced some major hindrances, including the development of
off-flavour. In addition, the use of irradiation is also hin-
dered by legal requirements, as several countries have estab-
lished a specified dose limit of food irradiation.
Furthermore, the market for irradiated foods is also nega-
tively affected by trade agreements and labelling require-
ments (Grandison 2012). The large-scale application of
nonthermal technologies is limited due to lack of knowledge
on the full-scale equipment design and scale up. Thus, fur-
ther studies are required for the optimisation of suitable pro-
cessing conditions, for the best-optimised inactivation of
microbes and enzymes.
The combination of nonthermal processes and the tradi-
tional techniques has proven to be effective, with promising
results, including the control of the harmful microbes in
food products. Therefore, studies on the combined effects of
these technologies are necessary, aimed at a better under-
standing and new preservation strategies.
CONCLUSIONS
Different nonthermal technologies (US, HPP, PEF and irra-
diation) have been studied on the inactivation of various
milk enzymes, with good results for the enzyme inactiva-
tion. However, more research efforts are required to opti-
mise processing conditions. All the technologies discussed
in the present research mainly affect the enzymes by dena-
turing the protein structure through energy deposition, free
radical production, chemical changes and pressure. When
the treatments are used alone, high intensities and conditions
are required for enzyme inactivation, while the combination
of different nonthermal treatments and mild heat treatment
seems to be more effective, with better results at a reduced
treatment time. However, future studies are necessary,
focused on the effective combinations between treatments
and technologies. The enzyme AP is the most resistant to
the nonthermal technologies, and its complete inactivation
requires higher energy inputs; thus, it can be used as a bio-
marker. Milk AP is inactivated slightly above the pasteurisa-
tion conditions and has been used as a biomarker in the
dairy industry to check the inadequate pasteurisation. It is
worth mentioning that the enzyme inactivation by nonther-
mal processing does not guarantee the destruction of patho-
gens, which should be taken into account when discussing
the substitution of heat by other methods. Despite the bene-
fits of inactivating a number of enzymes and improving
quality of the processed products, the nonthermal treatments
have not been adopted by the dairy industry, probably due
to the high cost and large production volumes, which cannot
be accommodated in the current scenario. Further studies

Table 5 Effect of TS treatment on milk enzymes

Enzyme Sample Treatment Plan Percent reduction Reference

AP Whole milk Amplitude: 120 µm, 20 kHz, 102.3 sec, 75.5 °C 100 Villamiel and de Jong (2000)
Skimmed milk 100
c-GTP Whole milk 100
Skimmed milk 100
LPO Whole milk 69.2
Skimmed milk 52.8
Lysozyme Human milk US: 60 W, 45 °C, 10 min 51 Czank et al. (2010)
US: 60 W, 50 °C, 10 min 61
BSSL US: 60 W, 45 °C, 10 min 91.7
US: 60 W, 50 °C, 10 min 95.2
Plasmin Skim milk US amplitude 152 µm, 3 min, 60 oC 80.9 Vijayakumar (2012)
Plasmin Skim milk US amplitude 133 µm, 3 min, 60 oC 83.24
TS, thermosonication; US, ultrasound; AP, alkaline phosphatase; LPO, lactoperoxidase; c-GTP, c-glutamyltranspeptidase; BSSL, bile salt-stimu-
lated lipase.

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are needed on affordable technologies to be used in large-
scale production, aimed to meet the needs of the dairy
industry.
ACKNOWLEDGEMENTS
The authors are grateful to the Higher Education Commission
(HEC), Islamabad, Pakistan, for supporting this project from
National Research Program for Universities (NRPU-7366) and
CNPQ, CAPES and FAPERJ.
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