Ultrasonics Sonochemistry 86 (2022) 105999

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Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultson

Review

Thermal treatment alternatives for enzymes inactivation in fruit juices:

Recent breakthroughs and advancements
Muhammad Umair a, b, Sidra Jabeen c, Zekai Ke d, Saqib Jabbar e, Faiqa Javed c,
Muhammad Abid f, Kashif-ur Rehman Khan g, Yu Ji h, *, Sameh A. Korma i,
Mohamed T. El-Saadony j, Liqing Zhao a, *, Ilaria Cacciotti k, Clara Mariana Gonçalves Lima l,
Carlos Adam Conte-Junior m
a
Department of Food Science and Engineering, College of Chemistry and Engineering, Shenzhen University, 518060 Shenzhen, Guangdong, China
b
Key Laboratory of Optoelectronic Devices and Systems, College of Physics and Optoelectronic Engineering, Ministry of Education and Guangdong Province, Shenzhen
University, Shenzhen 518060, Guangdong, China
c
National Institute of Food Science and Technology, University of Agriculture, Faisalabad 38000, Pakistan
d
Department of Orthopaedics, Shenzhen University General Hospital, Shenzhen University Clinical Medical Academy, Shenzhen, Guangdong, China
e
Food Science Research Institute (FSRI), National Agricultural Research Centre (NARC), Islamabad, Pakistan
f
Institute of Food and Nutritional Sciences, Pir Mehr Ali Shah, Arid Agriculture University Rawalpindi, Pakistan
g
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, The Islamia University of Bahawalpur, 63100 Bahawalpur, Pakistan
h
Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, Aachen 52074, Germany
i
Department of Food Science, Faculty of Agriculture, Zagazig University, Zagazig 44519, Egypt
j
Department of Agricultural Microbiology, Faculty of Agriculture, Zagazig University, Zagazig 44519, Egypt
k
Department of Engineering, INSTM RU, University of Rome “Niccolò Cusano”, Roma 00166, Italy
l
Department of Food Science, Federal University of Lavras, Minas Gerais, Brazil
m
Center for Food Analysis (NAL), Technological Development Support Laboratory (LADETEC), Federal University of Rio de Janeiro (UFRJ), Cidade Universitária, Rio
de Janeiro 21941-598, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Fruit juices (FJs) are frequently taken owing to their nutritious benefits, appealing flavour, and vibrant colour.
Fruit juices The colours of the FJs are critical indicators of the qualitative features that influence the consumer’s attention.
Enzymes Although FJs’ intrinsic acidity serves as a barrier to bacterial growth, their enzymatic stability remains an issue
Polyphenol oxidase
for their shelf life. Inactivation of enzymes is critical during FJ processing, and selective inactivation is the
Peroxidase
primary focus of enzyme inactivation. The merchants, on the other hand, want the FJs to stay stable. The most
Non-thermal technologies
Quality prevalent technique of processing FJ is by conventional heat treatment, which degrades its nutritive value and
appearance. The FJ processing industry has undergone a dramatic transformation from thermal treatments to
nonthermal treatments (NTTs) during the past two decades to meet the requirements for microbiological and
enzymatic stability. The manufacturers want safe and stable FJs, while buyers want high-quality FJs. According
to the past investigation, NTTs have the potential to manufacture microbiologically safe and enzymatically stable
FJs with low loss of bioactive components. Furthermore, it has been demonstrated that different NTTs combined
with or without other NTTs or mild heating as a hurdle technology increase the synergistic effect for microbi­
ological safety and stability of FJs. Concise information about the variables that affect NTTs’ action mode has
also been addressed. Primary inactivates enzymes by modifying the protein structure and active site confor­
mation. NTTs may increase enzyme activity depending on the nature of the enzyme contained in FJs, the applied
pressure, pH, temperature, and treatment period. This is due to the release of membrane-bound enzymes as well
as changes in protein structure and active sites that allow substrate interaction. Additionally, the combination of
several NTTs as a hurdle technology, as well as temperature and treatment periods, resulted in increased enzyme
inactivation in FJs. Therefore, a combination of thermal and non-thermal technologies is suggested to increase
the effectiveness of the process as well as preserve the juice quality.

* Corresponding authors.
E-mail addresses: yu.ji@biotec.rwth-aachen.de (Y. Ji), qzhao@szu.edu.cn (L. Zhao).

https://doi.org/10.1016/j.ultsonch.2022.105999
Received 25 February 2022; Received in revised form 30 March 2022; Accepted 1 April 2022
Available online 4 April 2022
1350-4177/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
M. Umair et al. Ultrasonics Sonochemistry 86 (2022) 105999

1. Introduction However; resulting compounds are chemical toxicants that are carci­
nogenic and harmful for human body. The amount and nature of toxi­
Regulatory authorities demand FJs with a certain minimum level of cants depend on the type of thermal treatment used for processing and
quality and safety standards that can be considered as one of the most
important concerns for food scientists and consumers, and strict re­
quirements from the manufacturer’s perspective. In addition to Table 1
adequate safety and quality, the retailer wants the processed FJs to be IR processing effect on the enzyme activities in various fruit juices.
stable during storage [1]. The quality standards defines the quality of Fruit juices Dose/ PPO POD References
water for soft drinks should be free from any component that adversely fluency (residual (residual
effects the sensory (taste, odour and colour) and physicochemical (mJ/cm2) activity) activity)
properties of FJs. The organic matter should be < 1 mg/L, alkalinity and Apple-juice NC 99.51 97.00 [78]
dissolved solids should be 50 mg/L and < 500 mg/L respectively. The Apple-juice NC 0.00 0.00 [126]
sugar or sweetening agents such as glucose and fructose corn syrup (Sparkling)
Grape-juice NC 20.00 0.00 [127]
(72◦ Brix), artificial sweeteners i.e., saccharin, and granulated sugar also
(Dauphine)
affects the quality of FJs. Therefore, specific quality parameters (<1% Pear-juice NC 0.00 0.00 [128]
arsenic content, < 0.02% ash contents) have been established for FJs [2- Orange-juice 58.21 75.00 97.00 [129]
4]. To ensure the safety concerns of FJs, the microbial inactivation are Apple-juice 12,483 10.00 0.00 [130]
(Clear)
the most challenging tasks of modern food processing. In NTTs, PEF, UV
Nectarine- NC 60.00 40.00 [26]
irradiation, HPP and ultrasonication have been extensively studied for Juice
deactivation of L. innocua, S. aureus, E. coli, and spoilage yeast while, the Clear apple- 72.00 70.00 NA [131]
synergistic effects of NTTs are very promising for tackling the more juice
resistant spores and species [5-7]. Grape-juice 30.10 kJ/L 60.90 NA [132]
Apple-juice 8.10 kJ/L 86.10 NA [133]
Thermal (conventional) methods are used to preserve the food [3,8]
Apple-juice 1.50 kJ/L 114.50 NA [134]
and causes undesirable changes in food products due to the long term Mango nectar- 108 kJ/L 19.00 NA [125]
heat exposure results in the degradation of food quality. These methods juice
can effectively decontaminate the food and inactivate the enzymes PPO: polyphenoloxidase; POD: peroxidase; IR: irradiation

Fig. 1a. Flow diagram of the fruit juices processing steps using a gamma-radiation-assisted extraction system. Adapted from Kalaiselvan, Sugumar [46]
with permission.

2
M. Umair et al.
Fig. 1b. Gamma-irradiation theory of water molecules radiolysis in fruit juices. Adapted from Kalaiselvan, Sugumar [46] with permission.
cooking food [9]. It can also cause the loss of water from food, oxidation
of lipids that leads to the changes in the composition of fatty acids. The
consumers’ awareness regarding food quality and safety has been
increased and they demand food that is free from microorganism, high
nutritional and organoleptic properties with excellent mouth feel
[10,11].
In non-thermal technologies (NTTs), the food is treated at room
temperature for a very short period (~l min or less), maintaining its
nutritional composition, and ensuring unchanged mouth feels, without
damaging the product texture. There is no damage to food because heat-
sensitive nutritious materials are intact in food without increasing the
food temperature [3,6,12]. The NTTs that have been emerged over the
past few decades are: irradiation (IR), ohmic heating (OH), pulsed
electric field (PEF), high voltage electric field cold plasma (HVCP),
3
Ultrasonics Sonochemistry 86 (2022) 105999
ultrasonication (US), high hydrostatic pressure (HHP) processing etc.,
The significance of these technologies has been greatly increased due to
their applications in enzyme deactivation that are responsible for the
quality degradation and spoilage in various FJs [12,13]. In the appli­
cation of NTTs, the ohmic heating is used for the extraction of bio­
activecomponents, enzyme inactivation with great retention of desired
ingredients in FJs. It provides a rapid and uniform heat transformation
to the FJ viscous and heat-sensitive products with less colour degrada­
tion [14]. PEF processing is applied directly to FJs in order to destroy the
pathogenic bacteria, inactivation of enzymes, and recovery of bioactive
components, freezing and structure modification. For enzyme deacti­
vation the specific electric field intensity is used 15–60 kV cm− 1
[15,16], with specific energy 110–240 kJ kg− 1 for various FJs. However
the inactivation of most undesirable enzymes remains ineffective due to
M. Umair et al. Ultrasonics Sonochemistry 86 (2022) 105999

Fig. 2. Schematic diagram of the ohmic heating (OH) processing system; (1) power unit, (2) power analyzer, (3) computer, (4) thermocouple, (5) cell, (6) electrode,
(7) glass dome, (8) vacuum pump, (9) buffer tank, (10) valve, (11) vacuum gauge, (12) needle valve. Adapted from Fadavi and Salari [60] with permission.

Table 2
PEF processing effect on the enzyme inactivation in fruit juices.
Fruit juices Enzyme PEF system PEF parameters Reduction rate References

Apple-juice PPO Bench-scale and rectangular shape, bipolar-pulse, 38.50 kV/cm, 50 ◦ C 70% [135]
0.64 cm gap combined with 300 pps
Apple-juice PPO, P1: Pilot plant scale P2:40 ◦ C PEF P1: 48% for PPO [79]
POD P2: Bench scale P1: 60 ◦ C PEF (100 kJ/kg, 30 kV/cm) P2: 100% for PPO
and POD
Orange-juice PME Pilot plant scale, co field flow-tubular and PEF treatment chamber, the 35 kV/cm, 59 μs treatment time, 1.4 88% [136]
electrode gap is 1.0 cm with stainless-steel tubular-electrode pulse width, 600 pps, 98 mL/s
Orange-juice PME Co-field flow-tubular PEF treatment chamber system, electrode gap: 20–35 kV/cm, 2.2 or 2.0 pulse width, 90.2% [136]
0.2 cm Stainless-steel electrode 700 pps, 0.31, 0.42, mL/s
Tomato- PME Gene-electroporator (Bio Rad Laboratories) 24 kV/cm, 800ls of 93.5% [137]
juice treatment time with exponential
decay
Carrot-juice POD Continuous flow bench-scale PEF, co-field Flow-treatment chamber, 35 kV/cm for 1.000ls, 6ls pulse width 73.2% [138]
0.29 cm gap, square-wave bi-polar pulse at 200 Hz.
White POD, Bench scale, co-field flow-treatment square-wave bi-polar pulse 25–35 kV/cm at 50% POD, 100% [139]
Grape- PPO 200–1,000 Hz, 1–5 ms treatment for PPO
juice
PPO: polyphenoloxidase; POD: peroxidase; PME: pectinmethylesterase; PEF: pulse electric field

their requirement for high intensity electric field [17,18]. Ultra­
sonication is used for the extraction, synthesis, and preservation of FJs.
The combined effect of PEF and US is a novel non-thermal method,
which has shown its capability to enhance the quality and safety of FJs
with small nutrient losses. These methods reduced the processing time,
high through-put, less energy inputs, and are eco-friendly [5,19,20].
Cold plasma is a promising, novel non-thermal food processing treat­
ment in which reactive nitrogen species (RNS) (N2+, N•, NO•, NO etc.)
and reactive oxygen species (ROS) (OH–, •OH, H2O2, O2–-) are generated
due to the feed gas ionization in a high electric field [21]. It induced the
microbial and enzyme inactivation and reduced the anti-nutritional
components and contaminants in foodstuffs. Therefore, it enhances the
quality and stability by keeping heat-sensitive nutrients [4,22]. The
HHP processing is non-thermal energy-efficient technology used forthe
FJs treatment, killing the microorganisms and inhibiting the oxidative
enzymes, while masking the other chemicals and the quality degrading
mechanisms in FJs [23,24]. In this process, 100–1000 MP pressure is
applied to the FJs with or without heat. HPP works on force compression

4
M. Umair et al.
Fig. 3a. Pulsed electric field effect on the enzyme deactivation. Adapted from Zhang, Sun [80] with permission.
principle which applied to surrounding fluid of the product. Pressure has
a partial effect on covalent-bonds of low-molecular-mass compounds
such as pigments, vitamins, and volatile substances as compared to high-
molecular-mass molecules including enzymes or proteins. The three
dimensional structure of enzymes is stabilised due to covalent/non-
covalent interactions therefore, series of reactions involve in the
destruction and formation of new linkages, folding and unfolding thus
causes to bring changes in natural structure of enzymes [3,9,23,25].
Enzymes naturally present in FJs, including peroxidase (POD),
polyphenol oxidase (PPO), pectin methyl esterase (PME), and lip­
oxygenase (LOX), causesquality degradation FJs. These enzymes cata­
lyze the phenolic components to o-quinones and melamines after
polymerization, resulting in the development of off-flavours. For
instance, POD and PPO are responsible for the oxidation of a large
number of compounds in the presence of hydrogen peroxide (H2O2),
such as the phenolics, contributing to the enzymatic-browning [14,26-
28]. On the other hand, PME is a hydrolytic enzyme of the cell wall and
is also used for the FJs clarification process to reduce their viscosity.
However, in the processing of de-pectinate and clarified FJs, it is
important to leave these enzymes to eliminate the starch and pectin
[29]. However, in some purees and juices that contain a suspended pulp,
like citrus juices, it is important to inhibit the activities of endogenous
5
Ultrasonics Sonochemistry 86 (2022) 105999
enzymes to avoid the cloud loss in the final product. The PME can also
lower the FJs viscosity, contributing to reduce the consumer acceptance
and the FJs overall quality. PME hydrolyses the pectin in FJs, causes the
instability of cloud and decreases the viscosity of FJs by the degradation
of pectin chain. PME is present in all citrus fruits as a bounded cell wall
enzyme that forms a complex with pectin and induces the electrostatic
linkages. During FJs extraction, PME is released, hydrolysing the pectin
and changing it gradually into the pectic-acids and tolowmethoxy
pectin. These insoluble compounds interact with calcium ions [30,31],
induces the precipitation of the pectin and loss of cloud in FJs [9,32,33].
Another FJs natural enzyme, namely LOX, is responsible for the pro­
duction of volatile flavours and free radicals in many FJs, causing colour
and nutritional losses through the oxidation of polyunsaturated fatty
acids (PUFA) into hydroperoxides. Therefore, in FJs, the inactivation of
endogenous enzymes is very important for the production of high-
quality products [22,24,34,35].
It has been reported that the degree of enzyme inactivation varies
depending on the food type and processing conditions [36-42]. For same
fruit or cultivar, the processing time and temperature can be different
depending on the type of technology used for enzyme inactivation. For
example, in apple puree, 80% inhibition of PPO occurred after 3 min, in
thermosonication treatment at 72 ℃ and 460 W/cm2 and for HPP-
M. Umair et al.
Fig. 3b. Pulsed electric field schematization. Adapted from Dziadek, Kopeć [83] with permission.
treatment 71℃, 50 min were selected at 600 MPa. The combination of
ultrasound and HPP with thermal treatment offers an advantage in terms
of PPO inactivation. Even though, the different forms of same fruit like
puree, juice, and concentrate can also affect the inactivation of PPO for
similar treatments [6,43,44].
In addition to microbial safety and enzyme stability, the consumers
also demand minimally processed products with quality equivalent to
fresh produces. In this context, the application of NTTs attains a key role.
A comprehensive review about the effect of all NTTs on FJs enzymes
inactivation has not been reported yet. The present review is to focus on
the influence of all non-thermal processing techniques on FJs and pro­
vide discussions about the general FJs spoilage, an overview on NTTs,
fundamental procedures, factors affecting NTTs, enzymes inactivation
mechanisms, and NTTs effect on various FJs quality attributes.
2. Irradiation (IR)
In FJs, the IR processing is an established method to inactivate their
enzymes. It involves the use of ionizing radiations with sufficient energy
to emit the electrons from the product molecules. The general standards
of Codex Alimentarius allow the use of food irradiations including the
radioactive isotopes of cesium-137 (Cs 137) and, cobalt-60 (Co 60), as
well as high energy electrons beam [45]. These electrons are produced
by a machine, applying maximum energy to generate β-rays of 10
million electron-volts (MeV, while X-rays are produced by applying the
6
Ultrasonics Sonochemistry 86 (2022) 105999
energy of 5 MeV (Fig. 1a) [45,46]. These rays have sufficient energy to
convert the electrons into ions of food molecules which are electrically
charged particles and reactive oxygen species (ROS). During IR pro­
cessing, the rays break down the ionized molecules and chemical bonds
present in the enzymes [47]. The dose of irradiation acquired by the
food during IR processing is measured in kilo grays (kGy), and one Gy
equals 1 J of energy absorbed by 1 kg of food. The penetration power of
gamma and X-rays into the food matrix is different: the X-rays pene­
tration through the water equivalent matter is 23.00 cm while for the
gamma rays 3.90 cm [48].
The ultraviolet light electromagnetic spectrum includes radio waves,
microwaves, infrared radiation, X-rays visible light, and γ-radiations. In
high-energy UV radiations, the electromagnetic spectrum in the
100–400 nm range, categorized as UV-A, UV-B, and UV-C from 320 to
400 nm, 280 to 320 nm, and 200 to 280 nm, respectively, are used for
the treatment of different food products [49,50]. In particular, the UV
radiation of 254 nm is extensively used in FJs during IR processing [51].
Several studies have been carried out on the UV-radiation of FJs to study
its effect on the microbial inactivation. However, few studies reported
the UV radiations effect on the enzyme activities that are responsible for
browning reactions in FJs containing polyphenolic compounds
(Table 1). The PPO, POD, and PME activities in apple juice were
monitored when exposed to a 400 W mercury lamp that emits radiations
of 250 and 740 nm [51]. Results revealed that the PPO activity was
inhibited after 100 min, that of POD completely inactivated in 15 min,
M. Umair et al.
Fig. 4a. Represents low power ultrasound principles. Adapted from Dolas, Saravanan [27] with permission.
and that of PME (in one variety) in 40 min after the exposure to UV
radiation [51]. Moreover, authors observed no modifications in pH,
total phenolic content, sugars, and soluble solids in the treated and
controlled apple juice samples [51].
The action mode of ionizing radiations on enzymesis originated from
the biochemical changes in the biological system (Fig. 1b) [46]. The
exposure of irradiation, x-rays gamma rays, and high-speed electrons
induces chemical changes in the enzyme molecule atomic groups, with
consequent inhibition of enzyme activities in FJs [52]. It has been re­
ported that the gamma rays and x-rays exposure up to 100 Gy could
create a significant enzyme reduction activity in FJs [53]. Moreover, the
x-rays exposure could inactivate enzymes that contain the active sulf­
hydryl group or thiol group, due to the oxidation of the sulfhydryl
groups or thiol group, with disulfides inhibition [54].
3. Ohmic heating (OH)
The OH processing consists in the passing of an alternating current
through liquids and food substances. It presents an advantage over the
traditional thermal processes, being able to extend the shelf life of
pomegranate, melon, apple, and orange juices [55]. This NTT works
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Ultrasonics Sonochemistry 86 (2022) 105999
faster with 4.6 to 5.3 times lower energy consumption than that of the
conventional heating [55]. Furthermore, OH processing has revolu­
tionized the FJs industry for its effect on FJs bioactive components,
sensory properties, enzyme and microbial deactivation, and loss of the
physicochemical attributes [56]. Moreover, it plays a significant impact
on dehydration or concertation, sterilization, blanching, pasteurization,
and recovery of phenolic components and their antioxidant potential
[57]. Several studies have been greatly focused on developing OH pro­
cessing as an established and economical (being based only on elec­
tricity) alternative to the thermal technologies, including controlled
agitation processing, reciprocation processing, radio-frequency, and
microwave heating [58,59].
Concerning the OH processing action mode, it is based on electro­
magnetic methods such as, i.e., radioactive dielectric, capacitive
dielectric, radioactive magnetic, and inductive heating ones (Fig. 2)
[60]. Moreover, OH processing is similar, to some extent, to the mi­
crowave heating systems at different frequencies [61]. In the direct
resistance OH processing, liquid food and solid substances are simulta­
neously heated by means of passing an electric current through the
samples, whereas in continuous OH processing, a generator and a power
supply are used to generate the electric current [14,62]. The electrodes
M. Umair et al. Ultrasonics Sonochemistry 86 (2022) 105999

Table 3 of 32 V/cm was carried out [68]. Another research group of Bhat, Saini
US processingeffect on the enzyme inactivation in various fruit juices. [70] investigated the OH processing and thermal treatment effect on
Fruit juices Ultrasonic Effect on enzyme References various quality features such as colour and total phenolic content (TPC)
System deactivation with optimal of bottle guard juices at 60 to 90 ◦ C for 1 to 5 min. The authors evidenced
conditions better colour retention and higher TPC valuesas primary OH processing
Orange-juice System-A, D-19 • 62% PME inactivation at [140] outcomes, due to the higher enzymes deactivation than in thermally
mm and UP- 1500 optimal conditions F-20 treated (90 ◦ C for 5 min) juice samples [69].
Watt kHz, TI-20 min, AD-1.0 W/ A study reported that the grape juice preservation was increased by
cm 2
Orange-juice System-Aa, D-3 • 91% PME inactivation at [141]
increasing the voltage gradient duringthe OH processing, demonstrating
mm and UP-200 optimal conditions F-24 a higher PPO inhibition at 40 V/cm than at 20 and 30 V/cm. It could be
Watt kHz, TI-9.8 min, AD-80 W/ possible due to the rapid electric conductivity increment that induces
cm2, T-63 ◦ C greater PPO inhibition at a higher voltage gradient. Furthermore, at
Mosambi System-B, UP-400 • 96.8% PME inactivation at [142]
60 ◦ C with a constant voltage gradient during OH processing, a non-
juice Watt optimal conditions F-50
kHz, TI-20 min, AD-400 W/ significant increase in PPO activity was observed [70]. Baysal, Demi­
cm2, T-80 ◦ C rdöven [64], studied the OH processing application in the grape juice
Pineapple- System-A and D- • 20% PPO inactivation at [143] yield by grape pulp hot pressing.
Juice 13 mm and UP- optimal conditions F-19
500 Watt kHz, TI-10 min, AD-376 W/
cm 2
4. Pulsed electric field (PEF)
Apple-juice System-Aa, D-13 • 93.83%, 91% and 92.91% [144]
mm and UP- 750 PPO, POD and PME The PEF processing has been used as an emerging technology in the
Watt inactivation at optimal food industry over the last few decades for the processing of various FJs
conditions F-20 kHz, TI-10
such as orange, apple, carrot, and tomato juices. It is used for the high-
min, AD-0.3WmL− 1, T-
60 ◦ C quality food products as it is efficiently inhibits the activities of different
Pear-juice System-A, D-12.7 • 95.6%, 96.73%, and [145] enzymes, including POD, PPO, PME, and LOX, in FJs and several other
mm and UP–750 98.08% POD, PME, and beverage products (Table 2). In comparison to the microbial inactiva­
Watt PPO inactivation of at
tion, high-energy inputs are required for enzyme inhibition. At low in­
optimal conditions F-20
kHz, AL-70%, T-10 min, T-
tensity, PEF processing can be also used for controlling the enzymes
65 ◦ C activities [71,72].
Grape-juice System-B, UP-420 • 91%, 90%, and 89% PME, [44] In PEF processing, an electric field is applied instead of heat, high
Watt PPO, and POD inactivation and short voltage pulses (10–80 kV/cm, lasting within seconds from
at optimal condition F-28
micro to milliseconds) are connected among two electrodes directed
kHz, TI-60 min, AD-294 W/
Cm 2, T-60 ◦ C towards the projected foodstuff [73]. A PEF processing consists of a
Purple cactus System-Aa, D-13 • PME activity decreased at [146] monitoring and control system, high electrical power source, pulse
pear- juice mm, UP-1500 W F-20 kHz, AL-80%, TI-25 initiator, an assembly room (treatment chamber), a cooling scheme to
min, T-50 ◦ C check and control the heat escalation, compartments of raw and treated
Soursop- System-A, D-1.3 • 14.73% PPO inactivation of [147]
juice cm, UP-500 Watt PPO at optimal condition F-
foodstuff [74]. An energy input of 10–20 kJ/kg, for pre-treating to raise
19 kHz, TI-9 min, AD-330 the temperature 40 ℃, is usually required in a typical PEF processing
W/cm 2 [75,76]. And, therefore, the resulted product is a minimally processed
Bayberry- System-A, D-13 • 90% POD and PPO [148] juice. This type of product was characterized as pasteurized and fresh
juice mm, UP-600 Watt inactivation at optimal
juice to meet the consumers’ expectations of freshness and shelf life.
condition F-20 kHz, TI-6.7
min and 2.5 min AD-452 A study reported the influence of PEF processing on freshly prepared
W/cm2, AL 100% apple juice in the PPO and POD deactivation. A study revealed that the
PPO: polyphenoloxidase; POD: peroxidase; PME: pectinmethylesterase; US: reduction in the POD and PPO activity up to 68% and 71%, respectively,
ultrasonication was achieved when a preheating (50 ℃), and a PEF processing (at 40
kV/cm for 100 μs) were applied together [77,78]. This level of POD and
are in direct contact with the food material and pass the current through PPO inhibition was significantly greater (P < 0.05) when compared with
the food particles [61]. The distances among the electrodes are so conventionally pasteurized juices, characterised by a POD and PPO in­
adjusted, to generate the optimum electric field [61]. The heat genera­ hibition of only 48% and 46%, respectively. Schilling, Schmid [80], also
tion and the overall enzymes inactivation mechanism are strongly investigated the PEF processing influence on the apple juice quality,
affected by the electrical field strength and the residence time [55]. A reporting that the PPO and POD complete inhibition was obtained when
greater production of grape juice up to 50–70% was noted with ohmi­ the PEF processing was combined with preheating process of juices to
cally heated grape mash than conventionally heated one. This increase 60 ◦ C. They observed the PPO deactivation up to 48% at 40 ◦ C applying
in the yield has been recorded: 10.14% for OH and 5.23% for conven­ 30 kV/cm, 100 kJ/kg as optimal PEF process parameters [79].
tionally heated grape mash compared with the untreated group (control) As enzymes are proteins in nature, the PEF processing action mode to
[6,63,64]. The generator produces the electrical current that passes from inhibit the enzymes activities involves the alterations of the secondary
the first electrode and flows through the food matrix that lies between structure (α-helix and β-sheets), spatial conformation changes in the
the electrode gaps < a food particle resists the electric-current flow and tertiary structure, and modifications in the arrangements and number of
induces the instant volumetric heat generation by following the Joule protein subunits in the quaternary structure, therefore, losing the en­
first-law of heating. The current continuously passes to the second zymes functionality (Fig. 3a) [80].
electrode, then back to the power supply to close the circuit. The insu­ The PEF processing principle is based on two fundamental proced­
lator caps near the electrodes control the entire system [65,66]. ures, i.e. an electric-breakdown and electroporation (electric pulse used
The OH processing is efficient to inhibit the PPO and POD activities to form the temporary pores in the cell membrane), both coordinated to
at a lower temperature with a short time without damaging the FJs destroy the microorganisms and inactivate the enzymes [81]. The
quality [67]. Some studies reported that the PPO activity in sugarcane electric energy is uniformly transferred to whole food products due to
juice was decreased up to 97.80% at 90 ◦ C for 5 min when OH processing the presence of charged particles [36,82]. The PEF processing efficiency
depends on several components, including intensity, field strength, the

8
M. Umair et al.
Fig. 4b. Cavitation mechanisms for: (a) stable cavitation, (b) transient cavitation. Adapted from Dolas, Saravanan [27] with permission.
food particles conductivity, pH and temperature, the involved enzymes
and pathogen nature, the pulse rate and amount of energy, the time
interval, and the polarization (Fig. 3b) [74,83,84]. No studies have been
reported the chemical changes induced in the enzymes primary struc­
ture by the PEF process [85]. However, the PEF processing parameters
and conditions must be optimized to obtain desired enzyme inactivation
[86].
5. Ultrasonication (US)
Among NTTs, US is widely employed in the FJs industry [87]. Low-
energy US processes are used for non-invasive purposes with sound in­
tensities < 1 W/cm2 and frequencies > 100 kHz [88]. These ultrasound
waves are applied during the fermentation by the imitation of living cell
activity through degassing, for the monitoring of physicochemical
properties such as composition, particle size, and flow rate (Fig. 4a)
[27]. High-energy US, characterised by sound intensities < 1 W/cm2 and
frequencies from 18 to100 kHz, are particularly employed for enzymes
inactivation [89]. Furthermore, when US processing is combined with
heat or pressure, it has been found to be greatly effective, particularly for
FJs and beverages [44,90].
9
Ultrasonics Sonochemistry 86 (2022) 105999
Some studies have reported the efficacy of US processing (Table 3),
particularly in combination with non-thermal and thermal processing
methods such as the high pressure and pH differences application [89].
On the basis of the frequency ranges, it is possible to identify two main
US categories: high-intensity US (100–200 kHz, >1 W/cm2) and low-
intensity US (>100 kHz and 0–1 W/cm2) [91,92]. Low-intensity US
waves are commonly used for the non-destructive investigation of the
physicochemical changes in compounds during the food processing
[93].
The US processing action mode related to the enzyme inactivation is
based on the cavitation effects that originate during the food processing,
including evolution, inception, and implosion or disintegration of small
gas bubbles in a target food product. These cavitations consist in a
mechanism of creation of high-power sound waves due to the formation
of vapour or gas bubbles that are continuous and violently and rapidly
implode in the sample solution. This implosion occurs at extremely high
temperatures up to 1000 ℃ and high pressures from 50 to 500 MPa
[88,94]. This cavitation process originates due to the external stress
which has been found to be very effective to bring the conformational
secondary and tertiary structures changes through hydrogen bonds
cleavage (Fig. 4b) [27,95]. Moreover, the cavitation also produces free
M. Umair et al.
Fig. 4c. Represent the enzyme inactivation mechanism of FJs by ultrasound. Adapted from Dolas, Saravanan [27] with permission.
radicals due to the water hemolytic breakdown [96]. This results in free
radicals of hydroxyl (OH–) and hydrogen (H+) species, able to react with
free amino acids, responsible for the proteins and enzymes destabiliza­
tion, leading to alterations in the biological compounds activity [88].
High-intensity US waves can be used to destroy the microorganisms and
inhibit the enzyme activity by altering the proteins secondary structure
(Fig. 4c) [27], thus leading to changes in their nutritional and functional
properties, as highlighted earlier [97].
6. High voltage electric field cold plasma (HVCP)
In recent years, HVCP processing has been extensively used as a NTT
for the enzymes inactivation in FJs [7,87,98-102]. It is based on protons,
electrons, atoms, and ions in their excited and ground states. In function
of the electron’s temperature, plasma is divided into high-temperature,
i.e., Te (electron temperature) ¼ 106 to 108 K and low-temperature Te ¼
104 to 105 K plasma [103], which is further classified into non-thermal
and thermal plasma, on the basis of the thermodynamic equilibrium
[104]. Non-thermal plasma produces the non-equilibrium thermody­
namic effect in which Te can reach about 104 K, which is far greater than
the whole gas temperature. Therefore, the HVCP system can be retained
at lower temperatures and is well known as cold plasma [105,106].
10
Ultrasonics Sonochemistry 86 (2022) 105999
The HVCP is produced by supplying an adequate energy amount to a
neutral gas, causing its ionization, and, thus producing the chemically
active components, such as charged particles, non-excited or excited
molecules, UV radiations, and free radicals (e.g., reactive oxygen species
(ROS), and reactive nitrogen species (RNS)) [87,101,102,107]. The
energy used to produce the plasmas is classified as alternating current
(AC) discharges and direct current (DC) discharges, which are operated
in pulsed mode or continuously [98,108].
The HVCP plays a significant role in lowering the nutritional losses
that can alter the macro-molecules, thus expanding its applications in
the FJs industry [109,110]. In particular, extensive HVCP applications
have been reported for the pathogenic microorganisms and enzymes
deactivation [98,111,112], being the HVCP major advantage the FJs
processing at ambient temperature (Figs. 5a, 5b) [7,99].
The HVCP action mode in the enzyme deactivation is based on its
effect on the enzymes secondary structure (Fig. 5c) [113]. The ROS and
RNS species can induce the enzyme inhibition by preventing the binding
of coenzyme/substrate to their subsequent catalysis that occurs due to
the conformational changes in the enzymes active site [53]. During
HVCP processing, under the electric field effect, the primary target of
HVCP is α-helix and β pleated sheets of enzyme’s protein [114]. Radical
exposure of these proteins causes chemical and physical changes such as
M. Umair et al. Ultrasonics Sonochemistry 86 (2022) 105999

Fig. 5a. (A) Schematization of DBD-cold atmospheric plasma (CAP) machine; (B) Schematization of a DBD-CAP machine with the indication of its components; (C)
Picture of a DBD-CAP machine; (D) Treatment chamber of a DBD-CAP machine. Legend; 1, Indicator; 2, Exhaust fan; 3, Touch screen control panel; 4, Frequency
converter; 5, Power distribution box; 6, Electric voltage regulator; 7, Inductance 1; 8, Inductance 2; 9, Wheels; 10, Power cable inlet; 11, High voltage transformer;
12, lower electrode; 13, upper electrode; 14, lifting mechanism; 15, Frame; 16, Sample position. Adapted from Nasiru, Frimpong [99] with permission.

the cleavage of backbone or fragmentation, side-chain oxidation,
unfolding, cross-linking, changes in conformation, and hydrophobicity,
thus altering the susceptibility to proteolytic enzymes [115]. Some au­
thors reported that the α-helix structure of POD and PPO disrupted from
34.90% to 5% and 36.90% to 17.80% while β-sheets improved from
15.60% to 39.90% and 15.20% to 29.40%, respectively during HVCP
processing when treatment time was increased up to 360 s. This change
in the enzymes secondary structure occurs due to the protein polymers/
plasma-induce reactive species interaction [7,98].
A researcher [116], has reported the effect of HVCP on the cloudy
apple juice quality parameters, maintaining optimal conditions with a
power input of 10.5 kV for 5 min. The physicochemical properties and
enzyme inhibition were dependent on the treatment duration: a signif­
icant PPO activity reduction was achieved after 5 min and a complete

11
M. Umair et al.
Fig. 5b. Description of different sources used for the plasma generation. (A) plasma generation-ionization processing, (B) resistive barrier discharge (RBD), (C)
atmospheric pressure plasma jet (APPJ), (D) corona discharge system, (E) configuration of the DBD-BASED diffuse glow discharge atmospheric pressure. Adopted
from Umair, Jabbar [7] with permission.
PPO inactivation after 24 h. A remarkable pH decrement was noticed
due to the reactive species development after HVCP processing [116],
whereas the juice colour was also improved after long treatment.
However, the treated FJs were lighter than untreated juice samples that
were maintained during the storage period (Table 4).
7. High hydrostatic pressure (HHP)
The HHP processing is a another NTT that extensively used to pre­
serve the quality of different food products including liquid food
(bottled), high-moisture solid food (vacuum packed), and medium or
low moisture solids and semisolid foods (vacuum packed) [117]. To
lower the post-contamination risks, these products are packed before the
processing, placed in baskets, then lowered into the vessel [9,23], which
is filled with water. The HHP processing (800 MPa) is uniformly spread
in the vessel in order to assure the thorough processing of the product
[24,35,118-120].
12
Ultrasonics Sonochemistry 86 (2022) 105999
The effect of HHP processing on the enzyme inactivation involves the
unfolding of the endogenous enzymes promote in structure, changes in
the cell membrane fluid it’s phase transition, changes in intracellular
pH, and ribosomes rupture (Fig. 6) [24]. All these combined effects ul­
timately cause the complete or partial cellular components denaturation
[119]. However, the HHP processing effectiveness depends on the pH,
pressure, and temperature [6,121].
In addition to enzyme inhibition, HHP processing destroys the mi­
croorganisms up to a safe level [35,52,118,122], inactivates food
pathogens and the oxidative shelf-life is more than twice as compared to
the other NTTs [122]. The HHP processing is more applicable in food
products that have high moisture content with flexible vacuum pack­
aging. Orange juice, salsa, colourful products, flavoured fruit smoothies,
ready-to-eat meats, purees are examples of HPP processed products
[123].
It is evident that HHP processing has limited effects to achieve the
desired enzyme inhibition in FJs [121], and its effect depends on the
M. Umair et al.
Fig. 5c. Schematization related to cold plasma treatment effects on the enzyme conformation, reactive oxygen species (ROS); reactive nitrogen specie (RNS).
Adapted from Han, Cheng [113] with permission.
enzyme nature, the juices physicochemical properties, the medium pH,
elevated temperature, ionic strength, total soluble solids, and so on
[121]. It is possible to obtain high-quality products by applying the HHP
processing at 100 MPa to 1000 MPa for a short duration typically
ranging from a few seconds to several minutes, extending the shelf-life of
various FJs (Table 5). These products are additive-free, with very slight
alterations of their physical characteristics, sensory features, as well as
nutritional properties [121]. On the other hand, the application of
moderately HHP processing conditions, i.e., 200 to 600 MPa at ambient
temperature, often results in the activation of the native enzymes (POD,
PPO, and PME) in non-clarified FJs and fruit purees [24,124,125]. This
can result in the development of undesirable modifications in FJs colour,
flavour, texture, and nutritional quality during the processing and
storage period [120]. However, these parameters can be controlled with
proper refrigerated storage, appropriate packaging, the use of enzyme
inhibitors, and oxygen scavengers. Therefore, with HHP optimization of
FJs, it is vital to consider its effect on overall quality parameters,
particularly on health-promoting bioactive components.
13
Ultrasonics Sonochemistry 86 (2022) 105999
8. Conclusions and future perspectives
The NNTs employment potentially ensures to maintain the FJs
safety, quality, and nutritional aspects for a long storage period. For this
reason, there is a growing interest towards the use of NNTs in the current
global markets, taking into account the actualregulatory authorities and
manufacturers demand for FJs processing with a certain minimum level
of quality and safety standards. However, a combination of these tech­
nologies with other methods may be required in order to achieve a
synergistic enzyme inactivation effect. The primary action mode of NTTs
consists in inducing structural changes in proteins tertiary and second­
ary structures to deactivate enzymes in FJs. Nevertheless, these modi­
fications greatly depend on the food matrix processing intensity, and on
the properties of enzymes and proteins present in the different FJs.
Therefore, an appropriate optimization of both the equipment and the
operation parameters involved in NTTs should also be done in order to
guarantee an adequate control on the enzyme activities and a maximum
efficiency. In addition, it is pivotal to consider the NTTs impacts on the
safety and food quality. Despite the explored effects, NTTs have not been
M. Umair et al. Ultrasonics Sonochemistry 86 (2022) 105999

Table 4
HVCP effects on the enzymes inactivation in different fruit juices.
Fruit Enzyme Plasma Results References
Juices

Apples PPO DBD, 15 kV, 12.7 kHz, 10–30 min, Air, 1.5 Linear inactivityreductiondue to the treatment time
• [107]
m/s Residual activity of 42%, 68%, and 88% after 30, 20, and 10 min of treatment
•
Apples PPO DBD, 150 W, 15 + 15, 30 + 30 min, Air, 1.5 A significant decrease in superficial browning not proportional to the treatment time
• [149]
m/s Varying impact on the PPO activity
•
Cultivar dependent strict effects
•
Melon POD DBD, 15 kV, 12.5 kHz, 15 + 15, 30 + 30 min Air residue activity of 91% and 82% after treatment for15 + 5 and 30 + 30 min,
• [150]
respectively
Melon PME DBD, 15 kV, 12.5 kHz, 15 + 15, 30 + 30 min • not effective 15 + 15 min air treatment [150]
• 94% residual activity after 30 + 30 min treatment
Carrot PPO DBD, 70 kV, 3 min, air • 33–43 % residual activity during 28 days storage at 4 ℃ [121]
PPO: polyphenoloxidase; POD: peroxidase; PME: pectinmethylesterase; HVCP: high voltage electric field cold plasma

Fig. 6. Hypothesised mechanism of high-pressure effect on the enzyme conformational structure. Adapted from Chakraborty, Kaushik [24] with permission.

marketed for FJs processing industries. The major reasons could be the
product acceptability, unavailability of advanced machinery, optimized
process parameters and investment in scale-up process. Therefore,
further efforts are required to commercialize these NTTs with an
extensive array of desired benefits, taking into account the associated
risks.
Declaration of Competing Interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
Zhao Liqing reports financial support was provided by This work was
supported by the National Key R&D Program of China
(2021YFA0910800) and the Special Fund for Development of Strategic
Emerging Industries in Shenzhen (JCYJ20190808145613154,
KQJSCX20180328100801771).

14
M. Umair et al. Ultrasonics Sonochemistry 86 (2022) 105999

Table 5
HHP processing effect on the enzymes inactivation in fruit juices and purees.
Fruit juice Enzyme Range given as (MPa/min/◦ C/ Max. inactivation at (MPa/min/◦ C/ Results References
others, if any) others, if any)

Apple PPO 250–450.00/0–60.00/25.00–50.00 450/60/50 (91%) • P-T interaction at > 400/>40 ◦ C MPa [34]
Kiwifruit juice POD 200–600/0–30/10–50 600/30/50 (30%) • Activation at 200 MPa/10 min/30 ◦ C [151]
Nectarine puree PPO 400, 600/5/20–25/ (without or with 600/5/25/without ascorbic acid • Activity retention of at 400 MPa/ in the [118]
ascorbic acid) (60%) presence of ascorbic acid
Peach juice PPO 400–600/5–25/25 600/25/25 (79%) • Activation (maximum107) at 400 MPa/15 min [35]
Plum puree PPO 400–600/0–5/20–25 20% (600/2.5/25) • Non-significant effect for all treatments (P > [124]
0.05)
Strawberry PPO 400–800/5–15/18–22 800/15 (100%) • Total deactivation at 800 MPa/15 min [122]
Strawberry PPO 400–600/5–25/25 600/25/25 (48%) [123]
pulp
POD 400–600/5–25/25 600/5/25 (64%) • Activation at 600 MPa > 5 min [123]
Strawberry PPO 100–690/5–15/24–90 690/5, 15/90 (23%) • Deactivation from 16% to 23% [152]
puree
Watermelon PPO 200–600/5–60/25 600/60/25 (88%) [22]
juice
POD 200–600/5–60/25 600/60/25 (42%) [22]
PPO: polyphenoloxidase; POD: peroxidase; PME: pectinmethylesterase; HHP: high hydrostatic pressure

Acknowledgements [19] S. Natarajan, V. Ponnusamy, A review on the applications of ultrasound in food

This work was supported by the National Key R&D Program of China
(2021YFA0910800) and the Special Fund for Development of Strategic
Emerging Industries in Shenzhen (JCYJ20190808145613154,
KQJSCX20180328100801771).
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