Innovative Food Science and Emerging Technologies 57 (2019) 102192
Contents lists available at ScienceDirect
Innovative Food Science and Emerging Technologies
journal homepage: www.elsevier.com/locate/ifset
Effects of high pressure, microwave and ultrasound processing on proteins
and enzyme activity in dairy systems — A review
T
Masooma Munira,b,c, Muhammad Nadeema, Tahir Mahmood Qureshif, Thomas S.H. Leongb,d,e,
⁎
Charitha J. Gamlathb,d,e, Gregory J.O. Martind,e, Muthupandian Ashokkumarb,d,
a
Institute of Food Science and Nutrition, University of Sargodha, Sargodha, Pakistan
School of Chemistry, The University of Melbourne, Parkville, Victoria 3010, Australia
c
Food Science Research Institute, National Agricultural Research Centre, Islamabad, Pakistan
d
The ARC Dairy Innovation Hub, The University of Melbourne, Parkville, Victoria 3010, Australia
e
Department of Chemical Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia
f
Department of Food Sciences, Cholistan University of Veterinary & Animal Sciences, Bahawalpur, Pakistan
b
A R T I C LE I N FO
A B S T R A C T
Keywords:
Emerging technologies
Protein denaturation
Enzyme activity
Dairy product quality
High-pressure processing (HPP), microwaves (MW) and ultrasound (US) are used for pasteurization with
minimum heat input. They also alter physico-chemical properties of milk proteins and enzymes. This article aims
at identifying the important changes in milk proteins imparted by these three processing technologies. HPP
dissociates casein micelles at low pH (< 6.7) and concentrations (< 4% w/w), while β-LG is the most pressure
sensitive whey protein due to the presence of free thiol groups. Milk enzyme activity is inhibited at higher
pressures (> 400 MPa). MW treatment denatures whey proteins rapidly, even below their thermal denaturation
temperatures. High-power MW treatment (e.g. 60 kW) deactivates enzymes by denaturing them. However, lowpower controlled MW irradiation (e.g. 30 W) improves enzyme activity. Ultrasound can homogenize protein
aggregates in dairy systems and cause whey protein denaturation. Sonication under applied pressure and heat
(e.g. 3.5 kg/cm2, 126.5 °C) causes enzyme inhibition while mild sonication conditions can improve enzyme
activity.
Industrial relevance: HPP, MW and US are gaining popularity in the dairy industry due to their ability to pasteurize and functionalize dairy streams with minimal heat input. This review offers insights into how these
technologies can be used in isolation or in combination to alter milk proteins and enzyme activity for different
academic and industrial applications. However, to fully understand the potential of HPP, MW and US treatment
on dairy systems, further research is required in several areas including health related nutritional changes in
milk and milk products caused by these technologies.
1. Introduction
Bovine milk is rich in carbohydrates (mainly lactose), fat (fatty
acids, phospholipids), casein proteins (αS1-casein, αS2-casein, β-casein,
κ-casein), whey proteins (immunoglobulin, β-lactoglobulin, α-lactalbumin, bovine serum albumin, lactoferrin), enzymes (lactoperoxidase,
catalase, proteinase, xanthine oxidoreductase, lipoprotein lipase, alkaline phosphatase, salolase and amylase), vitamins (thiamin, riboflavin,
vitamin A, B12, D, E and K) and minerals (calcium, phosphorus, magnesium, potassium, zinc and selenium) (McSweeney & Fox, 2013; Walstra,
1990). It is commonly contaminated with microorganisms during milking
(unhygienic milking utensils, unclean water and teats, dung and dust particles) and transportation (unhygienic vehicles). Due to its high nutritional
⁎
value and water content, milk provides a favorable ground for pathogenic microbial growth, and therefore is quickly subjected to spoilage.
Conventionally, thermal pasteurization is used to eliminate pathogenic microorganisms and ensure milk is suitable for human consumption. However,
during thermal treatment, heat sensitive milk constituents undergo many
physical and chemical modifications leading to deleterious effects on sensorial characteristics (taste and flavor) and nutritional value (Tamime,
2009). These limitations are the drive to use novel technologies with shorter
processing times and minimal heating, in research and industrial environments. High pressure, microwave and ultrasound are three such emerging technologies in the dairy industry that can be used to improve shelf
life and processability of milk (Rodríguez et al., 2003).
In 1969, the possibility of microwave (MW) treatment to replace
Corresponding author at: School of Chemistry, The University of Melbourne, Parkville, Victoria 3010, Australia.
E-mail address: masho@unimelb.edu.au (M. Ashokkumar).
https://doi.org/10.1016/j.ifset.2019.102192
Received 15 February 2019; Received in revised form 8 July 2019; Accepted 8 July 2019
Available online 09 July 2019
1466-8564/ © 2019 Elsevier Ltd. All rights reserved.
Innovative Food Science and Emerging Technologies 57 (2019) 102192
M. Munir, et al.
techniques and process conditions, for the application of interest. From
the literature, it appears that such a comparison has not yet been reported. Therefore, in this paper, we aim to compare and summarize the
recent developments in the application HPP, MW and US during dairy
processing with an emphasis on the mechanisms by which milk proteins
and enzyme activity are affected.
thermal pasteurization was put forward by Hamid and coworkers
(Hamid, Boulanger, Tong, Gallop, & Pereira, 1969). Microwaves generate heat through molecular vibration in food and provide faster microbial deactivation compared to conventional conductive or convective heating. Due to its higher energy efficiency, reduced processing
times and easy operation, MW heating is preferred in continuous food
processing systems (Martins et al., 2019). A recent study by MorenoVilet, Hernández-Hernández, and Villanueva-Rodríguez (2018) reported that MW treatment is the most widely studied food processing
technology in the world at present.
Alternatively, high-pressure processing (HPP) can effectively inactivate microorganisms at ambient temperature (Jermann, Koutchma,
Margas, Leadley, & Ros-Polski, 2015), while cavitation induced shear
forces generated by high intensity ultrasound (US), have also shown
promise in deactivating microorganisms in dairy products via nonthermal mechanisms (i.e. shear) (Balthazar et al., 2019; Guimarães
et al., 2018; Monteiro et al., 2018). A recent survey done in North
America and Europe concluded that HPP and MW treatment are comparable to each other in terms of their application in industry and
academia in these two regions (Jermann et al., 2015). Roselli et al.
(2018) reported that customers are interested in purchasing US treated
food (at a comparable price to traditionally prepared food), predicting
an increased application of US food processing, when customers are
better informed about the benefits of US processing in the future.
However, all three technologies create extreme conditions (high
pressure during HPP, strong shear, localized pressure and temperature
during US treatment and molecular vibration during MW treatment) at
the micro scale in dairy systems while processing. Such conditions can
alter the physicochemical characteristics of sensitive milk constituents.
From a colloidal perspective, milk is a stable suspension of proteins
(caseins and whey proteins), emulsified fat droplets, lactose and minerals in an aqueous medium (Jenness, 1999). Caseins are present in
milk in the form of casein micelles. These roughly spherical aggregates
are poly-dispersed in size (50–300 nm in diameter) and consist of a
hydrophobic core (rich in αS1, αS2 and β-casein) covered by a hydrophilic (κ-casein rich) hairy layer. Caseins are held together in the micelle by colloidal calcium phosphate (CCP) and hydrophobic interactions, while the electrostatic and steric repulsion created by the ‘hairy
layer’ keep the micelles uniformly dispersed in the milk serum (Lucey,
2002). Compared to caseins, whey proteins are relatively hydrophilic in
their native conformation and are present as small (~3–7 nm) globular
proteins (Laiho, Ercili-Cura, Forssell, Myllärinen, & Partanen, 2015;
Rosa, Sala, Van Vliet, & Van De Velde, 2006). Upon drastic environmental changes (i.e. pH, temperature, shear) milk proteins denature to
expose hydrophobic sites and reactive amino acid residues (e.g., thiol
groups) that are buried within the native structure and participate in
protein-protein interactions.
In order to improve the shelf life of milk constituents, milk is widely
converted to a variety of milk products such as milk powder, cheese,
butter and condensed milk. Sometimes, conversion of milk into other
milk products involves addition of enzymes. For example, during cheese
production, addition of enzyme (chymosin) destabilizes the casein micelles leading to their aggregation and formation of a coagulum (Lucey,
2002). Further, during cheese maturation and intestinal digestion of
milk products, enzymes play a key role in breaking down complex
proteins into smaller peptides and amino acids that could be easily
absorbed in the human body (Segura-Campos, Chel-Guerrero, BetancurAncona, & Hernandez-Escalante, 2011; Voigt et al., 2012). As dairy
enzymes are also proteins, extreme processing conditions of HPP, MW
and US can alter their activity. Such effects can ultimately alter nutritional quality and the functionality of different dairy streams and products.
With the increasing popularity of more sustainable and efficient MW
(Atuonwu et al., 2018), HPP (Misra et al., 2017) and US dairy processing technologies, a thorough understanding of their key effects on
dairy proteins and enzyme activity, could help in selecting optimum
2. High-pressure processing
2.1. Mechanism of high-pressure processing
High-pressure processing (HPP) is the application of pressure in the
range of 100–600 MPa applied at ambient temperature. It is used, primarily to deactivate pathogenic microorganisms including vegetative
bacteria, yeast and moulds. In industrial applications, treatment times
may vary from 2 to 30 min depending on the food type (Ghasemkhani
et al., 2014). Although the pressure is applied at ambient temperature,
a 3–9 °C per 100 MPa increase in temperature (depending on the pressure-transmitting fluid and treatment duration) occurs due to adiabatic
heating (Balasubramaniam, Ting, Stewart, & Robbins, 2004). Highpressure processing can be operated in batch, continuous or semi-continuous modes. During batch processing, pre-packed food is introduced
into the pressure chamber which is then sealed. Water enters the
chamber to displace any air and pressure is built up in the chamber
until the specified limit is reached. After being pressurized for a specific
time interval, the chamber is depressurized, and the processed food is
removed. Batch (static) HPP is preferred over other forms of processing
due to its efficiency and simplicity. Contrastingly, during continuous
processing (also known as dynamic high-pressure processing) (de
Oliveira, Augusto, da Cruz, & Cristianini, 2014), liquid food is sent
through a narrow gap using a moving piston/s (Singh & Yousef, 2001).
Unlike batch processing where packed food is held at a constant pressure, during semi-continuous processing, a liquid food flow is introduced and held in a similar holding chamber for a specific period of
time, after which the depressurized liquid food is transferred to sterile
tanks for storage or shipment (Balasubramaniam, Barbosa-Cánovas, &
Lelieveld, 2016). In this review, we mainly focus on the effects of more
popular batch HPP.
2.2. Effect of high-pressure processing on dairy proteins
Milk proteins are varied complex entities that are affected differently by pressure. Their primary structure is held together by covalent
bonds while hydrogens bonds, electrostatic interactions and hydrophobic effects govern the secondary and tertiary structures. The order
of sensitivity of different bonds to HPP is in the order of hydrophobic
interactions > electrostatic interaction > hydrogen bonds > covalent
bonds. As covalent bonds are least affected by HPP, the protein primary
structure remains unchanged while the secondary and tertiary structures alter (Goyal, Sharma, Upadhyay, Sihag, & Kaushik, 2018). Out of
the two classes of milk proteins, caseins are more resilient in extreme
environmental conditions due to their micellar structure, held together
by hydrophobic bonds, hydrogen bonds and colloidal calcium phosphate (CCP) interactions (Lucey, 2002). Whey proteins are more susceptible to denaturation as hydrophobic interactions and hydrogen
bonds predominantly govern their tertiary structure (Chandrapala,
Zisu, Palmer, Kentish, & Ashokkumar, 2011). As Huppertz, Kelly, and
Fox (2002) have extensively reviewed the reports of the effects of HPP
on milk proteins until 2002, we discuss more recent findings in the
following section.
Depending on the intensity and duration of applied pressure, pH and
temperature of the system, high-pressure processing can alter the resilient casein micellar structure (Gebhardt, Doster, & Kulozik, 2005;
Orlien, Boserup, & Olsen, 2010). When treated with HPP, hydrophobic
bonds inside the casein micelle are disrupted and water penetrates into
the micellar structure (hydration). As a result, CCP starts to solubilize
2
Innovative Food Science and Emerging Technologies 57 (2019) 102192
M. Munir, et al.
Pressure >50 MPa, ~10 min
Casein
Micelle
Hydration
Disruption of
hydrophobic
interactions
Lower pH Low pressure thresholds
Higher pH High pressure thresholds
Release of casein
submicelles
Dissociation of
colloidal
calcium
phosphate
Prolonged
pressurization
250-300 MPa,
1-3 hr
Submicelle
reassociation
Prolonged
pressurization
300-500 MPa,
1-3hr
> 300MPa
No reassociation
of submicelles
Fig. 1. A schematic representation of the dissociation of the casein micelle under high pressure, based on the works of Orlien et al. (2010), Needs, Stenning, Gill,
Ferragut, and Rich (2000), Cadesky, Walkling-Ribeiro, Kriner, Karwe, and Moraru (2017) and Chawla, Patil, and Singh (2011).
contents (Baier, Schmitt, & Knorr, 2015; Cadesky et al., 2017; Needs
et al., 2000), at high protein concentrations (> 10% w/w) casein micelles aggregate and even form soft gels during HPP (Anema, 2008;
Cadesky et al., 2017). The mechanism of pressure induced aggregation
of casein micelles is yet to be fully understood.
In contrast to studies on high-pressure induced changes in casein
that were focused mostly on the dissociation of the micelles, studies on
whey proteins, rather focus on protein unfolding (i.e. changes in secondary and tertiary structures). β-Lactoglobulin (β-LG) is a pressure
sensitive globular whey protein (with two intramolecular disulphide
bridges and one free thiol group) that is present as a dimer in milk
(Goyal et al., 2018; Huppertz et al., 2002). β-LG denaturation under
pressure was initially believed to be a simple one-step process (Fig. 2a),
where native β-lactoglobulin permanently unfolded at neutral pH,
when subjected to a pressure of 350 MPa (Dufour, Hoa, & Haertlé,
1994). The effect of pressure was observed to be less at lower pH (3.0)
(Valente-Mesquita, Botelho, & Ferreira, 1998). Subsequent work revealed that the quaternary structure of native β-LG changes to an ‘inaccurately folded conformation’ under pressure, allowing the formation
of non-native disulphide bonds (Fig. 2b) (Valente-Mesquita et al.,
1998). However, according to the model proposed by Stapelfeldt and
Skibsted (1999), the denaturation of β-LG includes three major steps:
(a) pressure induced reversible unfolding, (b) SeS bond formation and
(c) subsequent irreversible aggregation leading to gelation (Fig. 2c).
Orlien, Olsen, and Skibsted (2007) proposed a detailed mechanism for
β-LG under pressure. When pressure is applied, the native β-lactoglobulin structure unfolds making the protein backbone more flexible for
conformational fluctuations and allowing water (in the medium) to
penetrate into the hydrophobic interior of the globule (hydration). The
exchange of water between the solvent and proteins under high pressure alters the protein conformation into a molten globule that does not
have any specific quaternary structure. The molten globule structure
may persist provided a constant pressure is maintained but with increasing pressure, it denatures to form aggregates (Fig. 2d).
Goyal et al. (2018) and Huppertz et al. (2002) summarized the
varying extents of β-LG denaturation reported in literature depending
on the physico-chemical properties of the protein solution, applied
leading to weaker intra-micellar bonds (Desobry-Banon, Richard, &
Hardy, 1994; Gaucheron et al., 1997). Particularly at low pH
(pH < 6.7), CCP readily dissociates into ionic calcium, resulting in relatively weaker micelles (Orlien et al., 2010). Thus, casein micelles are
more pressure resistant at natural or higher pH (pH > 7) but have
lower pressure threshold at low pH (Fig. 1). At a particular pH, Orlien
et al. (2010) observed that the pressure threshold of casein micelles in
reconstituted skim milk did not vary significantly when the temperature
increased from 5 °C to 40 °C. However, Gebhardt et al. (2005) observed
that over a broader temperature range (10 °C–80 °C), the pressure
threshold of 3% casein micelle solutions indeed increased with an increase in temperature.
Pressure-induced casein micelle dissociation is followed by observable increases in the calcium content in the aqueous phase possibly
due to dissociation and solubilization of CCP under HPP (Cadesky et al.,
2017; Chawla et al., 2011; Law et al., 1998). Upon dissociation, relatively large casein micelles (150–200 nm) form smaller submicelles
(~40 nm) (Needs et al., 2000). Lopez-Fandino, De La Fuente, Ramos,
and Olano (1998) reported that the types of casein submicelle released
to the serum depend on the phosphate content of individual proteins. As
phosphorylated residues in the peptide chain directly participate in
forming CCP, submicelles rich in caseins with more phosphorylated
residues (αS1, αS2) retain inside the micelle, while those with less
phosphorylated residues (κ, β) leach into the serum. Although micelles
dissociate when moderate to high pressures are applied for a short time
(e.g. 50–500 MPa for 10 mins (Orlien, Knudsen, Colon, & Skibsted,
2006), or 150–350 MPa for 15 mins (Cadesky et al., 2017), prolonged
HPP (e.g. 250–300 MPa for 1–3 h) results in reassociation of dissociated
submicelles. A similar yet less significant reassociation (detected only
by soluble protein quantification) was observed by Cadesky et al.
(2017) at low treatment durations (15 min) but at a higher applied
pressure (450 MPa). However, Orlien and coworkers who used turbidity
measurements to track the micellar dissociation did not observe a reassociation of casein micelles at higher pressures (300–500 MPa) when
treated up to 3 h (Fig. 1) (Orlien et al., 2006)
Interestingly, while varying extents of casein micelle dissociation
are reported in literature in milks with low (< 4% w/w) protein
3
Innovative Food Science and Emerging Technologies 57 (2019) 102192
M. Munir, et al.
Pressure (350 MPa)
Natural pH
Native beta-lactoglobulin
a.
Denatured beta-lactoglobulin
One step denaturation model of beta-lactoglobulin adapted from Dufour, et al., 1994
Pressure
Native beta-lactoglobulin
Inaccurately folded
conformation due to
S-S (non native)
bonds formation
Denatured betalactoglobulin
b. Extension of one step denaturation model of beta-lactoglobulin adapted from Valente-Mesquita, et al.,
1998
Pressure
Native beta-lactoglobulin
Thiol exchange resulting in
irreversible gelation and
aggregation (denaturation)
Reversible unfolding
(Dissociation)
c. Three step denaturation of beta-lactoglobulin adapted from Staplefeldt & Skibsted, 1999
Pressure
(A)Native beta-
Denaturation of Protein
Hydration
Unfolding
Molten Globule without
tertiary structure
Conformational
alterations of proteins
Water Penetration in the
hydrophobic Interior
Aggregation
Fig. 2. (a), (b), (c), (d) Development of the pressure induced denaturation model of β-lactoglobulin.
its relatively high conformational rigidity. Denaturation further increased to 50% when treated at 800 MPa for 30 min.
Bovine serum albumin (BSA) consists of 17 desulphated bridges and
one free thiol group. It is composed of 56.8% alpha-helices, 5.8% betasheets, 14.1% beta-turns and 23.9% random structures (Ye, Qin, Yang,
She, & Xing, 2007). Due to the large number of disulphide linkages and
high degree of helix structures, BSA can withstand pressures up to
600 MPa (Hayakawa, Kajihara, Morikawa, Oda, & Fijio, 1992). Felipe,
Capellas, and Law (1997) reported that immunoglobulin is more susceptible to pressure induced denaturation compared to α-LA, although
the reason for this behavior is not clear. As denaturation of whey proteins commonly leads to protein aggregation (due to the exposed thiol
group interactions and hydrophobic bonds), a decrease in the noncasein/soluble nitrogen content in milk is commonly observed (Goyal
et al., 2018; Johnston, Austin, & Murphy, 1992).
HPP-induced changes in the protein structure cause profound effects
on the functionality (e.g. foaming and emulsifying properties) of milk
pressure and the method of protein isolation/concentration. Goyal et al.
(2018) stated that β-LG unfolds when subjected to pressures between
100 and 400 MPa, exposing the free thiol groups to the surroundings. At
400 MPa, extensive denaturation of β–LG occurs. Exposed thiol groups
tend to form disulphide bridges with other milk proteins (κ-casein, αS2casein, β–LG and α-LA) (Goyal et al., 2018; Huppertz et al., 2002).
However, β-LG tends to renature during storage (up to 2 days) at
20–40 °C. At lower storage temperatures (5 °C), reassociation does not
take place due to the reduced effect of hydrophobic interactions (Goyal
et al., 2018).
Compared to β–LG (with two intramolecular disulphide bridges and
1 free thiol group), α-LA has a more rigid structure due to the presence
of four intramolecular disulphide bridges with no free thiol groups
(Huppertz et al., 2002; Lopez-Fandino et al., 1998). In comparison, Ye,
Anema, and Singh (2004) observed that only 10% of the α-LA in milk
denatured at 600 MPa when applied for 30 min. The lower susceptibility of this protein to pressure-induced denaturation was attributed to
4
Innovative Food Science and Emerging Technologies 57 (2019) 102192
M. Munir, et al.
concerned, there is a minimum pressure below which enzymes inactivation is not significant. As pressure increases, the extent of inactivation increases reaching complete inactivation at a certain pressure. The range of inactivation pressure is highly dependent upon the
enzyme type, composition of medium, temperature and pH
(Eisenmenger and Reyes-De-Corcuera, 2009; Sakharam et al., 2011).
Some enzymes have pressure sensitive (unfolded) and pressure resistant
portions (globular) in their active 3D structure. The pressure-sensitive
portion becomes irreversibly inactive at its pressure threshold while the
remaining potion continues to be active (Curl & Jansen, 1950).
Due to the above reasons, dairy enzymes express varying sensitivity
to high pressure. Lactoperoxidase, lipase and xanthine oxidase were
found to be resistant to pressures up to 400 MPa (Naik, Sharma, Rajput,
& Manju, 2013). While γ-glutamyl transferase, phosphohexose isomerase and alkaline phosphatase in milk were partially inactivated at
pressures exceeding 350, 400 and 600 MPa respectively, they were fully
inactivated at pressures of 550, 630 and 800 MPa respectively
(Sakharam et al., 2011). Similarly, alkaline phosphatase is not affected
by pressure treatment up to 400 MPa for 60 min, but is completely inactivated when treated at 800 MPa for 8 min (Naik et al., 2013).
Cheese production from milk is also affected by enzyme activity.
While intestinal proteases like chymosin and rennet are used to convert
liquid milk to a coagulum, lactic acid bacteria-derived proteases such as
pepsin and plasmin participate in post coagulation ripening and subsequent flavor development in cheese (Fox, Guinee, Cogan, &
McSweeney, 2000). The proteolytic activity of chymosin was reduced
after pretreatments at pressures over 400 MPa, although the activity
increased after exposure to moderate pressures < 275 MPa (Júnior,
Tribst, & Cristianini, 2017). Júnior, Tribst, Bonafe, and Cristianini
(2016) reported that the proteolytic activity and milk clotting activity
of calf rennet increased by 23% and 17%, when subjected to pressures
up to 175–285 MPa for 14–23 min at 25 °C. The increased rennet clotting activity of chymosin treated at 280 MPa for 20 min caused an 8.3%
reduction in rennet clotting time. Even after storage (for 24 h) the improved proteolytic activity of the enzyme was significant.
In the meantime, Zobrist et al. (2005) observed that mildly pressuretreated (200–250 MPa) milk tended to have reduced rennet coagulation
times. However, treatment at 400–600 MPa resulted in increased rennet
coagulation times. They speculated that the availability of dissociated
casein micelles with higher surface area for interaction, in moderate
pressure treated milk, could be the reason for faster gel formation.
Reassociation of the dissociated micelles at higher pressures was believed to cause increased rennet coagulation times. Similarly, Juan,
Ferragut, Buffa, Guamis, and Trujillo (2007) reported an increased
proteolysis in cheese treated at low pressures (300 MPa, 10 min holding
time), while at 500 MPa (for 10 min) treatment decreased the rate of
proteolysis. Contrastingly, Chawla et al. (2011) reported in their review, an improved proteolytic activity in Mozzarella, Gouda and ewes'
milk cheeses, exposed to even higher pressures (400–600 MPa). In a
complementary study, Delgado-Martínez, Carrapiso, Contador, and
Ramírez (2019) used HPP (600 MPa, holding time 5 and 20 min) in
cheese and observed a decrease in bitterness in ripened cheese due to
the decreased rate of ripening/proteolysis. They further reported improved sensory charactertics of cheese after HPP due to the reduction in
undesirable bitterness.
and dairy streams. For example, whey protein isolates (WPI) and concentrates (WPC) pressurized at 600 MPa had varied foaming properties
compared to control samples. It was found that high-pressure treatments can improve the foaming behavior of WPI (higher foam expansion and foam volume stability) than the unpressurized samples due to
the increase in the amount of available surface-active residues.
However, the foaming ability of pressurized WPC was reduced due to
formation of aggregates. Aggregation reduces the amount of protein
available for film formation, but the films that are formed, are considered to be thicker and more stable, facilitating the formation of a
network structure in the protein film, thereby increasing foam stability.
Therefore, foams formed with high-pressure treated WPC and WPI exhibited significantly prolonged stability compared with un treated
control samples (Krešic, Lelas, Herceg, & Režek, 2006).
High-pressure processing has shown a detrimental effect on the
emulsifying properties of both WPI (pressurization at 600 MPa), WPC
(pressurized at 300 MPa). Effect of pressure on emulsifying properties
of whey proteins could be due to conformational changes and aggregation of whey proteins under pressure, that reduces the proportion
of proteins which could be adsorbed at the oil-water interface (Krešic
et al., 2006).
The changes caused to milk proteins during HPP could affect the
final quality of products made from the treated milk. While studying the
ripening profile of cheddar cheese made from milk treated at 400 and
600 MPa, Voigt et al. (2012) reported an increased incorporation of βLG in the cheese curd. This is because β–LG denatures and aggregates at
pressures higher than 400 MPa and consequently becomes incorporated
into the cheese curd. The cheese produced from the HPP treated milk
had higher amounts of non-casein nitrogen and phosphotungstic acid
(PTA) soluble nitrogen content during 180 days of ripening, indicating
a higher proteolytic activity and protein hydrolysis compared to the
control cheese (Voigt et al., 2012). A higher proteolytic activity could
occur as denatured/unfolded whey proteins and disrupted casein micelles provide more sites for protein-enzyme interactions compared to
their protected native confirmation. They further reported that after
treating cheese milk with high pressure (400–600 MPa), the initial
whiteness (L-value) decreased due to the dissociation of casein micelles.
However, the L-value in pressure-treated ripened cheeses was higher
compared to the control, possibly due to differences in the protein
matrix and in fat distribution. Similarly, as the relatively smaller submicelles formed by casein micelle dissociation at 400 MPa have a faster
collision rate and higher probability of crosslinking, a 11% reduction in
the coagulation time (time required to reach a storage modulus of
50 Pa) was achieved (Voigt, Donaghy, Patterson, Stephan, & Kelly,
2010). Further, cheddar cheese milks treated at 600 MPa produced
more soft, less chewy and gummy cheeses, although their melt-ability
was significantly reduced (Voigt et al., 2012).
2.3. Effect of high-pressure processing on dairy-related enzyme activity
Enzymes are proteins whose biological activity depends on specific
active sites that arise from the 3-D configuration of the molecule.
Therefore, any change in their structure could alter enzyme activity
(Tsou, 1986). High pressure application imposes a dual effect on enzymes' biological activity. Lower pressures (generally 20–350 MPa depending on the enzyme type and treatment temperature) are documented to activate enzymes, while higher pressures (> 400 MPa) cause
inactivation (Sakharam, Prajapati, & Jana, 2011). Further, when untreated enzymes are added to HPP milk, they show a higher activity in
milk processed at moderate pressures whereas in those treated at higher
pressures their activity is reduced (Zobrist, Huppertz, Uniacke, Fox, &
Kelly, 2005).
Increased enzyme activity rates after pressurization, are attributed
to pressure induced conformational flexibility in enzymes and substrate
proteins' partial unfolding, allowing greater interactions (Eisenmenger
& Reyes-De-Corcuera, 2009). As far as enzyme inactivation is
3. Microwave processing
3.1. Mechanism of microwave processing
Microwaves (MW) are generated in food processing ovens by the
application of an alternating electric field. These waves typically have a
wavelength of < 1 cm and a frequency of 2.45 GHz. It is a form of nonionizing radiation and does not break covalent bonds (Anantheswaran
& Ramaswamy, 2001; Gomaa, 2010). MW can induce changes in milk
either thermally or non-thermally (Gomaa, 2010; Marani & Feirabend,
5
Innovative Food Science and Emerging Technologies 57 (2019) 102192
M. Munir, et al.
amino acid residues (Anantheswaran & Ramaswamy, 2001). Previous
studies therefore report that protein denaturation and aggregation due
to MW reduce the soluble amino acid content in milk with no change in
the total amino acid content. For example, Albert, Pohn, Mándoki,
Csapó-Kiss, and Csapó (2009) observed that the free/soluble amino acid
content was reduced under MW treatment (2.45 GHz, 68 °C 40 s), while
the total amino acid content remained unchanged.
Compared to conventional thermal pasteurization, microwave
heating requires shorter treatment times to achieve the same microbial
deactivation due to the faster cellular protein degradation. Therefore
MW-assisted pasteurization can reduce the extent of many adverse
biochemical phenomena occurring in milk during thermal pasteurization. For example, Villamiel, López-Fandiño, Corzo, Martínez-Castro,
and Olano (1996) reported satisfactory milk quality, when pasteurized
by microwaves, due to lower enzyme activity and lower degree of deterioration in milk consituents. Improved sensory attributes were also
reported by Clare et al. (2005), with respect to odor, caramelization,
astringency and fatty flavors as compared to UHT treated milk.
1994). Under the influence of microwaves, water dipoles try to rearrange themselves with those of the microwaves, resulting in friction
between the molecules. The changes in milk due to the localized heat
generated by the molecular friction are known as thermal effects, while
other changes occurring due to molecular rearrangement alone are
known as non-thermal effects (Gomaa, 2010; Tong, 1996). As molecular
rearrangement and heat generation occur simultaneously, their relative
impact on proteins and enzyme activity is still under debate.
3.2. Effect of microwaves on dairy proteins
Molecular rearrangement during MW treatment changes the intermolecular spacing between proteins and alters their quaternary and
tertiary structures (El Mecherfi et al., 2011). Depending on the duration, form, frequency and strength of the applied waves, MW can denature sensitive whey proteins molecules as well as resilient casein
micelles (Bi et al., 2015; Bohr & Bohr, 2000; Villamiel, Corzo, MartinezCastro, & Olano, 1996). As heat generation is inevitable during MW
processing, many reports compare the effects caused by MW treatment
to those caused by conventional heat treatments (e.g. pasteurization,
ultra-high temperature treatment) (Clare et al., 2005; Petrucelli &
Fisher, 1994; Villamiel, Corzo, et al., 1996).
From our literature survey, it appears that reports of the direct effects of MW on isolated casein micellar suspensions or caseins in milk
are limited compared to those on HPP. However Bi and coworkers have
studied MW and heat-induced folding and unfolding of casein proteins
in the presence of saccharides. They reported that at the same bulk
temperature, MW treatment caused casein unfolding (observed by an
increase in exposed tryptophan residues), whereas conventional
heating did not. Further, acid gels formed with MW treated casein solutions were harder and had a more compact microstructure compared
to those made with heated caseins, possibly due to more interactions
and crosslinks between thiol groups and hydrophobic residues in unfolded caseins (Bi et al., 2015).
Whey proteins also unfold and denature when treated with MW.
Villamiel, Corzo, et al. (1996) reported that at temperatures above
70 °C, whey proteins denature when subjected to either MW or conventional heating. At the same temperature, whey proteins tend to
denature more rapidly (measured over 30 min) in milk heated with
microwaves compared to conventional heating. However, at very short
processing times (≤20 s), the extent of whey protein denaturation
caused by MW heating and UHT treatment (at comparable thermal
energy inputs) remained similar (Clare et al., 2005). Extensive MWinduced whey protein denaturation could lead to disulphide crosslinking and aggregate formation (Iuliana, Rodica, Sorina, & Oana,
2015). de Pomerai and coworkers were able to form aggregates and
amyloid fibrils from BSA and bovine insulin at 60 °C, by supplying a
15–20 mW kg−1 MW irradiation (de Pomerai et al., 2003). Denaturation and aggregation of proteins during MW treatment often leads to
reduced soluble protein contents (Iuliana et al., 2015) in dairy streams.
While MW's ability to denature and unfold whey proteins is widely
documented, Bohr and Bohr (2000) reported an interesting dual folding
and unfolding effect of microwaves on cold-denatured β-LG. In their
study, denatured β-LG in urea was subjected to a heating and cooling
cycle between 48 °C and 4 °C, where the protein folded when the temperature increased and unfolded when the temperature decreased.
When microwaves were applied at 20 °C (during temperature drop) and
8 °C (during temperature rise) a significant enhancement in unfolding
(during temperature drop) and folding (during temperature rise) was
observed.
Although conformational changes in proteins could alter physical
attributes of dairy streams, their nutritional value depends on the
quality and the quantity of amino acids and peptides produced from
enzymatic digestion of proteins inside the human body. Although energy supplied by MW irradiation can disrupt weaker hydrophobic interactions, it is not adequate to break covalent bonds between or within
3.3. Effect of microwaves on dairy-related enzyme activity
Microwaves could be used to alter enzyme activity in dairy streams
in three ways. (a) Pre-treatment of thermally unstable enzymes with
MW leads to their inhibition (due to heat denaturation of enzymes). (b)
Controlled application of MW to protein-enzyme mixtures could make
specific sites of proteins more susceptible to enzymatic hydrolysis, due
to the continuous rearrangement of molecules and protein unfolding,
and accelerate proteolysis (c) Milk protein unfolding (without aggregation) using a MW pretreatment could improve enzymatic activity
due to the increase in the number of exposed specific sites. Recent case
studies of all three applications are discussed below.
Clare and colleagues were able deactivate naturally occurring
plasmin in milk by a continuous flow, high-power (60-kW) MW treatment (Clare et al., 2005). La Cara et al. (1999) demonstrated that βgalactosidase that survives water bath heating at 70 °C for 1 h, could be
deactivated by exposure to 1.1 or 1.7 W/g microwave irradiation while
heating (70 °C for 1 h)..
Contrastingly, Izquierdo, Alli, Gómez, Ramaswamy, and Yaylayan
(2005) used controlled MW irradiation (30 W at 40 °C) during proteinenzyme incubation to improve pronase-induced β–LG hydrolysis. In a
similar study it was demonstrated that the activity of multiple proteases
(pronase, chymotrypsin, papain, corolases 7089 and PN-L 100, alcalase
and neutrase) in whey protein concentrate could be improved by controlled application of MW irradiation (213 W at 40–50 °C) (Izquierdo,
Peñas, Baeza, & Gomez, 2008). More recently, Chen and Hsieh (2016)
in their attempts to polymerize caseins using microbial transglutaminase (MTGase), found that MW could greatly accelerate MTGase-induced polymerization reactions in milk. They were able to reduce the
reaction time (from 3 h to 1 h) using a 30 W microwave treatment.
Alternatively, Gomaa (2010) observed that when milk was preheated
with microwaves (to attain a temperature of 60 °C within 5 mins) the
activity of various added proteolytic enzymes (pepsin, trypsin, chymotrypisn and gastrointestinal enzymes) became more pronounced
during incubation.
4. Ultrasound processing
4.1. Mechanism of ultrasound processing
Ultrasound (US) refers to sound waves of frequency > 18 kHz.
When ultrasound waves pass through a medium, they create mechanical vibrations, acoustic streaming and acoustic cavitation. Mechanical
vibrations have the potential to alter the structure and size of solid
particles, while acoustic streaming can enable or improve mass transfer
through a medium (Tho, Manasseh, & Ooi, 2007). When applied to a
liquid medium, US causes pre-existing micro-bubbles in the liquid to
6
Innovative Food Science and Emerging Technologies 57 (2019) 102192
M. Munir, et al.
properties of milk/milk protein streams is provided in Fig. 5 and
Table 1.
Physical effects of US such as shock waves and microjets increase
aggregate interactions in a suspension, causing them to collide with a
greater force (Thompson & Doraiswamy, 1999). Protein aggregates can
disintegrate upon collision if bound by weaker hydrophobic interactions or Van der Waal bonds, giving US the ability to homogenize
protein suspensions. For instance, whey protein concentrates (WPC)
produced from cheese whey generally contain protein aggregates
(0.2–10 μm) held together mainly by hydrophobic interactions. Zisu
et al. (2011) sonicated reconstituted 5% (w/w) WPC solutions at 20 kHz
and 31 W and reduced the mean particle size from 200 nm to 125 nm
within 60 min. Similarly, O'Sullivan and colleagues were able to reduce
the aggregate size of reconstituted whey protein isolate suspensions by
50% by applying 20 kHz 34 W cm−2 US for 2 min (O'Sullivan et al.,
2014a). Similar aggregate reductions are also reported while sonicating
milk protein concentrate solutions (20 kHz, 300 W electric power)
(Yanjun et al., 2014), sodium caseinate solutions (34 W cm−2, 20 kHz)
(O'Sullivan et al., 2014b) and denatured casein-whey protein mixtures
(Leong et al., 2018). Jambrak et al. (2014), while comparing the effect
of probe (20 kHz, 43–48 W/cm2) and bath (40 kHz, 1 W/cm2) sonication on the size reduction of aggregates in WPC, observed that probe
sonication, which has a narrower but more intense cavitation region,
had a more profound effect. Therefore, the energy dissipated per unit
area through cavitation is key in determining the extent of aggregate
size reduction.
At higher applied energy intensities US can affect protein confirmation. Madadlou, Mousavi, Emam-djomeh, Ehsani, and Sheehan
(2009a) while working with reconstituted micellar casein powder solutions between pH 6 and 12, reported that casein micelles were disrupted by cavitation-induced shear resulting in micelle size reduction
(35 kHz, 200 W output power, 6 h). Nguyen and Anema (2010) too
observed a reduction in the particle size of skim milk in the initial
stages of sonication (22.5 kHz, 50 W up to 10 min) and deduced that the
casein micelles could by dissociated by US. However, Chandrapala et al.
(2012) argued that observed size reduction in the reconstituted micellar
solutions and skim milk by previous researchers occurred solely due to
expand and contract in a process known as cavitation. In the case of
transient, or inertial cavitation, the bubbles grow during these oscillations until they reach their resonance size range, whereby they violently collapse (Ashokkumar & Mason, 2007; Yasui, 2002). Transient
cavitation occurs at low frequency ultrasound where the bubbles increase in size within few acoustic cycles and then collapse into fragments, producing extreme localized temperatures ranging between
2000 and 5000 K as well as high pressure and physical shearing
(Ashokkumar & Mason, 2007). Stable cavitation occurs mostly at higher
frequencies where the bubble size increases little, over a large number
of acoustic cycles, and results in relatively mild streaming effects.
In addition to physical effects, cavitation can induce chemical
changes as well. In an aqueous medium, water vapor and gas molecules
inside the bubble form highly reactive radicals as a result of the localized high temperature generated by cavitation. Hydrogen and hydroxyl
radicals are produced by the cleavage of water molecules while other
radicals may also be generated if other gaseous molecules are present
inside the bubble. Physical effects of US are prominent at low frequencies (20 kHz) while chemical effects are more prominent at high
frequencies (300–500 kHz) (Ashokkumar & Mason, 2007). These chemical and physical effects provide a range of different effects to milk
proteins and enzymatic activity that can be applied in the dairy industry. A schematic representation of the utilization of different effects
of ultrasound in the dairy industry is provided in Fig. 3 and is discussed
in detail below.
4.2. Effect of ultrasonic treatment on dairy proteins
Compared to HPP and MW, which predominantly alter the native
casein and whey protein confirmation, ultrasound has two added
functions; (a) dis-integration of already denatured protein aggregates in
suspensions due to shear generated by high-power low-frequency ultrasound, and (b) chemical degradation of proteins due to radicals
formed by low-power high-frequency ultrasound. Therefore, in this
section we first discuss the application of US for aggregate disintegration followed by conformational and chemical changes to proteins. A
summary of the physical and chemical effects of US in relation to the
Mechanical
vibration
Non-cavitational
Acoustic
streaming
Shear forces
Shock waves
Micro-jets
Cavitational
Temperature
Pressure
Chemical
effects
Homogenization
Pasteurization
Viscosity
reduction
Physical
effects
Applications
of
ultrasound
Emulsification
Highly reactive
radicals
Micro sphere
synthesis from
isolated milk
proteins
Enzyme
activation
/deactivation
Milk oxidation
Fig. 3. Schematic representation of the utilization of different effects of ultrasound in dairy.
7
8
Reduction in flowability
Reduction in turbidity
Increase in solubility
30 kHz, 73–78 W cm−2, up to 15 min
20 kHZ, 50 W, 1 h
20 kHz, 31 W/cm2 and 69 W/cm2,
20 min
20 kHz, 73–78 W cm−2, up to 30 min
20 kHz, 12.50 W, up to 5 mins
30 kHz 73–78 W cm−2, 10 min
Reconstituted whey protein isolate
Emulsification ability
20 kHz, 31 W/cm2, 20 min
20 kHz, 34 W cm−2, 2 min
Reconstituted milk protein concentrate
Reconstituted whey protein isolate
Turbidity
Solubility
Viscosity
Reconstituted whey protein isolate
Reconstituted sodium caseinate, whey
protein isolate and concentrate
Reconstituted whey protein isolate
Reconstituted whey protein concentrate
Reconstituted whey protein isolate
Reconstituted whey protein concentrate
Surface hydrophobicity
Skim milk
20 W/cm2, up to 90 mins
20 kHz, 31 W, up to 60 mins
20 kHz, 4.27 W, 20 min
20 kHz, 31 W, up to 60 mins
22.5 kHz, 50 W 10 min
20 kHz, 31 W, 1 h
20 kHz 286 kJ kg−1 power, 15 mins
Reconstituted Micellar casein
Bovine Serum Albumin
Reconstituted whey protein concentrate
35 kHz, 200 W output power, 6 h
Reconstituted whey protein isolate
Reconstituted Sodium caseinate
Reconstituted milk protein concentrate
Heat denatured casein-whey mixtures
Free-Sulfhydryl groups
Reduction in casein micelle size
No change in casein micelle size
Reduction in micelle size when sonicated at pH 8 and later
neutralized (pH 6.7)
Decrease in free Sulfhydryl groups
No change in free Sulfhydryl groups
No change in free Sulfhydryl groups
Increased until 5 mins. Reduced from 5 min to 60 min due
to aggregation
Increase in surface hydrophobicity
Reduction in viscosity
Probe (20 kHz, 43–48 W/cm2) and bath
(40 kHz,1 W/cm2)
20 kHz, 34 W cm−2, 2 min
20 kHz, 34 W cm−2, 2 min
20 kHz, 300 W electric power
20 kHz, 20.8 W, 1 min
Foam stability increased up to 15 mins of treatment
followed by a reduction from 15 to 30 mins
Emulsion stability index increased up to 1 min of
treatment followed by a reduction from 2 to 5 mins
Emulsion stability index decreased
Reduction in the mean particle size from 200 nm to
125 nm
Probe sonication causes a larger reduction in the aggregate
size
50% reduction in the particle size
Reduction in particle size
Reduction in particle size
Reduction in the aggregate size in heated casein: whey
80:20 and 50:50 systems
Reduction in casein micelle size
20 kHz, 31 W, 60 min
Reconstituted whey protein concentrate
Effects
Protein aggregate size
Ultrasonic treatment conditions
Working system
Properties in dairy streams
Table 1
Tabulated abstract for effects of ultrasound on different properties of milk proteins.
(Jambrak et al., 2011)
(Yanjun et al., 2014)
(Jambrak et al., 2008)
(Shen et al., 2011).
(Arzeni et al., 2012; O'Sullivan, Arellano, Pichot, & Norton,
2014a)
(Jambrak et al., 2011)
(Zisu et al., 2011)
(Shen, Shao, & Guo, 2017; Zisu et al., 2011).
(Gülseren, Güzey, Bruce, & Weiss, 2007)
(Chandrapala et al., 2011)
(Arzeni et al., 2012)
(Chandrapala et al., 2011)
(Madadlou, Mousavi, Emam-Djomeh, Ehsani, & Sheehan,
2009b)
(Nguyen & Anema, 2010)
(Chandrapala, Martin, Zisu, Kentish, & Ashokkumar, 2012)
(Liu, Juliano, Williams, Niere, & Augustin, 2014)
(Jambrak, Mason, Lelas, Herceg, & Herceg, 2008; Jambrak,
Mason, Lelas, Paniwnyk, & Herceg, 2014)
(O'Sullivan, Arellano, Pichot, & Norton, 2014b)
(O'Sullivan et al., 2014b)
(Yanjun et al., 2014)
(Leong et al., 2018)
(Zisu et al., 2011)
References
M. Munir, et al.
Innovative Food Science and Emerging Technologies 57 (2019) 102192
Innovative Food Science and Emerging Technologies 57 (2019) 102192
M. Munir, et al.
Fig. 4. Size-exclusion chromatography profiles of particle size distribution in milks; non-sonicated at pH 6.7 (solid black), ultrasonicated at pH 6.7 (solid gray),
adjusted to pH 8.0 and re-adjusted to 6.7 without sonication (dotted black), adjusted to pH 8.0, ultrasonicated and re-adjusted to 6.7 (dotted gray). [Peak 1 (particle
size > 200 nm), Peak 2 (particle size range 160–80 nm) and Peak 3 (particle size range 80–37 nm), Peak 4 represents whey proteins. Peaks 5 and 6 represent small
peptides and small aromatic molecules, respectively.
The diagram is reproduced from Liu et al., 2014.
sulphate), heat or an oil/water interface, proteins denature to expose
more thiol groups that can participate in disulphide bridging during
sonication (Avivi, Nitzan, Dror, & Gedanken, 2003; Cavalieri,
Ashokkumar, Grieser, & Caruso, 2008; Suslick, Grinstaff, Kolbeck, &
Wong, 1994). Suslick et al. (1994) and Avivi et al. (2003) denatured
BSA using an oil/water interface and formed oil-encapsulated chemically-crosslinked microbubbles using 20 kHz 150 W cm−2 US. AlmanzaRubio, Gutiérrez-Méndez, Leal-Ramos, Sepulveda, and Salmeron (2016)
used heat (< 63 °C) coupled with sonication (20 kHz 50-100 W) treatment to crosslink whey proteins with thiol-containing caseins, to increase cream cheese yields even below the generally reported denaturation temperature of whey proteins (~65 °C).
As previously described, high-frequency US (300–800 kHz) can
produce free radicals (depending on the applied energy density) capable of oxidizing milk proteins (Ashokkumar et al., 2008; Johansson
et al., 2016). However, at lower energy densities (< 230 kJ kg−1),
Juliano et al. (2014) did not observe milk oxidation with high frequency US (0.4–1 MHz) applied for up to 20 min at temperatures from
4 °C to 63 °C.
Particle homogenization and protein conformational changes
caused by US results in functional changes to dairy streams. For example, when the protein aggregate size is reduced, reconstituted protein suspensions become fully solubilized and decrease in viscosity and
turbidity (Shen et al., 2017; Zisu et al., 2011). US denaturation and
subsequent aggregation of protein is also reported to cause significant
changes in viscosity and solubility. For example Jambrak et al. (2011)
reported that the solubility and the flowability of whey protein isolate
solutions decreased as a result of sonication (30 kHz, 73–78 W cm−2, up
to 15 min).
However, US-induced (30 kHz, 100 W) conformational changes in
proteins change their hydrophilic lipophilic balance (HLB) and alter the
emulsification ability. Mild sonication of milk protein concentrates
(MPC) (e.g. 20 kHz, 12.50 W, 1 min) could improve the emulsification
ability of the proteins (Yanjun et al., 2014) due to protein unfolding,
while more extreme treatments could lead to a reduction (due to protein aggregation) (Jambrak et al., 2008; Jambrak et al., 2011). By effectively controlling US processing parameters, researchers have successfully formed emulsions with flax seed oil and skim milk that were
aggregate dissociation and fat droplet (available in small amounts in
skim milk) breakage. They further confirmed that ultrasound (20 kHz,
31 W) cannot affect casein micelle integrity in treatments up to 1 h.
Interestingly, Liu et al. (2014) later observed that when milk was sonicated (20 kHz 286 kJ kg−1 power, 15 min) at high pH and later
neutralized, the casein micelle integrity was indeed compromised
(Fig. 4). They proposed that casein micelles are more susceptible to the
mechanical forces of ultrasonic cavitation at elevated pH, as they are
enlarged due to electrostatic repulsion. Their explanation agreed with
the previously reported results of Madadlou, Mousavi, Emam-Djomeh,
Ehsani, and Sheehan (2009b) and Madadlou et al. (2009a,) who too
observed significant reductions in particle size of reconstituted micellar
casein solutions at alkaline conditions.
Although the effect of US on the structure of individual caseins is yet
to be fully investigated, clear structural changes were observed in whey
proteins treated with US. Chandrapala et al. (2011) reported that
acoustic shear (from US treatment at 20 kHz, 31 W) could unfold whey
proteins and expose buried hydrophobic residues to the surroundings at
relatively short treatment times (~5 min). Prolonged treatment
(> 10 min) led to whey protein aggregation via hydrophobic interactions (Chandrapala et al., 2011). Similar US-induced unfolding and
conformational changes of whey proteins were also reported (Arzeni
et al., 2012; Gülseren et al., 2007). More recently, Silva, Zisu, and
Chandrapala (2018) studied changes in the secondary structure of whey
proteins caused by ultrasound (20 kHz, 75.6 J/ml) in whey protein rich
systems. They observed that β-LG dimers in whey dissociated when
sonicated. The number of random coil structures in the monomers also
increased. In BSA isolate solutions, Gülseren et al. (2007) observed a
decrease in the free sulfhydryl groups with sonication. Interestingly,
neither Chandrapala et al. (2011) nor Arzeni et al. (2012) who worked
with whey protein concentrates, saw an increase in the exposed thiol
content as a result of protein unfolding. They hypothesized that the
reduced availability of the internally-situated thiol groups compared to
hydrophobic amino residues in the peptide chain and the complex
composition of WPC that contains a mixture of proteins rather than the
pure BSA, could be the reasons for observed constancy in the exposed
thiol content.
However, in the presence of chemicals (e.g. sodium dodecyl
9
Innovative Food Science and Emerging Technologies 57 (2019) 102192
M. Munir, et al.
Ultrasound
Low frequency
High frequency
(Shear forces, cavitation,
turbulence)
(Shear forces, turbulence, free
radicals)
Milk Proteins
Milk Proteins
Dissociation of
protein aggregates
Conformational
changes in proteins
Not suitable for food processing
(Higher radical production may
cause oxidation of milk
components)
Increased solubility
Reduced turbidity
and viscosity
Exposing hydrophobic
residues
Improved emulsification
properties
Protein aggregation
Fig. 5. Effects of low and high frequency ultrasound on milk proteins.
Cánovas, 2010) and cream cheese (Almanza-Rubio et al., 2016). As US
homogenizes the aggregates formed during thermo-sonication, the
cheese whey released retains better flow properties without sedimentation or phase separation (Jeličič, Božanić, Brnčic, & Tripalo,
2012). Similarly, probiotic whey beverages pasteurized with 19 kHz,
600 W US were stable against phase separation (Guimarães et al.,
2018). However, extensive denaturation of proteins and lipolysis during
sonication could cause off flavors in milk (Chouliara, Georgogianni,
Kanellopoulou, & Kontominas, 2010), and affect textural attributes such
as spreadability in subsequent cream cheeses (Almanza-Rubio et al.,
2016)
stable for up to 9 days without the addition of an external emulsifier
(Shanmugam & Ashokkumar, 2014). Similar submicron emulsions
made with olive oil and whey protein solutions have also remained
stable for up to 10 days (Kaltsa, Michon, Yanniotis, & Mandala, 2013).
Sonication-induced changes in milk proteins can be effectively used
to improve the yield, physico-chemical properties and sensory attributes of dairy products. For instance, when sonicated milk was used for
yogurt production, Tabatabaie, Mortazavi, and Ebadi (2009) observed
that the gel structure became more interconnected and regular compared to untreated milk. Similarly, firm rennet gels and faster gelation
rates have also been recorded in US-treated milk and protein suspensions. Such dense microstructures lead to gels with higher elastic
moduli (Yanjun et al., 2014). The increased hydrophobicity of proteins
and the breakage of protein aggregates under US are believed to accelerate gelation via hydrophobic and improved protein-protein interactions, leading to firmer curds (Chandrapala, Zisu, Kentish, &
Ashokkumar, 2013; Leong et al., 2018; Liu et al., 2014).
Gursoy, Yilmaz, Gokce, and Ertan (2016) utilized whey protein
casein crosslinking in milk, triggered by thermo-sonication (24 kHz,
100–150 W, 70 °C) to increase the yield and reduce the serum separation in yogurt drinks. Similar thermo-sonication treatments can also
increase the yields of soft cheese (Bermúdez-Aguirre & Barbosa-
4.3. Effect of ultrasound on dairy-related enzyme activity
Cavitation-induced physical effects as well as sonochemical products are responsible for altering enzyme activity in milk in three ways.
(a) Homogenizing effects (moderate treatments) of US and protein
unfolding can improve mass transfer and enzyme-substrate interactions
leading to higher enzymatic activity. (b) However, extensive US pretreatment combined with mild pressure or heat conditions could denature the enzymes leading to impaired activity (O'donnell, Tiwari,
Bourke, & Cullen, 2010; Uluko et al., 2013). Recent reports of all three
10
11
Faster milk coagulation and firmer curds (Tabatabaie et al., 2009;
Yanjun et al., 2014). Increased cheese yield by using thermosonication (Almanza-Rubio et al., 2016; Gursoy et al., 2016).
Better quality attributes in milk compared to conventional
pasteurization and UHT (Clare et al., 2005; Villamiel, LópezFandiño, et al., 1996).
Deactivation of thermally unstable enzymes by MW
pretreatment (Clare et al., 2005). Improved enzyme activity via
controlled MW irradiation during incubation (Izquierdo et al.,
2005, 2008). Improved enzyme activity via MW assisted
protein unfolding before incubation (Gomaa, 2010).
Denaturation of whey proteins and change in functionality (eg:
surface hydrophobicity, emulsification ability) (Arzeni et al.,
2012; Chandrapala et al., 2011; Gülseren et al., 2007).
Rapid denaturation and aggregation of whey proteins above
70 °C (Villamiel, Corzo, et al., 1996).
Changes in product
attributes as a result protein
denaturation
Enzymatic Activity
4
5
Protein denaturation
Decrease in casein micellar size at low protein concentrations
(Baier et al., 2015; Cadesky et al., 2017; Needs et al., 2000).
Increase in casein micellar size (aggregation) at high protein
concentrations (Anema, 2008; Cadesky et al., 2017). Increase in
the mean size of whey proteins due to denaturation and
aggregation (Goyal et al., 2018).
Dissociation of casein micelles at low concentrations and pH
(Orlien et al., 2010). Denaturation of β-LG, α-LA and
immunoglobulin (Felipe, Capellas, & Law, 1997; Goyal et al.,
2018; Huppertz et al., 2002).
Faster milk coagulation during cheese making at 400 MPa, while
slower coagulation at 600 MPa. Incorporation of more whey
proteins into cheeses. Improved whiteness in cheese (Voigt et al.,
2012, 2010).
Improved enzyme activity at moderate pressures and low enzyme
activity at high pressures (Eisenmenger & Reyes-De-Corcuera,
2009).
Cavitation, pressure, heat, shear forces (Ashokkumar & Mason,
2007; Yasui, 2002).
Decrease in aggregate size (Multiple references are provided in
Table 1).
Particle size
High pressure
2
Financial support was provided by the Institute of Food Science and
Nutrition, University of Sargodha, Sargodha, Pakistan and School of
Chemistry, The University of Melbourne, Parkville, Victoria 3010,
Australia. Authors are grateful for this support. This research was
supported
under
Australian
Research
Councils
Industrial
Transformation Research Program (ITRP) funding scheme (project
Mode of Action
Acknowledgement
1
None.
Properties of dairy streams
Declaration of Competing Interest
Sr #
Table 2
General summary of the different innovative processing technologies.
Microwave
The physical and functional properties of milk proteins can be significantly altered in a variety of ways by MW, HPP and US technologies.
A summary of the general changes to select properties relevant to different food systems is presented in Table 2. High-pressure processing is
a relatively mature technology that is widely used in food processing.
High-pressure processing of milk results in the denaturation of whey
proteins and disruption of casein micelles in the absence of a heat input.
Such changes are reported to improve cheese yields and sensory
properties. At high pressures, HPP can also partially or fully deactivate
pressure-sensitive enzymes.
Microwave processing has both thermal and non-thermal effects. It
leads to the rapid denaturation of milk proteins followed by aggregation. Controlled MW irradiation could be used to improve or reduce
enzyme activity by optimizing process parameters. Ultrasonic processing of dairy systems is a relatively novel technology that is gaining
increased attention. Although the protein confirmation changes caused
by US do not result in nutritional degradation, they lead to improved
emulsification ability and rheological parameters (viscosity, gel
strength) in dairy streams. Such improvements can lead to more efficient dairy processing and higher yields. When process conditions are
carefully designed, US can be used effectively either to activate or deactivate enzymes.
From the critical analysis of various studies presented in this review,
it can be concluded that processing of milk systems using HPP, MW and
US technologies is still in the early stages of development, despite
providing several positive attributes to the milk systems. The adaptation of these novel technologies by the dairy industry is a slow process
and further research is needed for efficient implementation of these
technologies in dairy and other food industries.
Localized heating and molecular rearrangement (El Mecherfi
et al., 2011; Gomaa, 2010; Tong, 1996).
Possible increase in the mean size of whey proteins due to
denaturation and aggregation (Iuliana et al., 2015).
5. Summary
Pressure (Jermann et al., 2015)
Ultrasound
instances are discussed below.
Uluko et al. (2013) investigated the enzymatic hydrolysis of USpretreated (800 W, 1–8 min) milk protein concentrate and found an
84% increase in trypsin activity and a 185% increase in alkaline protease activity. The authors proposed that this increase in hydrolysis
indicated a higher susceptibility of proteins to enzymes as a result of
protein aggregate dissociation. Similar improvements in the trypsin and
pepsin activity have been reported in sonicated (20 kHz,60 W/cm2) βLG solutions due to protein unfolding and the exposure of more cleavage sites (Ma, Wang, & Guo, 2018).
In an interesting study, Lopez and Burgos (1995a) reported that
mano-thermosonication (application of US with heat and pressure) can
inactivate the peroxidase enzyme. When compared to heat treatment
(126.5 °C), they observed a marked decrease in the enzyme activity at
neutral pH when enzymes were pre-treated with US (126.5 °C at
20 kHz) at elevated pressure (3.5 kg/cm2). They hypothesized that
splitting the enzyme's prosthetic heme group or apoenzyme denaturation under the provided conditions could be the reasons for enzyme
inhibition (Lopez & Burgos, 1995b). Similar mano-thermo-sonication
treatments to lipoxygenase have also shown promise in inhibiting the
enzyme activity (Lopez & Burgos, 1995a).
Improved enzyme activity using moderate treatments
(homogenization effects and protein unfolding) (Ma et al., 2018);
Uluko et al., 2013). Enzyme deactivation/denaturation by manothermosonication (Lopez & Burgos, 1995a, 1995b).
Innovative Food Science and Emerging Technologies 57 (2019) 102192
M. Munir, et al.
Innovative Food Science and Emerging Technologies 57 (2019) 102192
M. Munir, et al.
number IH120100005). The ARC Dairy Innovation Hub is a collaboration between The University of Melbourne, The University of
Queensland and Dairy Innovation Australia Ltd.
Desobry-Banon, S., Richard, F., & Hardy, J. (1994). Study of acid and rennet coagulation
of high pressurized milk. Journal of Dairy Science, 77(11), 3267–3274.
Dufour, E., Hoa, G. H. B., & Haertlé, T. (1994). High-pressure effects on β-lactoglobulin
interactions with ligands studied by fluorescence. Biochimica et Biophysica Acta
(BBA)-Protein Structure and Molecular Enzymology, 1206(2), 166–172.
Eisenmenger, M. J., & Reyes-De-Corcuera, J. I. (2009). High pressure enhancement of
enzymes: A review. Enzyme and Microbial Technology, 45(5), 331–347.
El Mecherfi, K. E., Saidi, D., Kheroua, O., Boudraa, G., Touhami, M., Rouaud, O., &
Chobert, J.-M. (2011). Combined microwave and enzymatic treatments for β-lactoglobulin and bovine whey proteins and their effect on the IgE immunoreactivity.
European Food Research and Technology, 233(5), 859.
Felipe, X., Capellas, M., & Law, A. J. (1997). Comparison of the effects of high-pressure
treatments and heat pasteurization on the whey proteins in goat's milk. Journal of
Agricultural and Food Chemistry, 45(3), 627–631.
Fox, P. F., Guinee, T. P., Cogan, T. M., & McSweeney, P. L. (2000). Fundamentals of cheese
science.
Gaucheron, F., Famelart, M., Mariette, F., Raulot, K., Michela, F., & Le Graeta, Y. (1997).
Combined effects of temperature and high-pressure treatments on physicochemical
characteristics of skim milk. Food Chemistry, 59(3), 439–447.
Gebhardt, R., Doster, W., & Kulozik, U. (2005). Pressure-induced dissociation of casein
micelles: Size distribution and effect of temperature. Brazilian Journal of Medical and
Biological Research, 38(8), 1209–1214.
Ghasemkhani, N., Morshedi, A., Poursharif, Z., Aghamohammadi, B., Akbarian, M., &
Moayedi, F. (2014). Microbiological effects of high pressure processing on food.
Journal of Biodiversity and Environmental Sciences, 4(4), 133–145.
Gomaa, A. (2010). An investigation of effects of microwave treatment on the structure, enzymatic hydrolysis, and nutraceutical properties of –lactoglobulin. McGill University.
Goyal, A., Sharma, V., Upadhyay, N., Sihag, M., & Kaushik, R. (2018). High pressure
processing and its impact on milk proteins: A review. Research & Reviews: Journal of
Dairy Science and Technology, 2(1), 12–20.
Guimarães, J. T., Silva, E. K., Alvarenga, V. O., Costa, A. L. R., Cunha, R. L., Sant'Ana, A.
S., & Cruz, A. G. (2018). Physicochemical changes and microbial inactivation after
high-intensity ultrasound processing of prebiotic whey beverage applying different
ultrasonic power levels. Ultrasonics Sonochemistry, 44, 251–260.
Gülseren, İ., Güzey, D., Bruce, B. D., & Weiss, J. (2007). Structural and functional changes
in ultrasonicated bovine serum albumin solutions. Ultrasonics Sonochemistry, 14(2),
173–183.
Gursoy, O., Yilmaz, Y., Gokce, O., & Ertan, K. (2016). Effect of ultrasound power on
physicochemical and rheological properties of yoghurt drink produced with thermosonicated milk. Emirates Journal of Food and Agriculture, 235–241.
Hamid, M., Boulanger, R., Tong, S., Gallop, R., & Pereira, R. (1969). Microwave pasteurization of raw milk. Journal of Microwave Power, 4(4), 272–275.
Hayakawa, I., Kajihara, J., Morikawa, K., Oda, M., & Fijio, Y. (1992). Denaturation of
bovine serum albumin (BSA) and ovalbumin by high pressure, heat and chemicals.
Journal of Food Science, 57(2), 288–292.
Huppertz, T., Kelly, A. L., & Fox, P. F. (2002). Effects of high pressure on constituents and
properties of milk. International Dairy Journal, 12(7), 561–572.
Iuliana, C., Rodica, C., Sorina, R., & Oana, M. (2015). Impact of microwaves on the
physico-chemical characteristics of cow milk. Romanian Reports in Physics, 67(2),
423–430.
Izquierdo, F. J., Alli, I., Gómez, R., Ramaswamy, H. S., & Yaylayan, V. (2005). Effects of
high pressure and microwave on pronase and α-chymotrypsin hydrolysis of β-lactoglobulin. Food Chemistry, 92(4), 713–719.
Izquierdo, F. J., Peñas, E., Baeza, M. L., & Gomez, R. (2008). Effects of combined microwave and enzymatic treatments on the hydrolysis and immunoreactivity of dairy
whey proteins. International Dairy Journal, 18(9), 918–922.
Jambrak, A. R., Lelas, V., Krešić, G., Badanjak, M., Brnčić, S. R., Herceg, Z., ... Grčić, I.
(2011). Rheological, functional and thermo-physical properties of ultrasound treated
whey proteins with addition of sucrose or milk powder. Mljekarstvo, 61(1), 79–91.
Jambrak, A. R., Mason, T. J., Lelas, V., Herceg, Z., & Herceg, I. L. (2008). Effect of ultrasound treatment on solubility and foaming properties of whey protein suspensions.
Journal of Food Engineering, 86(2), 281–287.
Jambrak, A. R., Mason, T. J., Lelas, V., Paniwnyk, L., & Herceg, Z. (2014). Effect of ultrasound treatment on particle size and molecular weight of whey proteins. Journal of
Food Engineering, 121, 15–23.
Jeličič, I., Božanić, R., Brnčic, M., & Tripalo, B. (2012). Influence and comparison of
thermal, ultrasonic and thermo-sonic treatments on microbiological quality and
sensory properties of rennet cheese whey. Mljekarstvo/Dairy, 62(3).
Jenness, R. (1999). Composition of Milk. In N. P. Wong (Ed.). Fundamentals of dairy
chemistry (pp. 1–38). Gaithersburg, Maryland: Aspen Publishers, Inc.
Jermann, C., Koutchma, T., Margas, E., Leadley, C., & Ros-Polski, V. (2015). Mapping
trends in novel and emerging food processing technologies around the world.
Innovative Food Science & Emerging Technologies, 31, 14–27.
Johansson, L., Singh, T., Leong, T., Mawson, R., McArthur, S., Manasseh, R., & Juliano, P.
(2016). Cavitation and non-cavitation regime for large-scale ultrasonic standing wave
particle separation systems–In situ gentle cavitation threshold determination and free
radical related oxidation. Ultrasonics Sonochemistry, 28, 346–356.
Johnston, D., Austin, B., & Murphy, R. (1992). Effects of high hydrostatic pressure on
milk. Milchwissenschaft (Germany), 47, 760–763.
Juan, B., Ferragut, V., Buffa, M., Guamis, B., & Trujillo, A. (2007). Effects of high pressure
on proteolytic enzymes in cheese: Relationship with the proteolysis of ewe milk
cheese. Journal of Dairy Science, 90(5), 2113–2125.
Juliano, P., Torkamani, A. E., Leong, T., Kolb, V., Watkins, P., Ajlouni, S., & Singh, T. K.
(2014). Lipid oxidation volatiles absent in milk after selected ultrasound processing.
Ultrasonics Sonochemistry, 21(6), 2165–2175.
Júnior, B. R.d. C. L., Tribst, A. A. L., Bonafe, C. F. S., & Cristianini, M. (2016).
References
Albert, C., Pohn, G., Mándoki, Z., Csapó-Kiss, Z., & Csapó, J. (2009). The effect of microwave pasteurization on the composition of milk. Krmiva: Časopis o hranidbi
životinja, proizvodnji i tehnologiji krme, 51(4), 213–222.
Almanza-Rubio, J. L., Gutiérrez-Méndez, N., Leal-Ramos, M. Y., Sepulveda, D., &
Salmeron, I. (2016). Modification of the textural and rheological properties of cream
cheese using thermosonicated milk. Journal of Food Engineering, 168, 223–230.
Anantheswaran, R. C., & Ramaswamy, H. S. (2001). Bacterial destruction and enzyme inactivation during microwave heating. Vol. 191. New York: Marcel Dekker.
Anema, S. G. (2008). Effect of milk solids concentration on whey protein denaturation,
particle size changes and solubilization of casein in high-pressure-treated skim milk.
International Dairy Journal, 18(3), 228–235.
Arzeni, C., Martínez, K., Zema, P., Arias, A., Pérez, O., & Pilosof, A. (2012). Comparative
study of high intensity ultrasound effects on food proteins functionality. Journal of
Food Engineering, 108(3), 463–472.
Ashokkumar, M., & Mason, T. J. (2007). Sonochemistry. Kirk-Othmer encyclopedia of
chemical technology.
Ashokkumar, M., Sunartio, D., Kentish, S., Mawson, R., Simons, L., Vilkhu, K., & Versteeg,
C. K. (2008). Modification of food ingredients by ultrasound to improve functionality:
A preliminary study on a model system. Innovative Food Science & Emerging
Technologies, 9(2), 155–160.
Atuonwu, J. C., Leadley, C., Bosman, A., Tassou, S. A., Lopez-Quiroga, E., & Fryer, P. J.
(2018). Comparative assessment of innovative and conventional food preservation
technologies: Process energy performance and greenhouse gas emissions. Innovative
Food Science & Emerging Technologies, 50, 174–187.
Avivi, Nitzan, Y., Dror, R., & Gedanken, A. (2003). An easy sonochemical route for the
encapsulation of tetracycline in bovine serum albumin microspheres. Journal of the
American Chemical Society, 125(51), 15712–15713.
Baier, D., Schmitt, C., & Knorr, D. (2015). Effect of high pressure-low temperature processing on composition and colloidal stability of casein micelles and whey proteins.
International Dairy Journal, 43, 51–60.
Balasubramaniam, V., Barbosa-Cánovas, G. V., & Lelieveld, H. (2016). High pressure
processing of food. Food Engineering Series.
Balasubramaniam, V., Ting, E., Stewart, C., & Robbins, J. (2004). Recommended laboratory practices for conducting high-pressure microbial inactivation experiments.
Innovative Food Science & Emerging Technologies, 5(3), 299–306.
Balthazar, C. F., Santillo, A., Guimarães, J. T., Bevilacqua, A., Corbo, M. R., Caroprese, M.,
& Raices, R. S. (2019). Ultrasound processing of fresh and frozen semi-skimmed sheep
milk and its effects on microbiological and physical-chemical quality. Ultrasonics
Sonochemistry, 51, 241–248.
Bermúdez-Aguirre, D., & Barbosa-Cánovas, G. V. (2010). Processing of soft Hispanic
cheese (“Queso Fresco”) using thermo-sonicated milk: A study of physicochemical
characteristics and storage life. Journal of Food Science, 75(9).
Bi, W., Zhao, W., Li, X., Ge, W., Muhammad, Z., Wang, H., & Du, L. (2015). Study on
microwave-accelerated casein protein grafted with glucose and β-cyclodextrin to
improve the gel properties. International Journal of Food Science & Technology, 50(6),
1429–1435.
Bohr, H., & Bohr, J. (2000). Microwave-enhanced folding and denaturation of globular
proteins. Physical Review E, 61(4), 4310.
Cadesky, L., Walkling-Ribeiro, M., Kriner, K. T., Karwe, M. V., & Moraru, C. I. (2017).
Structural changes induced by high-pressure processing in micellar casein and milk
protein concentrates. Journal of Dairy Science, 100(9), 7055–7070.
Cavalieri, F., Ashokkumar, M., Grieser, F., & Caruso, F. (2008). Ultrasonic synthesis of
stable, functional lysozyme microbubbles. Langmuir, 24(18), 10078–10083.
Chandrapala, J., Martin, G., Zisu, B., Kentish, S., & Ashokkumar, M. (2012). The effect of
ultrasound on casein micelle integrity. Journal of Dairy Science, 95(12), 6882–6890.
Chandrapala, J., Zisu, B., Kentish, S., & Ashokkumar, M. (2013). Influence of ultrasound
on chemically induced gelation of micellar casein systems. Journal of Dairy Research,
80(2), 138–143.
Chandrapala, J., Zisu, B., Palmer, M., Kentish, S., & Ashokkumar, M. (2011). Effects of
ultrasound on the thermal and structural characteristics of proteins in reconstituted
whey protein concentrate. Ultrasonics Sonochemistry, 18(5), 951–957.
Chawla, R., Patil, G. R., & Singh, A. K. (2011). High hydrostatic pressure technology in
dairy processing: A review. Journal of Food Science and Technology, 48(3), 260–268.
Chen, C.-C., & Hsieh, J.-F. (2016). Microwave-assisted cross-linking of milk proteins induced by microbial transglutaminase. Scientific Reports, 6, 39040.
Chouliara, E., Georgogianni, K., Kanellopoulou, N., & Kontominas, M. (2010). Effect of
ultrasonication on microbiological, chemical and sensory properties of raw, thermized and pasteurized milk. International Dairy Journal, 20(5), 307–313.
Clare, D., Bang, W., Cartwright, G., Drake, M., Coronel, P., & Simunovic, J. (2005).
Comparison of sensory, microbiological, and biochemical parameters of microwave
versus indirect UHT fluid skim milk during storage. Journal of Dairy Science, 88(12),
4172–4182.
Curl, A. L., & Jansen, E. F. (1950). Effect of high pressures on trypsin and chymotrypsin.
Journal of Biological Chemistry, 184(1), 45–54.
Delgado-Martínez, F. J., Carrapiso, A. I., Contador, R., & Ramírez, M. R. (2019). Volatile
compounds and sensory changes after high pressure processing of mature “Torta del
Casar” (raw ewe's milk cheese) during refrigerated storage. Innovative Food Science &
Emerging Technologies, 52, 34–41.
12
Innovative Food Science and Emerging Technologies 57 (2019) 102192
M. Munir, et al.
lactoglobulin solutions under high hydrostatic pressure. Journal of Agricultural and
Food Chemistry, 55(11), 4422–4428.
O'Sullivan, J., Arellano, M., Pichot, R., & Norton, I. (2014a). The effect of ultrasound
treatment on the structural, physical and emulsifying properties of dairy proteins.
Food Hydrocolloids, 42, 386–396.
O'Sullivan, J., Arellano, M., Pichot, R., & Norton, I. (2014b). sh. Food Hydrocolloids, 42,
386–396.
Petrucelli, L., & Fisher, G. H. (1994). D-aspartate and D-glutamate in microwaved versus
conventionally heated milk. Journal of the American College of Nutrition, 13(2),
209–210.
de Pomerai, D. I., Smith, B., Dawe, A., North, K., Smith, T., Archer, D. B., ... Candido, E. P.
M. (2003). Microwave radiation can alter protein conformation without bulk heating.
FEBS Letters, 543(1–3), 93–97.
Rodríguez, J. J., Barbosa-Cánovas, G. V., Gutiérrez-López, G. F., Dorantes-Álvarez, L.,
Yeom, H. W., & Zhang, Q. H. (2003). An update on some key alternative food processing technologies: Microwave, pulsed electric field, high hydrostatic pressure, irradiation and ultrasound. Food science and food biotechnology, 279–312.
Rosa, P., Sala, G., Van Vliet, T., & Van De Velde, F. (2006). Cold gelation of whey protein
emulsions. Journal of Texture Studies, 37(5), 516–537.
Roselli, L., Cicia, G., Cavallo, C., Del Giudice, T., Carlucci, D., Clodoveo, M. L., & De
Gennaro, B. C. (2018). Consumers' willingness to buy innovative traditional food
products: The case of extra-virgin olive oil extracted by ultrasound. Food Research
International, 108, 482–490.
Sakharam, P., Prajapati, J., & Jana, A. (2011). High hydrostatic pressure treatment for
dairy applications. National seminar Indian Dairy Industry-Opportunities and challenges,
2014 (pp. 176–180). .
Segura-Campos, M., Chel-Guerrero, L., Betancur-Ancona, D., & Hernandez-Escalante, V.
M. (2011). Bioavailability of bioactive peptides. Food Reviews International, 27(3),
213–226.
Shanmugam, A., & Ashokkumar, M. (2014). Ultrasonic preparation of stable flax seed oil
emulsions in dairy systems–physicochemical characterization. Food Hydrocolloids, 39,
151–162.
Shen, X., Shao, S., & Guo, M. (2017). Ultrasound-induced changes in physical and functional properties of whey proteins. International Journal of Food Science & Technology,
52(2), 381–388.
Silva, M., Zisu, B., & Chandrapala, J. (2018). Influence of low-frequency ultrasound on
the physico-chemical and structural characteristics of milk systems with varying
casein to whey protein ratios. Ultrasonics Sonochemistry, 49, 268–276.
Singh, R. P., & Yousef, A. E. (2001). Technical elements of new and emerging non-thermal
food technologies. FAO report.
Stapelfeldt, H., & Skibsted, L. H. (1999). Pressure denaturation and aggregation of βlactoglobulin studied by intrinsic fluorescence depolarization, Rayleigh scattering,
radiationless energy transfer and hydrophobic fluoroprobing. Journal of Dairy
Research, 66(4), 545–558.
Suslick, K., Grinstaff, M., Kolbeck, K., & Wong, M. (1994). Characterization of sonochemically prepared proteinaceous microspheres. Ultrasonics Sonochemistry, 1(1),
S65–S68.
Tabatabaie, F., Mortazavi, A., & Ebadi, A. (2009). Effect of power ultrasound and microstructure change of casein micelle in yoghurt. Asian Journal of Chemistry, 21(2),
1589–1594.
Tamime, A. Y. (2009). Milk processing and quality management. John Wiley & Sons.
Tho, P., Manasseh, R., & Ooi, A. (2007). Cavitation microstreaming patterns in single and
multiple bubble systems. Journal of Fluid Mechanics, 576, 191–233.
Thompson, L., & Doraiswamy, L. (1999). Sonochemistry: Science and engineering.
Industrial & Engineering Chemistry Research, 38(4), 1215–1249.
Tong, C. (1996). Effect of microwaves on biological and chemical systems. MICROWAVE
WORLD-NEW YORK, 17, 14–23.
Tsou, C.-L. (1986). Location of the active sites of some enzymes in limited and flexible
molecular regions. Trends in Biochemical Sciences, 11(10), 427–429.
Uluko, H., Zhang, S., Liu, L., Chen, J., Sun, Y., Su, Y., ... Lv, J. (2013). Effects of microwave and ultrasound pretreatments on enzymolysis of milk protein concentrate with
different enzymes. International Journal of Food Science & Technology, 48(11),
2250–2257.
Valente-Mesquita, V. L., Botelho, M. M., & Ferreira, S. T. (1998). Pressure-induced subunit dissociation and unfolding of dimeric β-lactoglobulin. Biophysical Journal, 75(1),
471–476.
Villamiel, M., Corzo, N., Martinez-Castro, I., & Olano, A. (1996). Chemical changes during
microwave treatment of milk. Food Chemistry, 56(4), 385–388.
Villamiel, M., López-Fandiño, R., Corzo, N., Martínez-Castro, I., & Olano, A. (1996).
Effects of continuous flow microwave treatment on chemical and microbiological
characteristics of milk. Zeitschrift für Lebensmittel-Untersuchung und Forschung, 202(1),
15–18.
Voigt, D. D., Chevalier, F., Donaghy, J. A., Patterson, M. F., Qian, M. C., & Kelly, A. L.
(2012). Effect of high-pressure treatment of milk for cheese manufacture on proteolysis, lipolysis, texture and functionality of Cheddar cheese during ripening.
Innovative Food Science & Emerging Technologies, 13, 23–30.
Voigt, D. D., Donaghy, J. A., Patterson, M. F., Stephan, S., & Kelly, A. L. (2010).
Manufacture of Cheddar cheese from high-pressure-treated whole milk. Innovative
Food Science & Emerging Technologies, 11(4), 574–579.
Walstra, P. (1990). On the stability of casein micelles1. Journal of Dairy Science, 73(8),
1965–1979.
Yanjun, S., Jianhang, C., Shuwen, Z., Hongjuan, L., Jing, L., Lu, L., ... Wupeng, G. (2014).
Effect of power ultrasound pre-treatment on the physical and functional properties of
reconstituted milk protein concentrate. Journal of Food Engineering, 124, 11–18.
Yasui, K. (2002). Influence of ultrasonic frequency on multibubble sonoluminescence. The
Journal of the Acoustical Society of America, 112(4), 1405–1413.
Determination of the influence of high pressure processing on calf rennet using response surface methodology: Effects on milk coagulation. LWT-Food Science and
Technology, 65, 10–17.
Júnior, B. R.d. C. L., Tribst, A. A. L., & Cristianini, M. (2017). The effect of high pressure
processing on recombinant chymosin, bovine rennet and porcine pepsin: Influence on
the proteolytic and milk-clotting activities and on milk-clotting characteristics. LWTFood Science and Technology, 76, 351–360.
Kaltsa, O., Michon, C., Yanniotis, S., & Mandala, I. (2013). Ultrasonic energy input influence οn the production of sub-micron o/w emulsions containing whey protein and
common stabilizers. Ultrasonics Sonochemistry, 20(3), 881–891.
Krešic, G., Lelas, V., Herceg, Z., & Režek, A. (2006). Effects of high pressure on functionality of whey protein concentrate and whey protein isolate. Le Lait, 86(4),
303–315.
La Cara, F., Scarffi, M. R., D'Auria, S., Massa, R., d'Ambrosio, G., Franceschetti, G., & De
Rosa, M. (1999). Different effects of microwave energy and conventional heat on the
activity of a thermophilic ß-galactosidase from Bacillus acidocaldarius.
Bioelectromagnetics, 20(3), 172–176.
Laiho, S., Ercili-Cura, D., Forssell, P., Myllärinen, P., & Partanen, R. (2015). The effect of
dynamic heat treatments of native whey protein concentrate on its dispersion characteristics. International Dairy Journal, 49, 139–147.
Law, A. J., Leaver, J., Felipe, X., Ferragut, V., Pla, R., & Guamis, B. (1998). Comparison of
the effects of high pressure and thermal treatments on the casein micelles in goat's
milk. Journal of Agricultural and Food Chemistry, 46(7), 2523–2530.
Leong, T. S., Walter, V., Gamlath, C. J., Yang, M., Martin, G. J., & Ashokkumar, M. (2018).
Functionalised dairy streams: Tailoring protein functionality using sonication and
heating. Ultrasonics Sonochemistry, 48, 499–508.
Liu, Z., Juliano, P., Williams, R. P., Niere, J., & Augustin, M. A. (2014). Ultrasound improves the renneting properties of milk. Ultrasonics Sonochemistry, 21(6), 2131–2137.
Lopez, P., & Burgos, J. (1995a). Lipoxygenase inactivation by manothermosonication:
Effects of sonication on physical parameters, pH, KCl, sugars, glycerol, and enzyme
concentration. Journal of Agricultural and Food Chemistry, 43(3), 620–625.
Lopez, P., & Burgos, J. (1995b). Peroxidase stability and reactivation after heat treatment
and manothermosonication. Journal of Food Science, 60(3), 451–455.
Lopez-Fandino, R., De La Fuente, M., Ramos, M., & Olano, A. (1998). Distribution of
minerals and proteins between the soluble and colloidal phases of pressurized milks
from different species. Journal of Dairy Research, 65(1), 69–78.
Lucey, J. (2002). Formation and physical properties of milk protein gels. Journal of Dairy
Science, 85(2), 281–294.
Ma, S., Wang, C., & Guo, M. (2018). Changes in structure and antioxidant activity of βlactoglobulin by ultrasound and enzymatic treatment. Ultrasonics Sonochemistry, 43,
227–236.
Madadlou, A., Mousavi, M. E., Emam-djomeh, Z., Ehsani, M., & Sheehan, D. (2009a).
Sonodisruption of re-assembled casein micelles at different pH values. Ultrasonics
Sonochemistry, 16(5), 644–648.
Madadlou, A., Mousavi, M. E., Emam-Djomeh, Z., Ehsani, M., & Sheehan, D. (2009b).
Comparison of pH-dependent sonodisruption of re-assembled casein micelles by 35
and 130 kHz ultrasounds. Journal of Food Engineering, 95(3), 505–509.
Marani, E., & Feirabend, H. (1994). Future perspectives in microwave applications in life
sciences. European Journal of Morphology, 32(2–4 Suppl), 330–334.
Martins, C. P., Cavalcanti, R. N., Couto, S. M., Moraes, J., Esmerino, E. A., Silva, M. C., ...
Tadini, C. C. (2019). Microwave processing: Current background and effects on the
physicochemical and microbiological aspects of dairy products. Comprehensive
Reviews in Food Science and Food Safety, 18(1), 67–83.
McSweeney, P. L., & Fox, P. F. (2013). Advanced dairy chemistry: Volume 1A: Proteins: Basic
aspects. Springer Science & Business Media.
Misra, N., Koubaa, M., Roohinejad, S., Juliano, P., Alpas, H., Inácio, R. S., ... Barba, F. J.
(2017). Landmarks in the historical development of twenty first century food processing technologies. Food Research International, 97, 318–339.
Monteiro, S. H., Silva, E. K., Alvarenga, V. O., Moraes, J., Freitas, M. Q., Silva, M. C., ...
Cruz, A. G. (2018). Effects of ultrasound energy density on the non-thermal pasteurization of chocolate milk beverage. Ultrasonics Sonochemistry, 42, 1–10.
Moreno-Vilet, L., Hernández-Hernández, H., & Villanueva-Rodríguez, S. (2018). Current
status of emerging food processing technologies in Latin America: Novel thermal
processing. Innovative Food Science & Emerging Technologies, 50, 196–206.
Naik, L., Sharma, R., Rajput, Y., & Manju, G. (2013). Application of high pressure processing technology for dairy food preservation-future perspective: A review. Journal
of Animal Production Advances, 3(8), 232–241.
Needs, E. C., Stenning, R. A., Gill, A. L., Ferragut, V., & Rich, G. T. (2000). High-pressure
treatment of milk: Effects on casein micelle structure and on enzymic coagulation.
Journal of Dairy Research, 67(1), 31–42.
Nguyen, N. H., & Anema, S. G. (2010). Effect of ultrasonication on the properties of skim
milk used in the formation of acid gels. Innovative Food Science & Emerging
Technologies, 11(4), 616–622.
O'donnell, C., Tiwari, B., Bourke, P., & Cullen, P. (2010). Effect of ultrasonic processing on
food enzymes of industrial importance. Trends in Food Science & Technology, 21(7),
358–367.
de Oliveira, M. M., Augusto, P. E. D., da Cruz, A. G., & Cristianini, M. (2014). Effect of
dynamic high pressure on milk fermentation kinetics and rheological properties of
probiotic fermented milk. Innovative Food Science & Emerging Technologies, 26, 67–75.
Orlien, V., Boserup, L., & Olsen, K. (2010). Casein micelle dissociation in skim milk during
high-pressure treatment: Effects of pressure, pH, and temperature. Journal of Dairy
Science, 93(1), 12–18.
Orlien, V., Knudsen, J. C., Colon, M., & Skibsted, L. H. (2006). Dynamics of casein micelles in skim milk during and after high pressure treatment. Food Chemistry, 98(3),
513–521.
Orlien, V., Olsen, K., & Skibsted, L. H. (2007). In situ measurements of pH changes in β-
13
Innovative Food Science and Emerging Technologies 57 (2019) 102192
M. Munir, et al.
Ashokkumar, M. (2011). Effect of ultrasound on the physical and functional properties of reconstituted whey protein powders. Journal of Dairy Research, 78(2),
226–232.
Zobrist, M., Huppertz, T., Uniacke, T., Fox, P., & Kelly, A. (2005). High-pressure-induced
changes in the rennet coagulation properties of bovine milk. International Dairy
Journal, 15(6–9), 655–662.
Ye, A., Anema, S., & Singh, H. (2004). High-pressure–induced interactions between milk
fat globule membrane proteins and skim milk proteins in whole milk. Journal of Dairy
Science, 87(12), 4013–4022.
Ye, A., Qin, D., Yang, L., She, L., & Xing, R. (2007). Spectroscopic study on the effect of
crystallization of the hydroxyapatite on the secondary structure of bovine serum albumin. Guang pu xue yu guang pu fen xi= Guang pu, 27(2), 321–324.
Zisu, B., Lee, J., Chandrapala, J., Bhaskaracharya, R., Palmer, M., Kentish, S., &
14