Ultrasonido Paper
Ultrasonido Paper
Ultrasonido Paper
Copyright © 2013 Songül Şahin Ercan, Çiğdem Soysal. This is an open access article distributed under the Creative Commons At-
tribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is prop-
erly cited.
converts this energy into the sound energy at ultrasonic the food industry is generally used for analytical applica-
frequencies. Three main types of transducers are reported tions to get information about the physicochemical prop-
as fluid-driven, magnetostrictive and piezoelectric trans- erties of foods such as composition, structure and physi-
ducers [5]. cal state [8-10].
The fluid-driven transducer produces vibration at ul- High energy (high power, high-intensity) ultrasounic
trasonic frequencies by forcing liquid to thin metal blade applications are performed generally at frequencies be-
which can be used for mixing and homogenisation sys- tween 18 and 100 kHz and are intensities higher than 1
tems. The magnetostrictive transducer is made from a W/cm2 (typically in the range 10 - 1000 W/cm2) [10]. At
kind of ferromagnetic materials which change dimension this power, destruction can be observed due to the
upon the application of a magnetic field and these physical, mechanical or chemical effects of ultrasonic
changes produce sought after mechanical vibrations. The waves (e.g. physical disruption, acceleration of certain
efficieny of system is low somewhat 60% transfer to chemical reactions). High-intensity ultrasound has been
acoustic energy [6]. The piezoelectric transducers pro- used for many years to generate emulsions, disrupt cells
duce acoustic energy by changes in size produced by and disperse aggregated materials. More recently it is
electrical signals in piezoceramic materials such as lead used for many purposes such as modification and control
zirconate titanate, barium titanate and lead metaniobate. of crystallization processes, degassing of liquid foods,
The piezoelectric transducers are most commonly used enzyme inactivation, enhanced drying and filtration and
devices and are more efficient (80% - 95% transfer to the induction of oxidation reactions [9,11].
acoustic energy) [5,6].
In application system a coupler device is used to 4. METHODS OF ULTRASOUND
transfer ultrasonic vibrations to the sample. This is gen-
Ultrasound can be used for food preservation in com-
erally obtained by ultrasonic bath and probe system. In
bination with other treatments by improving its inactiva-
ultrasonic baths, generally the transducers are fixed to
tion efficacy. There have been many studies combining
the underside of the tank and most of the baths operated
ultrasound with either pressure, temperature, or pressure
at around 40 kHz [6,7]. In probe systems the horns or
and temperature.
probes are used to transmit or to amplify the ultrasonic
1) Ultrasonication (US) is the application of ultra-
signal. Their lengths must be half the wavelengths, or
sound at low temperature. Therefore, it can be used for
multiple, to maintain the resonant conditions of the sys-
the heat sensible products. However, it requires long
tem [5,7]. The horn shape defines the amplitude gain of
treatment time to inactivate stable enzymes and/or mi-
ultrasonic signal. If the probe is the same diameter along
croorganisms which may cause high energy requirement.
its length then no gain in amplitude will occur but the
During ultrasound application there may be rise in tem-
acoustic energy will be simply transferred to the media
perature depending on the ultrasonic power and time of
[6].
application and needs control to optimize the process
[11].
3. CLASSIFICATION OF ULTRASOUND 2) Thermosonication (TS) is a combined method of
APPLICATION ultrasound and heat. The product is subjected to ultra-
Nowadays, ultrasound is an attractive subject in the sound and moderate heat simultaneously. This method
food industry. Industries can use practically ultrasonic produces a greater effect on inactivation of microorgan-
equipments and it is known as green novel technology isms than heat alone (Table 1). When thermosonication
due to its role in the environment sustainability. Methods is used for pasteurization or sterilization purpose, lower
of ultrasound applications can be divided into three: 1) process temperatures and processing times are required
Direct application to the product, 2) Coupling with the to achieve the same lethality values as with conventional
device, 3) Submergence in an ultrasonic bath [3]. Also, processes [12,13].
ultrasonic applications in the food industry are divided 3) Manosonication (MS) is a combined method in
into two distinct categories according to the energy gen- which ultrasound and pressure are applied together. Ma-
erated by sound field. These are low and high energy nosonication provides to inactivate enzymes and/or mi-
ultrasounds which are classified by their sound power croorganisms by combining ultrasound with moderate
(W), sound energy density (Ws/m3) and sound intensity pressures at low temperatures. Its inactivation efficiency
(W/m2). Low energy (low power, low-intensity) ultra- is higher than ultrasound alone at the same temperature.
sound applications are performed at frequencies higher 4) Manothermosonication (MTS) is a combined me-
than 100 kHz and below 1 W/cm2 intensities. Small thod of heat, ultrasound and pressure. MTS treatments
power level is used for low intensity ultrasound so that it inactivate several enzymes at lower temperatures and/or
is nondestructive and no change occurs in the physical or in a shorter time than thermal treatments at the same
chemical properties of food. Low intensity ultrasound in temperatures [3]. Applied temperature and pressure
D value (min)
Temperature
Organism Manosonication/ References
(˚C) Heat Ultrasound Thermosonication
manothermosonication
1.5 (200 kPa)
Listeria monocytogenes Ambient - 4.3 - [30]
1.0 (400 kPa)
Yersenia entercolitica 30 - 1.52 - 0.2 (600 kPa) [16]
Saccharomyces cerevisiae 60 3.53 3.1 0.73 - [21]
Escherichia coli K12 61 0.79 1.01 0.44 0.40 (300 kPa) [64]
Cronabacter sakazakii 56 0.86 - - 0.28 [65]
55 17.40 5.06
Aspergillus flavus - - [66]
60 2.60 1.20
Penicillium digitatum 50 25.42 - 9.59 - [66]
Listeria innocua 63 30 - 10 - [67]
Lactobacillus acidophilus 60 70.5 - 43.3 - [9]
Enterecoccus faecium 62 11.2 30 1.8 - [68]
Staphylococcus aureus 50.5 19.7 - 7.3 - [68]
maximizes the cavitation or bubble implosion in the me- tion mechanisms of ultrasound is simply explained by
dia which increase the level of inactivation. Microorgan- cavitation phenomena that caused by the changes in
isms that have high thermotolerance can be inactivated pressure [16,17]. Earnshaw, [18] explained that the ex-
by manothermosonication. Also some thermoresistant tremely rapid creation and collapse of bubbles formed by
enzymes, such as lipoxygenase, peroxidase and poly- ultrasonic waves in a medium creates the antimicrobial
phenoloxidase, and heat labile lipases and proteases from effect of ultrasound. During the cavitation process, lo-
Pseudomonas can be inactivated by manothermosonica- calized changes in pressure and temperature cause break-
tion [14]. down of cell walls, disruption and thinning of cell mem-
branes, and DNA damage via free radical production
5. ULTRASOUND IN FOOD [4,19]. In fact, type of bacteria is an important criteria
PRESERVATION that changes the effectiveness of an ultrasound treat-
ment [19]. Different kinds of microorganisms have dif-
Consumer demand for fresher, higher quality and
ferent membrane structure. Such as, Gram-positive and
microbiologically safe and stable food has promoted
Gram-negative bacteria do not show same behaviour
research on nonthermal methods for the inactivation of
against ultrasonic waves due to their different cell and
microorganisms and enzymes. During nonthermal proc-
membrane structures. Gram-positive bacteria have a
essing, the temperature of foods is held below the tem-
thicker cell wall and lack of membrane and also Gram-
perature normally used in thermal processing; therefore,
negative bacteria have a thinner cell wall with an outer
a minimal degradation of food quality is expected [15].
membrane [16]. Drakopoulou, et al. [20] examined the
Ultrasound is one of the nonthermal process which has
disinfection capability of ultrasound irradiation in the
been continuously suggested for food preservation. How-
absence and presence of TiO2 particles on different bac-
ever, the high resistance of certain enzymes and bacterial
teria groups, namely total coliforms (TC), faecal coli-
spores to ultrasound treatment limit its application. To
forms (FC), Pseudomonas spp. (PS), faecal streptococci
increase its lethality, ultrasound can be combined with
(FS) and Clostridium perfringens species (CP), found in
pressure, with temperature or with both simultaneously.
actual municipal wastewaters. They reported that Gram-
negative bacteria are more readily susceptible to ultra-
5.1. Microbial Inactivation
sound inactivation than the gram-positive ones.
Thermal treatment (i.e. pasteurization, ultra high tem- Effect of ultrasound on microbial inactivation also de-
perature) is generally considered to be main method for pends on intensity and frequency of ultrasound applied.
the inactivation of bacteria but often result in some un- Generally, frequency range of 200 - 600 kHz enhanced
desirable results such as formation of unwanted flavors the effects of ultrasound on microorganisms. Wordon, et
and loss of nutrients. Nowadays, ultrasound is used for al. [21] suggested that high frequency of ultrasound was
inactivation of microorganisms to overcome the unde- more effective in irradiation of microorganisms. Micro-
sirable results of thermal processing. Microbial inactiva- bial inactivation using ultrasound has been investigated
for application to a range of liquid foodstuffs. Levels of lus subtilis spores by ultrasonic treatments under static
E. coli O157:H7 were reduced by 5 log cfu/mL with ul- pressure and a combined pressure and heat treatments.
trasound in apple cider and the inactivation of E. coli They showed that manosonication treatment at 500 kPa
K12 was enhanced using ultrasound at ambient tempera- and 117 µm of amplitude for 12 min inactivated ap-
tures. In the same study levels of Listeria monocytogenes proximately 99% of the B. subtilis spore population.
in milk were reduced by 5 log cfu/mL when processed They reported that the ultrasound amplitude was also
with ultrasound under mild heat conditions [22]. Kap- very effective on microbial inactivation. While manos-
turowska, et al. [23] investigated the use of sonication as onication treatment (20 kHz, 300 kPa, 70˚C, 12 min) at
an alternative method to inactivate yeast cells. Cells of 90 µm inactivated 75% of the B. subtilis spore popula-
Saccharomyces cerevisiae 2200 strain were sonicated in tion, the same treatment at 150 µm inactivated 99.9% of
a 20 kHz horn-type sonicator. They found that the time, this population. The manosonication treatments at tem-
duty cycle, and power of ultrasounds significantly im- peratures higher than 70˚C (manothermosonication) led
pacted the cell inactivation. After sonication, the count of to more spore inactivation. In the range 70˚C - 90˚C, the
live yeast cells decreased by 100 to 1000 times compared combination of heat with a manosonication treatment (20
to their initial count expressed as cfu/cm3, this effect can kHz, 300 kPa, 117 mm, 6 min) had a synergistic effect on
be intensified by combing the activity of ultrasounds spore inactivation.
with a thermal factor. Application of ultrasound (20 kHz, 117 μm) to Listeria
Inactivations of microorganisms (especially spores) monocytogenes under sublethal pressure (200 kPa) caused
are resistant to environmental factors so that their inacti- reduction of pH from 7.0 to 4.0. The acidic conditions
vation is relatively difficult. Bacillus and Clostridium had a much greater effect on the organisms resistance to
spores were found to be more resistant to heat and simi- heat than its sensitivity to ultrasonication [30].
larly resistant to ultrasound [24]. To inactivate resistant
microorganisms generally ultrasound is applied with com- 5.2. Enzyme Inactivation
bination of pressure (manosonication), heat (thermosoni- Enzymatic reactions produce undesirable changes in
cation) or both pressure and heat treatments (manother- many foods during processing and storage periods. Heat
mosonication) [16]. Effectiveness of microbial inactiva- treatment to eliminate enzymes is the commonly used
tion by these methods is dependent on the amplitude of method but it also destroys nutrients and may cause loss
the ultrasonic waves, exposure/contact time, volume of of food quality. For this reason, nonthermal technologies
food being processed, the composition of the food and are being tested as an option for reducing the enzymatic
the treatment conditions [25]. When higher amplitudes activities in foods [31].
were used, higher inactivation rate was observed and it First enzyme inactivation by ultrasound was applied to
could be due to an increase in the number of bubbles pure pepsin almost 60 years ago and its inactivation
undergoing cavitation per unit of time [26] or to an in- mechanism was explained by cavitation. Since then, it
crease in the volume of liquid in which cavitation is li- has been proven that ultrasound is an effective method in
able to occur [27]. the inactivation of enzymes when it is used alone or with
D’Amico et al. [22] showed that ultrasound treatment temperature and pressure. There are many enzymes inac-
combined with mild heat (57˚C) for 18 min. resulted in a tivated with ultrasound such as glucose oxidase [32],
5-log reduction of L. monocytogenes in milk, a 5-log peroxidase [17,33], pectin methyl esterase [34], protease
reduction in total aerobic bacteria in raw milk, and a and lipase [35], watercress peroxidase [36] and poly-
6-log reduction in E. coli O157:H7 in pasteurized apple phenoloxidase [15]. Table 2 summarizes the ultrasound
cider. Juraga, et al. [28] work with high intensity ultra- application on enzymes.
sound to investigate inactivation of Enterobacteriae in Ultrasound creates continuous vibration and produce
raw milk. For ultrasounds treatment, they used three pa- stable cavitation bubbles which collapse due to the ex-
rameters: temperature (20˚C, 40˚C and 60˚C), amplitude treme local increase in pressure (1000 P) and tempera-
(120, 90 and 60 µm) and time (6, 9 and 12 min). They ture (5000 K) [37]. Also, because of shock waves strong
found that inactivation of microorganisms using ultra- shear and microstreaming the adjacent liquid is observed.
sound depends on the amplitude of the ultrasonic waves, All of these factors can cause modification of secondary
the exposure/contact time with the microorganisms, and and tertiary structure of protein due to the breakdown of
the temperature of treatment. The achieved results indi- hydrogen bonding or Van der Walls interaction in the
cate significant inactivation of microorganisms under polypeptide chains. These changes cause activity loss of
longer period of treatments with ultrasonic probe par- many enzymes. The extreme pressure and temperature
ticularly in combination with higher temperature and also lead to homolytic water molecule cleavage generat-
amplitude. ing high energy intermediates such as hydroxyl and hy-
Raso, et al. [29] investigated the inactivation of Bacil- drogen free radicals. The free radical formed may re-
Phosphate buffer 20 kHz, 50˚C - 80˚C, Application of external pressure and temperatures
Egg white lysozyme [14]
(pH 6.2) 200 kPa, 117 μm increased the inactivating effect of ultrasound.
act with some amino acid residues that participate in en- progressive oxidation of cystein by hydroxyl-free radi-
zyme stability, substrate binding or in the catalytic func- cals and aggregation of the enzymes. They observed that
tion with a consequent change in biological activity [31]. disulfide linked aggregates formed during ultrasonic
Such free radicals could recombine with amino acid treatment causes inactivation of enzymes. The involve-
residues of the enzymes. These residues are associated ment of free radicals in the inactivation of trypsin has
with structure stability, substrate binding and catalytic also been observed indirectly through the strong protec-
functions [36]. Distruption of tissue due to the ultrasonic tive effect of mannitol against ultrasound inactivation. It
application is an important criterion. As the amount of is a free radical scavenger, as well as the presence of
distruption tissue increases, surface area that contact with polypeptide fragments following sonications [39]. Also,
the enzymes and free radicals increases. For example, some researches concluded that the role of free radicals
oxidases are usually inactivated by sonication while ca- on the ultrasound inactivation of enzymes has also been
talyses are affected at low concentrations. Reductases indirectly confirmed through the effect of free radical
and amylases are highly resistant to sonication [7]. Pro- scavenging solutes in horseradish peroxidase, catalase
duction of free radicals during the ultrasonic application and glucose-6-phosphatase dehydrogenase [40].
is crucially important in enzyme inactivation that is sup- Inactivation of enzymes by ultrasonic treatment shows
ported by several studies. Barteri, et al. [38] studied the discrepancy amoung enzymes due to the different amino
inactivation of fumarase by ultrasound and explained that acid composition and the conformational structure of the
enzyme [41]. Lopez and Burgos [42] explained that inac- to manothermosonication tratment is independent on the
tivation of peroxidase by manothermosonication is due to medium of treatment, the substrates, small co-solutes,
the splitting its prosthetic heme group, as for the mecha- and other proteins [35]. Manothermosonication has an
nism of heat inactivation. However it was suggested that increased effectiveness of enzyme inactivation compared
lipoxygenase inactivated by a free radical mediated me- with ultrasound alone [4]. Firstly in 1994, a research
chanism [43] and possibly by denaturation of proteins [7]. group headed by Burgos started the study of the applica-
Some enzymes, such as catalase, yeast invertase and pep- tion of manothermosonication to enzymes releavent to
sin are resistant to ultrasound [19]. the food industry (peroxidase, lipoxygenase and poly-
The ultrasound stability of individual proteins varies phenoloxidase) in model buffer systems. Manothermoso-
between the enzymes [35,41,44,45] and also depends on nication treatments proved to be much more efficient
ultrasound treatment conditions [34], the composition of than heat treatment for inactivating these enzymes, espe-
treatment medium, treatment pH, and whether they are cially those which are more thermally labile (lipoxy-
bound (e.g., membrane-bound proteins) or free (e.g., genase and polyphenoloxidase) [52]. Also, it was ob-
cytoplasmic proteins). Enzyme inactivation generally served that manothermosonication is more effective than
increases with increasing ultrasound power, ultrasound heat treatment alone (within the temperature range of
freqency, exposure time, amplitude level, cavitation in- 110˚C - 140˚C) for the inactivation of lipoxygenase, per-
tensity, processing temperature and processing pressure, oxidase and polyphenoloxidase, heat-resistant lipase from
but decreases as the volume being treated increases Pseudomonas fluorescens [53]. However, the effective
[34,35,41,46]. improvement achieved using this combined treatment
Ultrasound does not inactivate all enzymes at mild decreased as the treatment temperature increased. Man-
temperature conditions. Such as, Villamiel and de Jong othermosonication could be useful to inactivate those
[47] reported that ultrasound treatment (20 kHz, 120 μm) enzymes within food materials that do not require such
at temperature less than 55˚C did not inactivate alkaline high temperatures for preservation [54].
phosphatase in milk, while only 22 and 14% inactivation Orange pectinmethylesterase was inactivated by heat
was observed in γ-glutamyltranspeptidase and lactoper- (72˚C, D value of 500 min) and manothermosonication
oxidase, respectively. Similar results were obtained for (72˚C, 20 kHz, 117 m, 350 kPa) and manothermosoni-
sonication (20 kHz, 7 - 40 W) of alkaline phosphatase in cation gave a much lower D value (1.2 min) [55]. Pectic
buffer [41]. enzymes of tomatoes, pectic methylestearase and the two
Thermosonication is also a good alternative to the heat endopolygalacturonase isozymes are also inactivated by
treatment for enzyme inactivation. Raviyan, et al. [34] MTS treatments with much higher efficiency, both in
indicate that when sonication is combined with heat, to- model systems [56] and in tomato juice [46]. General
mato pectin methylesterase is inactivated effectively com- trends arose from all these enzyme inactivation studies
pared to thermal treatment at the same temperature. Also, that thermolabile enzymes are more sensitive to ultra-
it was concluded that the thermosonication treatment is sound than those which are heat resistant [52].
more effective in inactivation of a number of enzymes Manothermosonication was reported to be effective for
including the pectinmethylesterase, polygalacturonase and inactivation of protease and lipase from psychrotrophic
peroxidase than heat treatment [17,48,49]. Pseudomanas [53] and pectin methyl esterase from orange
The rate of inactivation of tomato pectinmethyles- [55] and from tomatoes [56]. Vercet, et al. [35] reported
terase was greatly increased by combination of heat and that MTS is able to inactivate enzymes at a much higher
ultrasound, with increasing cavitation intensity dramati- rate than heat when these enzymes are not especially heat
cally increasing the rate of inactivation [34]. Similar re- stable.
sults were obtained for the inactivation of tomato per-
oxidase by heat and ultrasound [17]. De Gennaro, et al. 5.3. Effect of Ultrasound on Food Quality
[33] studied the effect of heat and thermosonication on
the activity of horseradish peroxidase and they found that Nowadays, food technology is aimed to reduce the nu-
the decimal reduction time of peroxidase at 80˚C, re- trients loss during the processing and storage. Ascorbic
duces from 65 to 10 min when ultrasound is applied. acid is not stable against heat treatment and usually con-
Peroxidase was inactivated by combinations of heat and sidered as an index of nutrient quality during processing
ultrasound at neutral [33] or low pH [50] and lipoxy- and storage of foods [57]. It acts as a valid criterion for
genase has been shown to be inactivated at low sonica- other sensorial or nutritional components, such as natural
tion intensities [51]. pigments and aromatic substances. Its concentration de-
Manothermosonication has reported to inactivate sev- creases during storage, depending on storage conditions
eral enzymes at lower temperatures and/or in a shorter such as temperature, oxygen content and light [58].
time than thermal treatments. Sensitivity of the enzymes Tiwari, et al. [59] found that vitamin C retention of
orange juice after ultrasonic treatment is higher when it Understanding physical inactivation processes: Combined
is compared to thermal processing. Also, Cruz, et al. [49] preservation opportunities using heat, ultrasound and
pressure. International Journal of Food Microbiology, 28,
found that ultrasonication was more effective in retention
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