Electrofusion

Electrofusion provides a convenient way to study fundamental mechanisms of membrane fusion, which may help to understand the fusion mechanisms in biological systems.

From: Handbook of Biological Physics, 1995

Structure and Dynamics of Membranes

D.S. Dimitrov, in Handbook of Biological Physics, 1995

7 Fusion in life processes involves specialized proteins but kinetically can be similar to electrofusion

Electrofusion provides a convenient way to study fundamental mechanisms of membrane fusion, which may help to understand the fusion mechanisms in biological systems. While the major requirements for electrofusion and biological fusion (close contact, destabilization, overcoming energy barriers and appropriate structural rearrangements) are the same, fusion in biological systems is tightly controlled by specific protein interactions and full understanding of its mechanisms requires understanding the structure and conformational changes of the fusion proteins.

The biological fusion process which is best understood in terms of molecular structure and conformational changes of fusion proteins is viral fusion [154, 155]. Viral fusion is caused by proteins which undergo conformational changes to expose hydrophobic peptides after activation by low pH or interaction with receptor molecules. The exposure of the hydrophobic fusion peptide to the water environment results in an increase in the free energy of the peptide and its immediate environment possibly including the surfaces of the interacting membranes. This leads to a high free energy state which could be similar to that of a fusogenic electropore in electrofusion. Therefore, the kinetic pathways of viral fusion after activation of the viral proteins could be similar to those of electrofusion. The existence of a high energy state of the viral fusion protein leads to local mutual approach of the lipid bilayers and destabilization of the membranes and of the liquid layer between them. The lipid matrix of the membrane then undergoes localized structural rearrangements resulting in lipid intermixing. An indication that this stage of viral fusion is also similar to that in electrofusion is the magnitude of the activation energy which in both cases is of the order of the activation energy of the lateral mobility of the lipid molecules (15-25 kcal/mol) [85, 156]. After the formation of fusion junctions and pores, they can either expand and lead to formation of giant cells or stabilize and preserve the morphology of aggregated cells [85, 100].

The comparison between electrofusion and virus fusion may lead to the discovery of new important parameters affecting the kinetics of viral fusion. For example, by analogy with electrofusion one might predict that delays in viral fusion should increase with increasing the viscosity of the liquid between the membranes. This was confirmed experimentally for fusion of Sendai virus [118]. A detailed comparison between electrofusion and viral fusion is made elsewhere [107].

The above considerations could be applied to a wide variety of biological processes involving fusion, including exo- and endocytosis, fertilization, cell division, intracellular transport and myoblast formation.

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Tetrahymena Thermophila

Eric Cole, Toshiro Sugai, in Methods in Cell Biology, 2012

4 Electrofusion

Electrofusion was first applied to Tetrahymena by Gaertig et al. (1988) as a means of creating parabiotic, live-cell fusions. Since then, live-cell fusion experiments have shed light on nuclear–cortical interactions during both vegetative development (Gaertig and Iftode, 1989) and conjugal development (Kaczanowski and Kiersnowska, 1996a; Gaertig and Cole, 2000; Cole et al., 2001). In one set of experiments, vegetative cells were parabiotically fused and these were then allowed to mate with normal partners. Nuclear configurations were examined by Giemsa staining. By examining a variety of cytogenetic configurations some surprising insights into conjugal development were revealed.

1.

In early meiosis, during the crescent stage, the crescent spindle becomes straight and not curved in double-long partners, suggesting the crescent spindle curvature is normally due to simple space constraints and not some intrinsic morphogenetic process.

2.

In “unipolar” matings (the anterior of one cell is fused to the posterior of an actively pairing second cell), both fusion cell MICs migrate toward the anterior cytoplasm after meiosis, but only the anterior end that is actively fused to a partner will capture a nucleus and bring about “nuclear selection.” There may be some residual “selection” activity in the region of the posterior fusion cell corresponding to the para-oral exchange plaque that may serve to shield adjacent nuclei from programmed nuclear degeneration (PND).

3.

The meiotic spindles will elongate to span a double-cell length on the fusion side of a mating pair.

4.

The second postzygotic division spindle remains short delivering nuclear products to the mid-body region between parabiotic fusions.

5.

Nuclei from the anterior cell of a mating unipolar fusion that are delivered to this mid-body region remain determined to form germline micronuclei.

This latter result was reported by both Gaertig and Cole (2000) and Kaczanowski and Kiersnowski (1996a) who fused already mating pairs together. Together, observations 2 and 5 strongly reinforce the idea that there are cortical determinants that influence nuclear fate. More specifically, the para-oral cortex (near the exchange junction) not only anchors “selected” meiotic nuclei, but may shield them from cytoplasmic signals that trigger PND. These “selected” nuclei become available to respond to subsequent mitotic triggers leading to the third gametogenic division. Similarly, there appear to be cortical determinants in the cell's posterior that shield nuclei derived from the second postzygotic division from cytoplasmic signals triggering differentiation of macronuclear anlagen. It is intriguing that in both cases, nuclei anchored to these sites are shielded from cytoplasmic signals that otherwise affect untethered nuclei. (This is a different interpretation from Nanney's in which anchorage was seen to drive a developmental event rather than “shield” nuclei from some cytoplasmic signal.)

In a very different set of electrofusion experiments, mating pairs were allowed to progress into conjugation, and then electrically fused to either vegetative cells or other mating pairs (Cole et al., 2001). These experiments revealed several things about the developmental program of mating Tetrahymena.

1.

Dividing cells produce some soluble activity that can inhibit conjugal development.

2.

This activity peaks during mitosis and drops during interphase.

3.

Mating pairs are sensitive to this arrest activity (division factor) up until the second postzygotic division. This is a “terminal commitment point” or point of no return.

4.

Pairs undergoing “genomic exclusion” [a form of abortive conjugal development triggered when diploid cells are mated to severely aneuploidy partners, (see below)] also exhibit a “conjugal arrest activity” blocking development when fused to normal diploid partners.

5.

The arrest activity expressed by genomic exclusion pairs peaks in strength just after meiosis I.

6.

Healthy diploid pairs are sensitive to this arrest activity (abort factor), up until the second postzygotic division (as with the division factor arrest).

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In Vitro Models for Nephrotoxiciry Screening and Risk Assessment

PETER H. BACH, ... STEPHEN BRANT, in In Vitro Methods in Pharmaceutical Research, 1997

2 New cell lines

Cell hybrids formed by electrofusion have already been used to produce monoclonal antibodies.223 No single cell type contains all of the factors that may predispose them to ‘target-selective’ injury. Thus the possibility of using the cell biological approach of fusing two different cells to combine desirable functions (such as a transport process and a metabolic activation system) is already being investigated. Combining human renal primary cells with stable lines could provide hybrids that reflect the characteristic of each cell type for nephrotoxicity testing. Hybrid cell lines expressing functions lost in primary cells are being developed by fusing permanent, non-tumorigenic cell lines with primary human or rat proximal tubule cells. Several clones have been produced, and now need a substantial investment to characterize them, to establish whether they are stable and immortal, and to clarify whether they are more useful for in vitro testing than cells that are currently available. Similarly, cells transfected with genes coding for specific characteristics (xenobiotic metabolism, organic anion transport, etc.) that are absent from the original cell line will be an area of important future advancement.

The ability to establish an array of tailored cells that represent each of the different regions of the kidney, by fusion, transfection or nuclear injection with selected properties will provide the potential to learn a great deal more about the mechanisms of injury in model chimeric systems. The newly tailored cell lines need to be rapidly characterized by molecular and cellular biology techniques such as immunocytochemistry and fluorescent probes to document biochemical, metabolic and transport properties, to establish their hormone responsiveness, handling of macromolecules, as well as to document their ultrastructural features.

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Tissue Culture Applications for the Genetic Improvement of Plants

Moacir Pasqual, ... Filipe Almendagna Rodrigues, in Biotechnology and Plant Breeding, 2014

Electrofusion

The following steps are used in protoplast electrofusion: (1) alternating current is used to transfer the protoplasts and to promote a close contact between the membranes, and (2) continuous short pulses are used to induce membrane disruption at the contact points. Despite the need for expensive and sophisticated equipment to generate alternating current fields and continuous current pulses, the electrofusion method has become increasingly popular because it is less damaging to protoplasts than chemical procedures.

Geerts et al. (2008) performed a study using protoplast fusion technology by somatic hybridization in Phaseolus. The success of interspecific breeding between Phaseolus vulgaris L. (PV) and the two donor species, Phaseolus coccineus L. (PC) or Phaseolus polyanthus Greenm. (PP), required the use of donor species as the female parents. Although the incompatibility barriers were post-zygotic, the success of the phase-F1 cross was very limited given the hybrid embryo abortion. The study described the use of protoplast fusion methods within the genera Phaseolus as an alternative to conventional crosses between PV, PC, or PP. A large number of heterokaryons were generated through different genotypes using protoplast fusion procedures based on both electrical (750 or 1500 V cm−1) and chemical fusion using polyethylene glycol (PEG 6000) as the fusing agent.

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Insulin-Secreting Cell Lines

Shanta J. Persaud, ... Peter M. Jones, in Cellular Endocrinology in Health and Disease, 2014

Electrofusion-Derived Insulin-Secreting-Cells

Three human insulin-secreting cell lines have been generated by electrofusion of human islet cells with a proliferative pancreatic epithelial cell line (PANC-1), all of which respond to elevated glucose with increased insulin secretion. The 1.4E7 line is of limited use as it showed only a 50% increase in insulin release, and the glucose concentration–response curve was left-shifted such that maximal stimulation was obtained at 5.6 mM glucose.45 However, the 1.1B4 line had more promising characteristics, showing a maximal 2.3-fold increase in insulin at 11.1 mM glucose, and these cells expressed the GLUT1 glucose transporter and glucokinase.45 Although the insulin content of 1.1B4 cells is maintained with prolonged culture, they contain at least 100-fold less insulin per cell than the recently developed EndoC-βH1 cells (see below).

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Micromanipulation Techniques for Cloning

Raymond L. Page, Christopher M. Malcuit, in Principles of Cloning (Second Edition), 2014

Couplet Fusion

Fusion may be accomplished by electrical stimulation, polyethylene glycol (PEG), or Sendai virus, with electrical stimulation by far the most popular method. For electrofusion, the cell–oocyte couplets are placed in a largely non-ionic, slightly hypotonic medium between two parallel electrodes, and a high-voltage DC pulse is delivered such that membrane breakdown is achieved (reviewed in Zimmermann and Vienken, 1982). The pulse strength and duration must be determined experimentally, as conditions will vary slightly between different laboratories. The electrical pulse results in inversion of membrane phospholipids (breakdown), thus creating holes in the membrane; as these heal, the membranes are fused. Therefore, too high a voltage for too long will result in lysis. However, if the voltage or duration is too low, poor fusion rates will result. A rule of thumb for bovine nuclear transfer is to adjust the fusion parameters such that about 10% of the oocytes will lyse. On average, fusion parameters are about 1.25–1.5 kV/cm for 10–50 μs. However, some investigators prefer to deliver several shorter pulses rather than one longer one.

A rule of thumb for bovine nuclear transfer is to adjust the fusion parameters such that about 10% of the oocytes will lyse.

Another variable is the fusion chamber used, which will affect the raw settings due to the dimensions and position of the electrodes. Suppliers of electrofusion equipment, such as BTX Instruments (San Diego, CA) and Eppendorf (Brinkman Instruments), supply several chambers to choose from and provide methods to calculate the electric field strength for each chamber. The temperature of the fusion medium will also affect results, so this should be kept constant either by using a heated stage for the stereomicroscope or by maintaining a constant base temperature. The latter may be more difficult, as the intensity of the light for transillumination will affect the base temperature and may be variable for different users. In addition, small volumes of fusion medium and higher temperature will result in rapid evaporation leading to increased osmolarity, which leads to couplet detachment. Therefore, if a small chamber is used, the fusion medium should be replaced often.

A common basis for fusion medium is 0.3-M mannitol, 0.1-mM MgSO4, and 0.05-mM CaCl2 (Iwasaki et al., 1989), with slight modifications. The osmolarity may be lowered by adjustment of the mannitol concentration to 0.25–0.28 M. If an activation stimulus at the time of fusion is not desired, then the calcium is deleted. The magnesium is important as it provides a divalent cation source to help maintain membrane contact between the cell and oocyte. To prevent the oocytes from adhering to the glass, 0.1 mg/ml of a macromolecule such as BSA, PVA, or PVP may be added. Better results may be achieved using a macromolecule with a molecular weight high enough that it will not cross the zona pellucida, as this can interfere with membrane contact between the couplets. Most investigators do not buffer the fusion medium, but this has been done using 0.5-mM Hepes (Wells et al., 1999) with good results.

Typically, the fusion medium is of higher density than the manipulation medium, so when oocytes are added they float until equilibrated. We prefer to use a four-well plate to prepare couplets for fusion. Well 1 contains manipulation medium, Well 2 contains a 1:1 mixture of manipulation medium, Well 3 contains fusion medium, and Well 4 contains manipulation medium. Couplets are transferred from the manipulation microscope to Well 1, then to Wells 2 then 3, and allowed to settle to the bottom in each well. We prefer to transfer five at a time, in overall batches of 20, to the fusion chamber from Well 3. Once fused, they are placed into Well 4. Once the batch of 20 is complete, all oocytes are placed into the incubator in culture medium, and a new batch is started. It is also useful to have a set-up with two stereomicroscopes side by side for the fusion operation. One microscope is used to hold and prepare the couplets, while the other has the fusion chamber attached. This allows for rapid transfer into and out of the fusion chamber, thus minimizing the time oocytes spend in the fusion medium. For maximum field strength at the site of membrane contact, the couplets are aligned in the chamber such that the area of membrane contact is parallel to the electrodes. For this reason couplets are generally fused in small batches of 10 or less, since it is difficult to align more than this number quickly in the field of few provided by a magnification large enough to visualize the cells. Oocyte activation and embryo culture are the next steps in the process. There are many choices for the method of artificial activation, and many theories regarding the optimal timing between fusion and activation as well as for embryo culture. Other chapters in this volume will deal with the nuances of species and theories regarding the cell cycle as it pertains to the timing of fusion and activation.

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Biophysical Techniques for Enhancing Microbiological Analysis

A.D. Goater, R. Pethig, in Encyclopedia of Food Microbiology (Second Edition), 2014

Are Cells Damaged?

To induce cell fusion, or indeed electrical breakdown of the cell membrane, a field strength of at least 10 times more than is typically used in DEP separations is required. Hybrid cells from electrofusion are viable, which suggests that cells having undergone exposure to normal DEP forces are not damaged. Further evidence includes the exclusion of trypan blue from dielectrophoretically separated erythrocytes and the successful culture of various cell types including yeast cells and CD34+ cells.

The fluid flow during a DEP separation procedure produces a maximum shear stress on the cell of around 3 dyn cm−2. T-lymphocytes and erythrocytes have been reported to be able to withstand a shear stress some 50 and 500 times this value, respectively. Therefore, almost insignificant levels of shear stress are experienced by these cells in DEP chambers.

The conductivity of suspending medium used is normally much below that of a normal physiological medium, however, as long as the osmolarity is of the right value, osmotically sensitive cells can be investigated. This is achieved by additives such as sucrose at 280 mM, which has little effect on the conductivity. An alternative approach has been to use sub-micrometer electrodes which minimize heating effects enabling the use of normal physiological strength media.

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Somatic (asexual) procedures (haploids, protoplasts, cell selection) and their applications

Tanya Tapingkae, ... Acram Taji, in Plant Biotechnology and Agriculture, 2012

Protoplast fusion methods

Protoplast surfaces bear strong negative charges, and intact protoplasts in suspension repel each other, hence fusion is accomplished by the addition of calcium ions or polyethylene glycol (PEG), or by using electric fields. Electrofusion is preferred over chemical fusion, as fusion conditions are much easier to control (Duquenne et al., 2007). According to work by Trigiano and Gray (2000), the yield of fusion products increases 20-fold when electrofusion is used. Even though binary chemical fusion is more efficient in some plants, it allows more efficient cell division and plant regeneration afterwards (Assani et al., 2005).

In recent years, somatic hybridization has been accomplished by electrofusion of protoplast, and characterizing the regenerated plantlets by flow cytometry and isozyme or DNA marker analysis. Electrochemical protoplast fusion is a process that combines the merits of both somatic hybridization and chemical methods (Olivares-Fuster et al., 2005). Olivares-Fuster et al. (2005) reported production of citrus somatic hybrids and cybrids via electrochemical protoplast fusion, where protoplasts of sweet orange and Mexican lime were induced to undergo fusion in the presence of PEG and electric impulses of direct current. High rates of embryogenesis were exhibited.

Since most of the hybrids obtained via symmetric protoplast fusion may contain numerous undesirable genes, repeated backcrossing and selection are required for elimination of undesirable traits. The obtained somatic hybrids often show chromosome loss, weakness, or sterility, probably due to somatic incompatibility (Fu et al., 2009). Chromosome elimination is an important issue and a complicated process influenced by many factors, such as genotype, type of irradiation rays, irradiation dose, and phylogenetic relatedness (Liu et al., 2005a; Yang et al., 2007).

In asymmetric fusion, metabolism inhibitors inactivate recipient protoplasts and simplify the selection of the regenerants. Iodoacetate (IOA) has been used to inactivate protoplasts of parents for fusion and has been found to be efficient in selectively producing somatic hybrids (Shimonaka et al., 2002). For transferring a part of the genome or cytoplasm, the donor protoplasts are usually irradiated with X- or γ-rays prior to fusion (Fu et al., 2009), but these two kinds of irradiating rays are dangerous and inconvenient to use. Therefore, UV has been increasingly used to break and fragment chromosomes of donors for production of asymmetric hybrids (Yang et al., 2007; Wang et al., 2008).

Effects of UV on donor chromosome elimination and fragmentation are dose dependent (Xiang et al., 2003). Using the viability, division percentage, and plating efficiency of UV-irradiated protoplasts as indicators, Fu et al. (2009) were able to enhance chromosome elimination by increasing the irradiation dose. Xiao et al. (2009) found that when UV dosage increased, the differentiation ability of colony formation and the frequency of plant regeneration decreased in protoplast fusion in banana. In addition, the UV treatment given to the donor protoplast influenced the growth and development of fused products, suggesting that optimal UV irradiation is a key factor for asymmetric protoplast fusion.

Many plant species have been used in intergeneric somatic hybridization, as shown in Table 10.1. Some important economic characteristics transferred through protoplast fusion, such as tolerance to disease, herbicides, and salt are shown in Table 10.2.

Table 10.1. Intergeneric symmetric and asymmetric fusions

Parents Fusions
A B Symmetric fusions Asymmetric fusions or microfusions
Atropa Nicotiana Yemets et al. (2000) Yemets et al. (2000)
Brassica Arabidopsis Yamagishi et al. (2002)
Camelina Jiang et al. (2009)
Crambe Wang et al. (2003)
Orychophragmus Hu et al. (2002b) Hu et al. (2002b)
Zhao et al. (2008)
Isatis Du et al. (2009) Du et al. (2009)
Tu et al. (2008)
Sinapis Hu et al. (2002a)
Citrus Fortunella Takami et al. (2004)
Poncirus Guo et al. (2002) Liu and Deng (2000)
Clausena Fu et al. (2003)
Microcitrus Liu and Deng (2002)
Xu et al. (2004)
Severinia Grosser and Chandler (2000)
Dendranthema Artemisia Furuta et al. (2004)
Daucus Panax Han et al. (2009)
Helianthus Cichorium Varotto et al. (2001)
Hyoscyamus Nicotiana Zubko et al. (2002)
Lathyrus Pisum Durieu and Ochatt (2000)
Raphanus Isatis Tu et al. (2008)
Triticum Avena Xiang et al. (2003)
Xiang et al. (2010)
Aeleuropus Yue et al. (2001)
Agropyron Cui et al. (2009) Xia et al. (2003)
Gao et al. (2010) Liu et al. (2009)
Bupleurum Zhou et al. (2006)
Lolium Cheng and Xia (2004)
Psathyrostachys Xing et al. (2001)
Setaria Xiang et al. (2004)
Haynaldia Zhou et al. (2001) Zhou et al. (2001)
Zea Szarka et al. (2002) Xu et al. (2003)

Table 10.2. Important economic characteristics transferred through protoplast fusion

Somatic hybrids Characters References
Musa acuminata cv. Mas + M. silk cv. Guoshanxiang Disease resistance (Fusarium) Xiao et al. (2009)
Brassica napus + Isatis indigotica Disease resistance Du et al. (2009)
Helianthus annuus L. + H. maximiliani Disease resistance Taski-Ajdukovic et al. (2006)
Triticum aestivum + Aeleuropus littorulis T. aestivum + Agropyron elongatum Salt tolerance Yue et al. (2001)
Xia et al. (2003)
B. napus + Crambe abyssinica Higher erucic acid content Wang et al. (2003)
Oryza meyeriana + O. sativa ssp. Japonica Bacterial blight resistance Yan et al. (2004)
Dendranthema × grandiflorum + Artemisia sieversiana Rust resistance Furuta et al. (2004)
Sinapsis arvensis + Arabidopsis thaliana Stem canker resistance Hu et al. (2002a)
Nicotiana plumbaginifolia + Atropa belladonna Amiprophos methyl resistance Yemets et al. (2000)
Citrus reticulata cv. Red Tangerine + Poncirus trifoliata Tolerant to CTV (citrus tristeza virus) and CEV (citrus exocortis virus) Guo et al. (2002)
T. aestivum + A. elongatum Higher protein content Gao et al. (2010)
Cui et al. (2009)
B. napus + Camelina sativa Higher level of linolenic and eicosanoic acids Jiang et al. (2009)
Citrus reticulata + C. limon Carotenoid compounds Bassene et al. (2009)
R. sativus + B. rapa Medicinal components Tu et al. (2008)
Daucus carota + Panax quinquefolius Ginsenoside Han et al. (2009)
Ipomoea batatas + I. triloba Storage root-bearing Yang et al. (2009)
Murcott tangor (Citrus reticulata Blanco × C. sinensis (L.) Osbeck) + Hirado Buntan Pink pummelo (HBP) (C. grandis (L.) Osbeck) Seedless Cai et al. (2010)
Citrus sinensis Osbeck cv. Yoshida navel orange + Citrus unshiu Marc cv. Okitsu satsuma mandarin Seedless An et al. (2008)
Bingtang orange (Citrus sinensis (L.) Osbeck) + Calamondin (C. microcarpa Bunge) Asiatic citrus canker-tolerant and ornamental citrus breeding Cai et al. (2010)
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Fluid Rheology in Novel Thermal and Non-Thermal Processes

P.J. Cullen, in Novel Thermal and Non-Thermal Technologies for Fluid Foods, 2012

3.4.1 Pulsed Electric Fields

PEF treatment is based on the application of high-intensity electric fields to a food as it flows through the confines of a treatment gap between two electrodes. Such electric fields induce transmembrane potential on cell membranes resulting in electroporation and electrofusion leading to bacterial inactivation. The field may, in turn, induce changes in the fluid structure. Garcia-Amezquita et al. (2009) examined the effect of PEF on the native size distribution of fat globules in bovine cheese-making milk. Although PEF processing did not modify the true mean diameter of milk-fat globules, it induced small globules to clump together, causing an apparent increment in the population of larger milk-fat globules. Sampedro et al. (2006) reviewed the application of PEF in egg and egg derivatives and concluded that treatment did not cause notable changes in proteins in a solution of ovalbumin and dialyzed fresh egg white. However, some structural changes and functional modifications were observed in fresh egg white as a result of PEF treatment. The texture and microstructure of gels were affected by the application of PEF, and therefore PEF treatment conditions in egg white must be optimized to minimize possible modifications. Aguilo-Aguayo et al. (2009) investigated the effects of pulse frequency (50–250 Hz), pulse width (1.0–7.0 mus), and polarity (monopolar or bipolar) of high-intensity PEF treatments (35 kVcm−1 and 1000 mus) on the viscosity and the pectin methylesterase (PME) and polygalacturonase (PG) activities of tomato and strawberry juices. Apparent viscosity of strawberry juices increased slightly when frequencies higher than 100 Hz and 1 mus monopolar pulses were applied to the juice. Tomato juice apparent viscosity increased within the range of the assayed conditions, achieving the highest values at 250 Hz and 7.0 mus in bipolar mode. At the same conditions the lowest residual PME (RAPME=10%) and PG (RAPG=45%) activities were observed in the juice. Treatments causing the greatest increase in strawberry juice apparent viscosity also led to the lowest RAPME (10%) and RAPG (75%) values. In contrast, viscosity loss was promoted under the rest of the assayed PEF conditions despite the low RAPME values (<20%) achieved. Aguilo-Aguayo et al. (2010) also reported that PEF-treated watermelon juices exhibit better physical properties such as color and viscosity when compared with thermally treated juices throughout storage.

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