Revista Mexicana de Ingeniería Química

ISSN: 1665-2738

amidiq@xanum.uam.mx

Universidad Autónoma Metropolitana Unidad

Iztapalapa

México

Castarena-García, J.H.; Martínez-Montes, F.J.; Robles-López, M.R.; Welti-Chanes, J.S.; Hemández-

Sánchez, H.; Robles-de-la-Torre, R.R.

EFFECT OF ELECTRIC FIELDS ON THE ACTIVITY OF POLYPHENOL OXIDASES

Revista Mexicana de Ingeniería Química, vol. 12, núm. 3, diciembre, 2013, pp. 391-400

Universidad Autónoma Metropolitana Unidad Iztapalapa

Distrito Federal, México

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Ingenieria de alimentos

Revista Mexicana de Ingeniería Química

Vol. 12, No. 3 (2013) 391-400

EFFECT OF ELECTRIC FIELDS ON THE ACTIVITY OF POLYPHENOL OXIDASES

EFECTO DE LOS CAMPOS ELÉCTRICOS SOBRE LA ACTIVIDAD DE LASPOLIFENOL OXIDASAS

J.H. Castarena-Garcíal ,2, El Martínez-Montesl, M.R. Robles-Lópezl ,3, J.S. Welti-Chanes4 ,

H. Hemández-Sánchez3, and R.R. Robles-de-la-Torrel

1 Centro de Investigaci6n en Biotecnología Aplicada, Instituto Politécnico Nacional,Carretera Tepetitla-Sta Inés, Tepetitla, 90700 Tlaxcala, México

2 Laboratorio de Biotecnología, Instituto Tecnológico del Altiplano de Tlaxcala.Carretera Fed. San Martín-Tlaxcala, Km. 7.5, San Diego Xocoyucan, 90122, Tlaxcala, México.

3Depto. Graduados e Inv. Alimentos, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional,Carpio y Plan de Ayala, México, DF, México.

4Departamento de Biotecnología e Ingeniería de Alimentos, Instituto Tecnológico y de Estudios Superiores deMonterrey, Ave. Eugenio Garza Sada 2501 Sur, Col. Tecnológico, 68489, Monterrey, N.L. México.

Received May 28, 2013; Accepted August 20, 2013

AbstractThe goal of lhis study was to evaluate the effect of Electric Fields (EF) on the activity of polyphenol oxidase (PPO)enzymes. EF were first applied to a commercial enzyme and then to an enzymatic extract from avocado. A fungalcommercial PPO was treated at 10 kVjcm far 0-6 min and frequencies from Oto 950 Hz; afler the treatments lheenzyme had a minimal residual activity (RA) of 12% afler 6 min at 260 Hz. The avocado extract was treated at18 kVjcm at frequencies from Oto 760 kHz, and it was shown lhat lhe PPO was insensitive to frequencies higherthan 1000 Hz, below tbis level, the enzyme was inactivated to a RA of about 15%. These results demonstrated lheeífectiveness of this Electric Field system on enzyme inactivation. It is important to mention that in this systemthere was no physical contact between the food sample and the electrodes and therefore the temperature remainedconstant throughout the processes.

Keywords : pulsed electromagnetic freId, electric freId, nonthermal techuologies, avocado, polyphenol oxidase.

ResumenEl objetivo de este estudio fue evaluar el efecto de los Campos Eléctricos (EF) en la actividad de las enzimaspolifenoloxidasas (PPO). Los EF se aplicaron primero a una enzima comercial y luego a un extracto enzimático deaguacate. Se trató una PPO comercial de origen fúngico a 10 kVjcm por 0-6 min a frecuencias que iban desde Oa 950 Hz; después de los tratamientos la mínima actividad residual (RA) de la enzima (12%) se tuvo después de 6min a 260 Hz. El extracto de aguacate se procesó a 18 kVjcm a frecuencias de Oa 760 kHz y se pudo demostrar queesta PPO fue estable a frecuencias por arriba de 1000 Hz, por debajo de este valor la enzima pierde actividad hastauna RA del 15%. Estos resultados demuestran la efectividad del sistema de EF para la inactivación enzimática. Esimportante mencionar que en este sistema no hay contacto físico entre la muestra de alimento y los electrodos y porlo tanto la temperatura permanece constante durante todo el proceso. Palabras clave: campos electromagnéticos

pulsantes, campo eléctrico, tecnologías no térmicas, aguacate, polifenol oxidasa.

* Corresponding author. E-mail: hhernan1955@yahoo.comTel. 57-29-60-00 ext 62461, Fax ext 62463

Publicado por la Academia Mexicana de Investigación y Docencia en Ingeniería Química A.C. 391

Caslorena-García el al./ Revisla Mexicana de Ingeniería Química Vol. 12, No. 3 (2013) 391-400

1 Introduction

Heal trealmenl preserves food 11uough lhedenaluration of lhe proleins and DNA of lhedeteriorative microorganisms present in it, however,heat also damages sorne of the foad components, suchas proteins, carbohydrates, vitamins, enzymes andfatty acids, and produces changes in lhe food qualilyattributes, e.g. color, fiavor and texture (Fennema1995). On lhe olher hand, lhe consumers demandfresher and less processed foad products. Since thesecond half of lhe 20th cenlury, researchers havesought to develop new fooo preservatian processeswilh less impacl on ils quality. The lasl len yearsof lhe 20th cenlury saw lhe emergence of newlechnologies lhal would lead lo lhe introductionof non-thermal foad preservatian. These emerginglechniques primarily include: high hydroslalicpressure, osmolic dehydration, pulsed lighl syslems,ultrasound, irradiatian, microwaves, and pulsedelectric fields, (Rodríguez el al., 2003; Sun, 2005).

Food preservation by Pulsed Eleclric Field orPEF lechnology has been lhe subjecl of considerableinvestigatian in research centers worldwide ayerlhe lasl lwenly years (Toepfl el al., 2005), beingused mainly for the inactivation of microorganismsand enzyme inhibition without altering the naturalattribules of lhe food (Barbosa-Cánovas el al., 2001;Martin-Belloso and Elez-Martínez, 2005). Theadvanlage of PEF trealmenl for foods is lhe shorl pulseduration. PEF trealmenl was initially applied onlylo liquids, allhough lhe syslem has now also beenlesled on semi-solid (Qinghua el al. 1994) and solidfoods, including meal and seafood (Gudmundssonand Hafsleinsson, 2005), and lhe process has alsobeen pul lo olher uses (Knorr, 2003), such as lheinhibilion of fruil fly (Hallman and Zhang, 1997). Inspile of lhe widespread research, by 2005 lhe processhad still nol been pul inlo commercial scale (Toepflel al., 2005); however, soon afterwards lhere werereports lhal PEF lechniques were being used for lhecommercial preservalion of juices (Clark, 2006).

A great deal of literature has now been writtenabout the effect of PEF treatment on microorganismsand there are sorne very comprehensive revisions(Silzmarm, 1995; Barbosa-Cánovas el al., 1998;Sun, 2005; Vega-Mercado el al., 2007). Anolherarea of inleresl on lhe use of PEF lechnology wasthe reduction of enzymatic activity. Other non­thermal teclmologies have also been used to inactivateenzymes (Sermenl-Moreno el al., 2012). Many papershave been written on the inhibition of enzymes from

different sources, e.g. alkaline protease, phosphatase,lactoperoxidase, lipase,-amylase, glucose oxidase,peroxidase, polyphenol oxidase, lizosyme, pepsin,laclale dehydrogenase and peclin melhyleslerase(Vega-Mercado el al., 1995; Grahland, 1996; Ho el al.,1997; Yeom el al. 2000; Giner el al., 2001; Espanchsel al., 2003). As far as lhe aclion mechanism ofPEF on enzyme inactivation is concemed, researchersgenerally agree lhal Ibis mechanism is nol fullyunderstood. However, more recent studies have shownlhal lhe PEF may have an impacl on lhe slruclure oflhe native prolein, and il has been suggesled lhal lhePEF acls on lhe weaker bonds lhal give slabilily lo lheprolein slruclure, such as lhe hydrogen bonds, logelherwith other types of non-covalent forces, such as theVan der Waals and electrostatic interaction, leading tolhe deslabilizalion of lhe enzyme and a loss of calalyticaclivily, (Ho el al., 1997; Yeom el al., 2002; Giner elal., 2002; Marlin-Belloso and Elez-Martínez, 2005).PEF induce moderate changes in the secondary andtertiary structure of proteins such as a-Iactalbumins,as delermined by circular dic1uoism (Robles-López elal., 2012).

Another research groups have focused on theuse of PEF treatment of plant tissue to induce poreformation and facilitate subsequent operations such asdehydration and solule exlraclion, resulting in yieldincreases (Knorr, 2003; Fincan el al., 2004; Lebovkael al., 2004; El-belghiti and Vorobiev, 2005; Amami elal., 2006).

All PEF-generating equipmenl consisls mainly oflwo melallic eleclrodes, where lhe liquid is pumped11uough lhem, having a close conlacl wilh lhe foodsample, and 11uough which lhe inlermittenl electriccurrenl or pulses are clischarged (Knorr el al., 1994;Martin-Belloso el al., 1994; Dunn, 1996). Thissimple description is not to be trivialized, because itaccuralely describes lhe type of syslem being used anddefines the type of treatment, namely, as mentionedabove, a discrete discharge of electrical current intolhe liquid, (Sitzman, 1995; McDonald el al., 2000;Vega-Mercado el al., 2007). However, parallel lo PEFlechnology lhere have been sorne arlicles on a PEFrelaled lechnology, named only as Electroslatic Fieldor Eleclric Field (EF). One of lhe firsl applicalionsof EF was electrophoresis. Electrophoresis is aseparations lechnique lhal is based on lhe mobililyof ions such as proteins or DNA in an electric field(Aguilar el al., 2009).

Actually, it is necessary to distinguish betweenthese two applications and understand the similarities

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Caslorena-García el al./ Revista Mexicana de Ingeniería Química Vol. 12, No. 3 (2013) 391-400

Treatment chamber

Frequency generator

SSR

Polarity change switch High voltage(ny-back)

Fig. 1. Diagram ofthe Electric Field system with constant voltage, (18 kV).

and fundamental differences of lhese two types oftechnology based on a field of forces generated byelectricity. The main observable difference betweenthe two systems is the contact between the foad sampleand the electrodes, and as a consequence of that,the time of treatment in every system. EF requireslonger application times for foad treatment than thoseof electromagnetic fields or PEF (Esaki el al., 1996).Anolher key difference is that in PEF lechnology,there are electric current discharges, while in the EFtechnique there are not; in the EF lhere is only a fieldof electrical forces, acting at a distance, similar to theforces in a gravitational fieId.

Sorne applications of electric or electrostatic fields(EF) on food processing includes: the effect on wheatdough and bread properties (Aibara el al., 1992), effecton evaporation and drying, (Hashinaga el al., 1995),induction of structural changes in proteins (Dong el

al., 1996), infiuence on the water activity of bread,(Esaki el al., 1996), changes in biological products andfood preservation (Wei el al., 2008; Wang el al., 2009).

The aim of this work was to evaluate the eífect of anew electric field generating equipment on natural andcommercial PPO enzymes.

2 Materials and methods

2.1 Electric field systems

Two Electric Field systems were used in this work,bolh of lhem built by personnel of the CIBA-IPN, oneof constant voltage (18 kV) shown in Figure 1 andanother with variable voltage, using a commercial andintegrated device of high voltage (Dielectric Test Set,Megger ®), which releases direct current (DC) in lherange of 5 to 80 kV (Figure 2). Bolh apparatus were

coupled wilh a function generator (BK precision ®) lovary the frequency and choose the waveform togetherwith a solid-state relay (SSR, Crouzet ®) which allowsto have, from the DC current, an alternate electric field.

The treatment chamber was made of acrylic with2 mm lhick for isolation, with external diameter of 10cm and 29 cm long, the chamber allows only a batchoperation. The electrodes were built of stainless steeland mirror polished with 0.505 cm lhick, diameter of9.6 cm, the array of these into the chamber was inparallel, one at lhe lop and lhe olher one at the bottom;each electrode was joined to an endless screw, whichallowed to close up or down the electrodes to placedifferent amounts of sample placed in a Petri dish. Thevoltage and frequency were chosen from the controlunit to treat the sample. The inlensity of the electricfield is equal to the ratio of lhe voltage applied loelectrcxies to the distance between them.

b

a 1...1'=::::=:=------Fig. 2. Electric Field system wilh variable voltage, (a)source of high voltage, from 5 lo 80 kV, (b) treatmentchamber with stain1ess steel circular electrodes, (c)generator and modulator of frequency from 5 to5,000,000 Hz, (d) oscilloscope, (e) control unit forvoltage.

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2.2 Test materials and effect ofEF

This "effect" is a modified residual activity inwhich the enzyme activity inhibition is indicated bya negative value in Eq. (2). A positive value willdesignate an activation effect.

Commercial polyphenoloxidase powder frammushroom (SIGMA Aldrich®) was dissolved intosoc1ium and potassium phosphate buffer pH 7.1. Next,

(3)

The effect of EF on lhe RA of PPO extract framavocado afler treatment at 9 kVjcm, 3 min andfrequency fram O to 760 kHz is shown in Figure 3,which praves that, with sorne exceptions, frequencieslower lhan 1000 Hz can inhibit or decrease lheactivity of polyphenoloxidase, whereas values higherthan lhat can activate the enzyme. This apparentlycontradictory result has also been reported by Martín­Belloso and Elez-Marínez (2005). They found thatsorne enzymes seem to activate with sorne PEFtreatments; Vega-Mercado el al. (2007) attributed lheactivation or deactivation of enzymes to the conditionsof treatments. With lhis, one can set the hypolhesisthat there will be a frequency at which a molecule willbe more affected while at other frequencies the samemolecule will present no sensitivity, or the moleculecould be activated; it is possible that each moleculepresent different conditions of frequency where itcan be active or inactive. The effectiveness of EFon the inactivation of enzymes is attributed to thepossible conformational changes in the structure of theenzyme after treatment. The inactivation results are inagreement with studies reported by Giner el al. (2001),who treated lhe enzyme PPO from extracts of apple(Golden delicious) and pear (Blanquilla), with PEFintensities up to 22.3 and 24.6 kVjcm, lhe minimumRA achieved was 97 % in apple extract and 28 % inpear extrac!. Giner el al. (2002) treated PPO extractfrom peach wilh PEF at intensities from 2.18 to 24.3kVjcm; lhe maximum RA of 30% was obtained withthe highest intensity electric field.

3.1 Effect of EF on the avocado enzymaticextract

Where RAo is the intercept of the curve, -k isthe kinetic constant of exponential decay of first order(min -1) and t is the treatment time (min) for first-orderkinetic model. Aflerwards, the individual values ofk were adjusted to a polynomial model to study lheeffect of frequency on the rate of enzyme inactivation.

3 Results and discussion

it was treated by EF at 10 kVjcm, time (O, 1,2, 3, 4,5 and 6 min.) and pulse frequency (O, 25, 100, 260,540, 730 and 950 Hz), square waveform. In lhis workexperimental data of each frequency were adjusted tofirst order kinetic model, accorc1ing to Eq. (3).

(1 )

(2)Effect ~ (~: - 1) x 100

(%)RA ~ (~:) x 100

Where Pe represents the units of enzymatic activityafter treatment with the EF, Po represents the units ofenzymatic activity of the enzyme without receiving EFtreatment, whereas RA sets the percentage of residualactivity. The kind of effect (inhibition or activation)was evaluated with Eq. (2):

Lyophilized and defatted avocado pulp was used as asource to extract the polyphenoloxidase (PPO). Sincea good correlation exists between PPO activity incrude homogenates of the fresh fruit and the enzymeextracted fram the acetone powder prepared framdifferent varieties, the concentratian of the enzyme inthe extract was not determined for these experiments(Kahn, 1975). The enzymatic extract was treated atconstant voltage (9 kVjcm) , treatment time (3 min)and wave square form, under a completely randomexperimental design, with frequency as the variable ofstudy, at levels of 0.00, 0.01, 0.03, 0.05, 0.055 0.0760.11, 0.14, 0.15, 0.25, 0.29, 0.38, 0.52, 0.56, 0.76,0.78, 0.99, 1.1, 2.5, 3.8, 5.2, 5.5, 7.6, 11, 25, 38,52, 76, 110, 380 and 760 kHz. The contral samplewas lhe 0.00 time. The sample was placed in a watchglass and taken into lhe chamber for treatmen!. Theresponse variable was the residual activity (RA) ofthe enzyme PPO. The RA was measured adapting amethodology used by González el al. (1999), whoused catechol dissolved in 0.1 M Tris-Hel buffer(pH 7.1) as substrate (mixture of reaction: 3 mI ofcatechol5 mM + 0.2 mL enzyme extract). Absorbancereadings were taken at 420 nm every 10 seconds for2 minutes in a UV-VIS spectrophotometer (HewlettPackard model 8453). A unit of enzyme activity wasdefined as lhe slope of the linear part of the curveof absorbance as a functian of time. The RA wascalculated according to Eq. (1) (Bendicho el al.,2005).

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"r---------------------------------------,

"Activation

." Inhibition

."L. J

~ ~ ~ ~ w :;; ~ ::; :¡; W N ~ ~ w ~ .., "l .. .., ." '" :: ::J w N ~ ~ " "" " " " " "., ", ", ..., ..., ., - N ~ ~ ~ ~

~ ~ ~ w ~

" " " " " " " " " " " " " " " " " - ~ ~

Frequency (kHz)

Fig. 3. Modified residual activily ("effecl") of polyphenoloxidase extracled from avocado as a funclion of lhe EFfrequency.

Zhong et al. (2007) trealed a mushroom PPOin solulion wilh PEF belween 15.1 lo 25 kVjcm,achieving aRA of 23.8 % al 25 kVjcm.

3.2 Effect of EF treatment on commercialpolyphenoloxidase from mushroom

Once the range of working frequencies was defined,commercial PPO was lrealed al 10 kVjcm of EF andfrequencies from O lo 950 Hz and times from O lo6 mino The results of RA of commercial PPO areshown in Table 1; the minimal activity was 12% after6 min and 260 Hz. The PPO inhibition by EF wasin lhe range reported by Yang et al. (2004) whoreached a RA of 38.2% wilh mushroom PPO al 33.6kVjcm- 1 while Zhong et al. (2007) reached 16.9% al25 kVjcm and 10 Hz. Effecls of lime and frequencyon lhe RA of PPO afler EF trealmenl (10 kVjcm-1)

are shown in Figures 4 and 5. Figure 4 shows anexponential decay of lhe RA of lhe PPO enzyme as afunctian of time for every frequency, the adjustmentof these data using equation 2 produces the valueof lhe rale of inactivation of lhe enzyme, (k). Thecomplete regression analysis is shown on Table 2. Gnlhe olher hand, frequency is considered lhe lhird faclorof importance afler lhe strenglh of lhe EF and timeof treatment. There are very few reports about theeífect of frequency on inactivation of enzymes withelectroslatic field lechnology; lhis work is an attempllo analyze lhe effecl of lhe frequency of lhe electricfieId on enzyme inactivation. Figure 5 shows the eífect

of lhe EF frequency on lhe RA of PPO lhrough lhek value, the first arder constant. The Figure shows aperiodical behavior. The polynomial model of k as afunction of frequency (í) was:

k ~ 0.18 + 1.44 x 1O-4! +4.79 x 1O-6!"

- 1.30 x 10-8!3 + 8.45 X- 12 t (4)

The adjusled delermination coefficienl was 0.9826.It is importanl lo emphasize lhal in lhe EF syslem used,there are no electrical pulses, there is no electricalcurrent being discharged into the sample, there is nophysical conlacl belween eleclrodes and sample al al!and therefore, the word frequency has arather differentmeaning lo lhe traditional PEF syslems; here, lwopieces of the equipment: the function generator andlhe SSR swilch, enables lhe EF syslem lo change lhepolarity al lhe eleclrodes, we cal! frequency lo lhischange. In the case of a PEF system, it measureslhe frequency of lhe electrical currenl lhal is beingdiscrelely discharged or pulsed inlo lhe sample Thephysical effecl of frequency during PEF lrealmenlshas been explained in terms of the polarizationof molecules, caused by lhe eleclromagnetic fieldapplied. If lhe change in lhe frequency applied is veryhigh, lhe dipoles formed by lhe PEF or jusI lhe EF willnol be able lo conlinue lo lhe exlernal field and lhepolarizatian of orientation will not be produced. Asaboye mentioned, frequency produced less eífect onPPO activity lhan lhe eleclric field strenglh and time.

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2

• ¿•••

•

•

1000600

•

600

•

400200

040

035

030,~

"l 025 •~ 020

~ o15..•u 010u'i" 005

'" 000

-o oso6

!••

* 100 Hz• 540 Hz.. 950 Hz

5

••=

4

t

•tT

• 25 Hz

" 260 Hz'll 730 Hz

3

•T

*•

•100

90

80

l 70

'" 60:~ü 50•.., 40u.¡;

• 30

'" 20

10

oo

Time (min) FreQuency (Hz)

Fig. 4. Residual aclivity of polyphenoloxidaseextracted fram avocado as a function of the time ofEF lrealmenl al 10 kVjcm.

Fig. 5. Behavior of lhe kinetic conslanl (min-1)from 25 lo 950 Hz.

Jeyamkondan et al. (1999) menlioned lhal !hefrequency of lhe pulse applied lo foods, plays animportanl role inlo !he energy added lo lhe mec1ium,an increase in pulse frequency implicates higherpower supply consumption of !he PEF syslem andconsequentIy, an increase in the temperature of foadas is usually observed. That same year, Fabregat el

al. (1999) menlioned lhal sinusoidal faclors producesinusoidal responses. Afterwards, Venkatesh andRaghavan (2005) commenled !hal lhe answer lo !hefrequency fram biological materials, is an electricalfeature in EF treatment and depend of composition andenvironment.

Table 1. Residual activity of polyphenoloxidase in the commercial enzyme preparatianunder differenl EF lrealmenl condilions al 10 kV.

Time (min) Frequency (Hz) (%) RA±s.d. Time (min) Frequency (Hz) (%) RA±s.d.1 25 67.0±3.61a 4 25 29.0±6.931 100 71.3±5.13 4 100 26.3±4.161 260 75.3±9.29 4 260 22.7±2.311 540 89.3±7.02 4 540 39.7±4.931 730 59.0±1.73 4 730 33.0±6.561 950 88.7±5.03 4 950 41.7±4.732 25 41.7±4.51 5 25 27.7±6.662 100 40.7±1.15 5 100 20.0±4.362 260 52.7±9.71 5 260 24.3±3.512 540 69.7± 1.53 5 540 23.0±2.652 730 47.3±7.51 5 730 32.3±3.512 950 59.0±4.36 5 950 18.7±6.663 25 66.7±13.65 6 25 28.0±2.653 100 41.7±4.16 6 100 22.7±3.213 260 40.0±5.57 6 260 12.0±4.583 540 44.7±4.04 6 540 18.7±2.083 730 41.0±5.57 6 730 25.7±7.093 950 51.7±2.31 6 950 18.0±3.46

(%)RA: Residual Activity percentage

s.d.: standard deviationa : Mean value oftree data

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Table 2. Regression analysis of the exponential decay kinetics of the polyphenoloxidase activity withtime of EF treatment at different frequencies

Treatmentfrequency

(Hz)

Regression Coefficient Coefficient 95% lowerstandard standard limit confidence

error probability limit

95% upperconfidence

limit

R

25100269540730950

0.18550.24010.35050.32280.16150.3378

0.28980.21310.24040.11610.15930.2280

0.03990.02940.03320.11610.02190.0315

0.00030.000012.8xl0-8

8.6xl0-13

1.64xl0-6

1.01xl0-8

0.10080.17780.28020.28880.11470.2711

0.27020.30240.42080.35680.20810.4045

0.75720.83630.92820.98080.87830.9371

Conclusions

By applying current knowledge, technology, andexperience, it was possible to design and build electricfieId generating equipment, which was eífective ininhibiting the growth of microorganisms, such asbacteria and fungi spores in real food (unpublishedresults), here, it was also possible to decrease theactivity of lhe enzyme polyphenol oxidase, pure andfram an avocado extracto As noted aboye, it is possibleto develop processes for foad preservatian by non­thermal methods, preserving the natural qualities offresh produce through treatments with sorne of themost promising emerging technologies such as theelectric fields. It is emphasized lhat in tbis equipmentthe concept of force field is elfectively appliec1, giventhe absence of physical contact between electrooe andsample, so that in this type of equipment, there is nota temperature rise and therefore, it does not requirecooling systems. The equipment built in this projectcan apply electric fields to liquid, semi-solids and solidfoods. The equipment applications can be expandedconsiderably, for example, to whole nuts, pieces ofmeat or dairy products and to similar products. Inaddition, having demonstrated the eífect on cellularproducts, enzymes, whole cells, bacteria, fungi, andfungal spores, it is possible to extend its application toother biological forms such as viruses.

Acknowledgments

The aulhors of lhis work acknowledge the financialsupport of FOMIX- Michoacán State Govemmentthrough the grant 2005-C01-015. Author Castorena­Garcia J Hugo would like to lhank CONACyT for lhescholarship for his graduate studies.

Nomenclaturef electric field frequency KHzk enzyme activity exponential decay

kinetic constant min-lPo enzyme activity of the untreated samplePe enzyme activity after electric field

treatmentRA residual activity of the enzyme %

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