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Enzyme and Microbial Technology 39 (2006) 10021007

Elimination of glucose in egg white using immobilized glucose oxidase


C. Sisak a, , Z. Csan di a , E. R nay b , B. Szaj ni c a o a
a

Research Institute of Chemical and Process Engineering, University of Veszpr m, Veszpr m, Hungary e e b SATO Egg Processing and Trade Ltd., Budapest, Hungary c Department of Genetics, E tv s L r nd University of Sciences, Budapest, Hungary o o oa Received 12 September 2005; received in revised form 7 February 2006; accepted 7 February 2006

Abstract A commercial glucose oxidase containing catalase (NOVOZYM 771) was immobilized on Amberlite UP 900 anion exchange resin using adsorption and subsequent cross-linking with glutaraldehyde. The maximum protein loading was 33.0 mg g1 dry support. The activity of the product was 10.2 units g1 dry solid. The immobilized enzyme was used for the elimination of glucose from egg white in a pilot-scale three-stage uidized-bed bioreactor with mechanical mixing, equipped with large porous surface for bubble-free transport of oxygen as co-substrate. The biocatalyst packing (10.9 dm3 ) was uniformly distributed between the stages. The best productivity was achieved using oxygen pressure of 1.5 bar and 2 h residence time at 25 C. 2006 Elsevier Inc. All rights reserved.
Keywords: Egg white processing; Glucose elimination; Glucose oxidase immobilized; Fluidized-bed bioreactor

1. Introduction Egg white as a spray dried powder is an important raw material for the food industry. The original liquid form contains about 4 g dm3 glucose. It is a general practice to remove glucose from the egg white before spray drying to avoid the browning of the product owing to the caramel formation. In addition, the elimination of glucose enhances the resistance of the product against the attack of microorganisms improving the storage stability of the egg white powder. At present, soluble glucose oxidase (GOD) ( -d-glucose: oxygen 1-oxidoreductase; EC 1.1.3.4) or microbes producing the enzyme are applied in the processes for the elimination of glucose [1,2]. Glucose oxidase catalyzes the oxidation of -d-glucose by oxygen to d-glucono1,5-lactone. For practical purposes the enzyme is mostly isolated from Aspergillus niger. During the catalytic oxidation of glucose, hydrogen peroxide arises. Under physiological conditions GOD is exposed to very low concentrations of H2 O2 , but in the industrial applications higher H2 O2 concentrations are arisen which can attack some residues of the proteins (methionine,

Corresponding author at: Research Institute of Chemical and Process Engineering, University of Veszpr m, Egyetem u. 10., H-8200 Veszpr m, Hungary. e e Tel.: +36 88 624 036; fax: +36 88 624 038. E-mail address: sisak@delta.richem.hu (C. Sisak).

cysteine, etc.) [3]. Therefore, it is inevitable to remove hydrogen peroxide, properly with another enzyme, catalase (hydrogen peroxide: hydrogen peroxide oxidoreductase; EC 1.11.1.6). The commercial GOD preparations usually contain catalase in a sufcient concentration for that purpose. The application of the soluble GOD makes possible only batch operations and the enzyme remains as contamination in the product. Therefore, it would be advisable if the enzyme were used in immobilized form. GOD has been immobilized using different methods. For instance, encapsulation within calcium alginate gel capsules [4,5], covalent attachment to nickel oxide [6], AlPO4 and AlPO4 /clay mineral Sepiolite system [7], alumina [8], magnesium silicate [9], glyoxyl agarose, epoxy sepabeads and glutaraldehyde-activated supports [10], polyacrylamide beads [11] as well as immobilization on polymer membrane [12]. Among the wide studies on the application of immobilized glucose oxidase only few ones propose egg white desugaring. Investigations carried out by DSouza and co-workers have shown that glucose can be removed from egg, using glucose oxidase and catalase which are co-immobilized either on polycationic cotton cloth [13] or in hen egg white foam matrix [14]. Glucose can also be removed by rapid heterogeneous fermentation of egg melange, using immobilized yeast [15]. Nevertheless, no report discusses the operational viewpoints of the egg desugaring process performed by immobilized biocatalyst.

0141-0229/$ see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.02.010

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The co-substrate of the enzyme is oxygen. The effective glucose elimination needs bubble-free oxygen supply to avoid the egg foam formation in the reactor. A number of innovations connected to the bubble-free oxygenation have been achievedrst of all in the eld of animal and plant cell cultivation [16,17]. Nevertheless, to harmonize the optimum operational conditions for a heterogenic enzymatic process and the simultaneous bubblefree aeration of a liquid phase seem to be a complex task. In the present paper, a novel egg white desugaring system operating with immobilized GOD is demonstrated. Taking into consideration of economical demands, the use of a relatively cheap industrial enzyme preparation and a simple way of immobilization appear to be justied. Therefore, we have chosen NOVOZYM 771 GOD isolated from A. niger containing catalase. For continuous desugaring, a three-stage uidized-bed reactor has been developed.
2. Materials and methods 2.1. Materials

added to 0.5 cm3 sample (glucose content between 0.1 and 0.4 mg cm3 ). The mixture was kept in a boiling water bath for 8 min then immediately cooled in an ice-bath and absorbance was measured at 620 nm with spectrophotometer of SPEKTROMOM 195D type (MOM, Budapest, Hungary). The o-toluidine reagent was made up from 15 cm3 o-toluidine and 235 cm3 concentrated acetic acid.

2.5. Assays of enzyme activities


For the determination of GOD activity, the decrease in glucose concentration was measured with the previously described o-toluidine method [24]. About 10 cm3 of 100 mg cm1 -d-glucose in 0.05 M acetate buffer (pH 5.1) used as substrate and reaction was started by adding free (3.47 g enzyme preparation) or immobilized GOD (450 mg solid). In order to provide a proper contact of substrate with immobilized GOD, reaction mixture was gently agitated on shaker. The determination of catalase activity was based on the change of the absorbance at 415 nm of the complex of H2 O2 with titanium sulfate [18]. Enzyme solution (25 L) or immobilized enzyme (450 mg) was added to 10 cm3 H2 O2 solution (8 mol cm3 in 0.1 M citric-phosphate buffer, pH 4.5), the reaction mixture was shaken for 2 min (soluble) or 30 min (immobilized) and 1 cm3 sample was added to 1 cm3 titanium(IV) sulfate reagent (SIGMA Co.). The absorbance was measured at 415 nm.

2.6. Heat stability tests


Filtered egg white liquor was obtained from SATO Egg Processing and Trade Ltd. (Budapest, Hungary). Glucose oxidase isolated from A. niger containing catalase (NOVOZYM 771) was purchased from Novo Nordisk (Bagsvaerd, Denmark). Total protein content of NOVOZYM 771 was 11.5 mg g1 , its GOD activity was 1.34 units mg1 . One unit of GOD activity was dened as the amount of enzyme that catalyzes the oxidation of 1 mol -d glucose per minute at pH 5.1 and 3 5 C [4]. The catalase activity of the preparation was 1.44 units mg1 . One unit of enzyme activity was assumed to be the amount of enzyme decomposing 1 mol H2 O2 per minute at 25 C and pH 4.5 [18]. Amberlite UP 900 anion exchange resin with macroporous styrenedivinylbenzene matrix purchased from Rohm and Haas (Philadelphia, USA) was applied as support for the immobilization with a particle size of 560700 m, pore size of 4075 nm, and a specic surface area of 2530 m2 g1 solid. Glutaraldehyde (25% aqueous solution) was bought from REANAL Factory of Laboratory Chemicals (Budapest, Hungary). All other chemicals were reagent grade commercial products. The experiments were performed at different incubation temperatures with 0.5 cm3 soluble enzyme or 0.45 g immobilized enzyme in 10 cm3 0.05 M acetate buffer (pH 5.1), respectively. After appropriate times of incubation, the samples were rapidly cooled in an ice-bath, and the residual activities were assayed by the standard methods.

2.7. Measurement of oxygen transfer through aeration membrane


Oxygen transfer through the silicone tube membrane was measured by a standard procedure [25]. A CHEMAP CBC 5 type bioreactor (working volume 2 dm3 ) was equipped with a stirrer of fork shape and with a silicon membrane tube screwed into two concentric cylinders made from stainless steel rods. The length, the diameter and the internal pressure of the tube were the variables. The reactor lled with distilled water or egg white was set to 25 C and 15 rpm then deoxygenated by bubbling with nitrogen. Afterwards constant oxygen (pure O2 ) pressure was adjusted in the silicone tube. The oxygen saturation % values of the liquid phase were recorded by oxygen electrode (Mettler-Toledo 19/150) in each minute. The time constant of the electrode was taken into consideration [26]. From the slope of the suitable section of the oxygen saturation % versus time curve, the oxygen transfer rate as well as the overall mass transfer coefcient of the membrane could be calculated.

2.2. Immobilization procedure


For economical reasons, we have chosen an ion exchange resin support for immobilization of the catalase containing commercial GOD preparation. The binding was stabilized with cross-linking using by glutaraldehyde [1922]. As the rst step of the two-stage immobilization method Amberlite UP 900 type anion exchange resin was shaken (120 rpm) in NOVOZYM 771 solution at pH 5.1 and 25 C for 2 h. Then the suspension was ltered and washed with deionized water. For the cross-linking the ion exchange resinprotein complex were shaken in glutaraldehyde/0.05 M acetate solution (pH 5.1). Then the suspension was ltered and washed aldehyde-free.

3. Results and discussion 3.1. Co-immobilization of GOD and catalase It was supposed that during the procedure not only GOD but catalase would be bound in a sufcient concentration, too. It is more advantageous to co-immobilize the two enzymes (GOD and catalase) than to x them separately on different support particles because the optimization of a mixed-bed operation is more complicated than that of the co-immobilized biocatalyst bed. The experiments for adsorbing NOVOZYM 771 onto AMBELITE UP 900 resin showed that the maximum loading was 33.0 mg protein g1 resin in the examined range (Table 1). The optimum protein loading (27.8 mg protein g1 resin) was found if 157 mg protein, i.e. 13.7 g NOVOZYM 771 was added to 1 g resin because the quantity of the bound protein tends to a limiting value over this protein/resin ratio.

2.3. Measurement of protein


Protein determination was performed according to Lowry et al. [23]. The amount of immobilized protein was calculated as the difference between the amount of protein introduced into the coupling reaction mixture and the amount of protein present in the ltrate and washing solutions after immobilization.

2.4. Determination of glucose concentrations


The glucose concentration was measured by photometric method using otoluidine (SIGMA Co.) as reagent [24]. About 4.5 cm3 o-toluidine reagent was

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Table 1 Effect of ratio of contacted protein on protein loading and on xed GOD/catalase using an anion exchange resin matrixa NOVOZYM 771 contacted (g g1 ) 6.06 8.44 13.7 22.6 32.6 Protein contacted (mg g1 ) 69.7 97.1 157 260 375 Protein bound (mg g1 ) 15.1 21.0 27.8 31.5 33.0 Bound protein yield (%) 21.7 21.7 17.7 12.1 8.80

a In the experiments 0.45 g aliquots of resin were shaken (120 rpm) in 2.7314.7 g enzyme solution for 2 h at 25 C.

The adsorbed enzymes were treated by glutaraldehyde to stabilize the xation by cross-linking. The effect of cross-linking parameters on the activity of immobilized GOD was also studied. The results are presented in Table 2. The highest catalytic activity of GOD (10.2 units g1 solid) was achieved using 0.25% glutaraldehyde for 15 min. Under these circumstances, the catalase activity of the preparation was 2.51 units g1 solid. The scale-up up to 400 g solid did not inuence the activities of immobilized GOD (9.89 units g1 solid) and catalase (2.33 units g1 solid). 3.2. Stabilities of immobilized enzymes The rates of heat inactivation of soluble and immobilized enzymes were investigated in the range of 4060 C. The time curves of the heat inactivation are presented in Fig. 1 for GOD and Fig. 2 for catalase. The graphs demonstrate that the heat stability of immobilized GOD improved to a great extent in comparison with that of free enzyme. The increase of stability could be observed especially at 60 C. The results are similar in case of catalase, too. The immobilized enzyme preparations were stored in pH 5.1 acetate buffer at 4 C. During a 2-year period any decrease in the activities was not experienced. 3.3. Optimum operational conditions for immobilized enzyme Determination of the optimum conditions for the immobilized enzyme was carried out by measuring the GOD activity.
Table 2 Dependence of activity of GOD adsorbed on anion exchange resin on the conditions of cross-linkinga Concentration of glutaraldehyde (%) Specic activity (U g1 dry solid) Cross-linking time (min) 15 0.032 0.26 0.52 1.04
a

Fig. 1. Heat inactivation of GOD activity in the case of soluble (A) and immobilized (B) preparation. Experiments with immobilized enzyme were carried out in 0.05 M acetate buffer (pH 5.1) at 40, 50, 55 and 60 C.

About 450 mg aliquots of air-dried immobilized enzyme were added to 10 cm3 of glucose (4 mg cm3 ) in 0.05 M acetate buffer of different pHs. The suspensions were shaken for 1 h at various temperatures. As it can be noted in Fig. 3 the optimum operational conditions for the immobilized enzyme are pH 5.1 and 25 C.

30 6.23 6.06 3.69

60 4.83 5.20 3.05 Fig. 2. Heat inactivation of catalase activity in the case of soluble (A) and immobilized (B) preparation. Experiments with immobilized enzyme were carried out in 0.1 M citric-phosphate buffer (pH 4.5) at 40, 50 and 60 C.

9.41 10.6 7.53 6.51

About 7080 mg aliquots of ion exchange resinprotein complex were shaken (120 rpm) in 1 cm3 glutaraldehyde/0.05 M acetate buffer (pH 5.1) at 25 C.

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3.5. Characterization of bubble-free aeration system Calculating the oxygen transfer rate from the slope of the oxygen saturation % versus time curve the applied relationships are as follows [25]: dPl Q =H = Ka(Pt Pl ) V dt (1)

Fig. 3. Optimum operational conditions for immobilized enzyme. In the experiments 450 mg aliquots of air-dried immobilized enzyme were added to 10 cm3 of 4 mg cm3 glucose in 0.05 M acetate buffer of different pHs. The suspensions were shaken (200 rpm) for 1 h at various temperatures.

Fig. 4. Elimination of glucose in egg white using batch system. In the experiments 10 cm3 of egg white (glucose content, 4.01 mg cm3 ) was shaken (200 rpm) with 450 mg air-dried immobilized enzyme at 25 C for 3 h.

3.4. Glucose elimination in a batch system The practical applicability of immobilized enzyme was tested in batch experiments. About 10 cm3 of egg white (glucose content: 4.01 g dm3 ) was shaken (200 rpm) with 450 mg immobilized enzyme at 25 C for 3 h. During this time, at least 98% of the initial glucose content was eliminated. Then the immobilized enzyme was ltered off and 10 cm3 of fresh egg white was added to it. The results of 11 cycles using the same enzyme aliquot are presented in Fig. 4. As it can be seen no considerable decrease in the productivity of immobilized GOD was experienced in the course of the operation.

where Q is the oxygen transfer rate (mg s1 ), V the reactor volume (cm3 ), H the Henrys constant for oxygen solubility in water at 25 C (mg cm3 Pa1 ), Pl the oxygen partial pressure in the bulk liquid (Pa), t the time (s), K the overall mass transfer coefcient (cm s1 ), a the outer surface area of the tubing per reactor volume (cm2 cm3 ) and Pt is the oxygen pressure inside the tube (Pa). After integration of Eq. (1) oxygen transfer rate can be calculated from the slope of the plot of ln [(Pt Pl,t=t )/(Pt Pl,t=0 )] versus time. The results of oxygen saturation measurements in silicone membrane equipped model system and the calculated transfer data are summarized in Table 3. The data show that the rate of oxygen saturation is affected rst of all by the oxygen pressure in the tube. The oxygen saturation in distilled water is about four times quicker than in egg white liquor. The presence of biocatalyst particles does not inuence the oxygen transfer. The transport does not depend signicantly on the thickness of the tube wall, probably because of the quality differences of the tubes of different diameter. Consequently, for set-up of the reactor to be applied to continuous glucose elimination the silicon tube of 4 mm internal diameter was selected, because of its higher mechanical strength than that of the thinner ones. If the 45 g dm3 glucose content of the egg white is to be eliminated in 2 h residence time, the oxygen demand of glucose oxidation is 0.124 mg O2 s1 dm3 . Since 800 cm2 dm3 specic surface area can be built into the reactor, it can be concluded from the data of Table 3 that sufciently high oxygen transfer can be achieved in the tube pressure range of 0.121.6 bar. 3.6. Three-stage continuous reactor for glucose elimination At the selection of suitable bioreactor conguration, the special characteristics of egg white have to be taken into

Table 3 Effect of silicon tube characteristics and oxygen pressure in the tube on the specic O2 saturation rate of liquid phase and the oxygen transfer rate Type of liquid phase Internal/external dia. of silicone tube (mm) 2.0/2.6 3.0/3.8 3.0/3.8 4.0/5.0 4.0/5.0 4.0/5.0 4.0/5.0 4.0/5.0 4.0/5.0 4.0/5.0 4.0/5.0 Tube pressure (bar) a/V = tube outer surface area/reactor volume (cm2 dm3 ) 180 160 160 210 210 790 790 790 790 81 81 O2 saturation rate related to a/V (103 O2 saturation % min1 cm2 dm3 ) 20.3 40.6 13.4 36.9 49.2 42.2 11.5 11.3 15.3 18.3 18.6 Oxygen transfer rate related to a/V (104 mg O2 s1 cm2 dm3 ) 1.79 3.58 1.18 3.25 4.34 3.72 1.01 1.00 1.01 1.35 1.61

Dist. water Dist. water Egg white Dist. water Dist. water Dist. water Egg white Egg white + catalyst Egg white Egg white Egg white + catalyst

0.6 0.6 0.6 0.6 0.8 0.8 0.8 0.8 1.2 1.6 1.6

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Table 4 Glucose elimination in a three-stage pilot-scale uidized-bed reactor operating in continuous regimea Oxygen pressure (bar) 0.8 0.8 1.2 1.5
a

Residence time (h) 2 4 4 2

Glucose residue (g dm3 ) 2.31 0.73 <0.01 0.103

Rate of glucose elimination (g h1 ) 47.28 38.68 45.60 87.82

Productivity (g h1 dm3 ) 4.34 3.55 4.18 8.18

Biocatalyst packing: 10.9 dm3 (6.04 g solid); initial glucose concentration of egg white: 4.86 g dm3 ; speed of stirrer: 15 rpm; temperature: 25 C; time of continuous operation: 133 h.

consideration. In the literature, there are several effective reactor types for glucose oxidation, e.g. a bubble column reactor described by Bao et al. [27] but egg white is susceptible to foaming therefore a bubble-free oxygen supply is desirable. On the other hand, egg white is a viscous uid therefore the xedbed reactor congurations are not suitable for the continuous treatment. On basis of these considerations, a special three-stage uidized-bed bioreactor has been constructed (Fig. 5). The col-

umn was sectionalized to reduce the axial back-mixing. The pilot-scale reactor of 45 dm3 working volume was equipped with a silicon membrane spiral supplying oxygen. The length of tube of 5 mm external diameter was 212 m, its total surface area was 3.33 m2 . Maximum allowable pressure inside the oxygenation system was 1.8 bar. The biocatalyst packing (10.9 dm3 ) was uniformly distributed between the stages separated by special plates with Venturi-throats and valves [28]. The plates are not sensitive to blocking. The stages were mixed gently with 2-2 fork shape stirrers xed onto a common shaft to improve the oxygen transfer patterns. 3.7. Glucose oxidation in continuous regime For the continuous elimination of glucose, the three-stage pilot-scale uidized-bed bioreactor was operated at 25 C, with 15 rpm speed of stirrer. The initial glucose concentration was 4.86 mg cm3 . The results of several continuous desugaring experiments are summarized in Table 4. Though the best productivity was achieved using oxygen pressure of 1.5 bar and residence time of 2 h but the residual glucose concentrations were somewhat lower when the reactor operated with 1.2 bar oxygen pressure and 4 h residence time. Since egg white with glucose content of 0.01 mg cm3 can be regarded practically sugar-free probably about 33.5 h residence time is enough to achieve this value in case of 1.5 bar oxygen pressure, on the basis of the experimental results. 4. Conclusions New enzymatic way applying immobilized glucose oxidase has been developed to eliminate the glucose content of egg white. Using a commercial glucose oxidase preparation containing catalase (NOVOZYM 771) and Amberlite UP 900 anion exchange resin as support a combined immobilization method has been elaborated. The enzyme molecules adsorbed on the surface of support were xed by cross-linking with glutaraldehyde. It is a simple and effective method, which results in a cheap solid-phase biocatalyst from a soluble commercial GOD product. The activity as well as stability values of the immobilized enzyme are suitably high for the industrial egg processing. Since the supply of oxygen as co-substrate requires a bubblefree oxygen transfer in order to avoid the foaming of the egg white a special pilot-scale uidized-bed bioreactor has been constructed with large oxygenator membrane surface. In the

Fig. 5. Pilot-scale three-stage uidized-bed bioreactor equipped with bubblefree oxygenator, developed for continuous desugaring of egg white liquor. (1) Lower elements of the reactor stages; (2) upper elements of the reactor stages; (3) conical bottom; (4) cover; (5) motor; (6) liquor feed; (7) liquor take-off; (8) stubs for liquor recirculation; (9) stubs for catalyst feed end discharge; (10) liquor discharge stub; (11) manometers for oxygen pressure measurement; (12) pump for liquor recirculation; (13) pump for disinfectant feed; (14) stub for internal pressure regulation; (15) to liquor feed tank.

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three-stage reactor packed with immobilized GOD, the continuous glucose elimination experiments performed resulted in a practically glucose-free egg white suitable for the spray drying. The desugaring method makes possible to get high-quality egg white powder which does not contains extraneous enzyme residues. Acknowledgements The nancial support provided by the National Committee for Technological Development (OMFB, Hungary) in the frames of a R&D grant (contract no. 02345/2000) is gratefully acknowledged. References
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