Food Hydrocolloids Vol.5 no.6 pp.541-548, 1992

The effect of irradiation on the functional properties of spray-driedegg white protein

D.C.Clark, I.F.Kiss1, P.l.Wilde and D.R.Wilson

Food Colloids and Biopolymer Science Department, AFRC Institute of FoodResearch, Norwich Science Park, Colney, Norwich NR4 7UA, UK and 1CentralFood Research Institute, PO Box 393, H-1525 Budapest, Hungary

Abstract. The foaming properties of spray-dried egg white powder were examined as a function ofdosage with ionizing radiation. Foam stability was studied by a micro-conductimetric method. Amarked increase in foam stability was observed in all the irradiated samples examined (2-16 kGy)when compared to a non-irradiated control. The drainage rate and minimum thickness of air­suspended thin liquid films stabilised with egg white protein was measured interferometrically. Therate of film thinning decreased with increasing dose suggesting a dose related change in the bulkviscosity. There were minor changes in the secondary and tertiary structure of the irradiated proteindetermined by circular dichroism, which may account for the observed changes in functionality.

Introduction

Microbiological quality is an important characteristic of food and may beinfluenced by the quality of raw material used, good manufacturing practice andhygienic measures. A number of methods are known for the elimination ofharmful microorganisms and for reducing the number of spoilage microbespresent in food. One such method is ionising radiation, an effective method formicrobiological decontamination that has already become legally accepted inseveral countries (1). Irradiation improves the microbiological quality of foodsand ensures their safety.

Microbiological contamination can be a problem in egg products. In thepreparation of egg white powder the drying process reduces microbiologicalcontamination, but some organisms survive the heat treatment. The recognisedstandards for microbiological contamination of egg white are for a totalorganism count between m = 5 x 104/g and M = 5 x 106/g, where m and Mrefer to the lower and upper limits respectively (2). In addition the productshould be essentially free from salmonella.

Earlier studies reported that the viable cell count of deep frozen whole liquidegg could be reduced to acceptable levels with a 3 kGy irradiation dose (3).More recently we have shown that the total count of viable organisms present indried egg white powder was reduced by 1.5-2.0 orders of magnitude by anirradiation dose of 2.5 kGy. Pathogenic organisms (coliform, enterococcus,S.aureus, salmonella, etc.) could not be detected in samples that had receivedsuch an irradiation dose (4). After irradiation it was noted that the functionalproperties of egg white were improved (5). Investigation of irradiated egg whitehas shown that in aqueous solution irradiation induces considerable cross-linkingof selected proteins (6). It should be noted that egg white is composed of acomplex mixture of at least 12 proteins of which ovalbumin (54%), conalbumin(13%), ovomucoid (11%) and lysozyme (5.5%) are the most abundant.

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In this paper we present the results of experiments that characterize thechanges in the foaming properties of spray-dried egg white as a function ofradiation dose.

Materials

Spray-dried egg white powder was an industrial product of Hungary. Afterseparation fresh egg white was pasteurised for one minute at a temperature of67-68°C. Potassium sorbate (0.3%) and citric acid (0.5%) were added aspreservatives. The initial temperature during spray-drying was 220°C and thedischarge temperature was 80-85°C. The product was packed in heat-sealedpolyethylene sachets. The moisture content of the product was 5.87 ± 0.11%,the nitrogen content was 84.60 ± 0.75% and the water activity was0.20 ± 0.009%.

Surface chemically pure water (surface tension >72.8 mN/m at 20°e) obtainedby steam distillation of deionised water from potassium permanganate was usedthroughout this study. All other chemicals were of 'AnalaR' grade from BDHChemical Co. (Dagenham, UK) and used without further purification.

Methods

Egg white samples were irradiated using a self-shielded RH-gamma-30 labora­tory Co-60 radiation source with an activity of 0.5 PBq at a dose rate of 6.5 kGy/h. The apparatus comprised a central sample chamber surrounded by severaltubes containing the radiation source. The dose rate distribution within thesample chamber was determined using a ethanol-chlorobenzene dose meter.The spray-dried egg white powder was introduced into the apparatus in 5 gsealed polyethylene pouches and exposed to the source for the time necessary todeliver the required dose. The temperature during irradiation was 18-22°C.

Foam stability studies were performed at 20°C using a conductimetric methodas described previously (7,8) but with minor modifications. A 2 ml sample of theegg white protein solution (0.5 mg/ml w/v) in 10 mM sodium phosphate buffer,pH 7.0, was introduced into the jacketted foaming column by pipette. Thesolution was foamed to a height of 5 ern by sparging with presaturated 'whitespot' grade nitrogen at a constant flow rate through a single microscopic jet. Thegas flow was then immediately shut off and the decay in conductivity of the foambetween two platinum electrodes , mounted flush against the column walls, wasmeasured using a Philips digital conductivity meter (Type PW9527) interfaced toa BBC microcomputer.

The conductivity decay curves provide information relating to film drainageand stability properties of the foam. Since these processes are difficult to defineanalytically (9), we present the complete decay curves for a series of differentsolution conditions to allow comparison .

Air-suspended thin liquid films of the solutions under investigation wereformed in a ground glass annulus as described in earlier papers (9,10,11) . Thedrainage of the films was monitored visually by viewing through a NikonDiaphot inverted microscope. The final thickness of the films was measured

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Effect of irradiation on egg white protein

interferometrically as described in earlier work (10,11). In addition the timedependence of the appearance of maxima and minima in the intensity of lightreflected from the film was recorded and used to calculate the drainage rate ofinterlamellar liquid from the suspended thin films (12).

Circular dichroism measurements

A stock solution of egg white protein was prepared by dissolving the spray-driedsample in 10 mM sodium phosphate buffer, pH 7.0. Samples for circulardichroism (CD) were prepared to have final concentrations of 0.1 mg/ml (w/v)for far-UV and 1 mg/ml (w/v) for near-UV measurements.

CD spectra were measured using a Jasco J600 spectropolarimeter (Tokyo,Japan) and data were recorded on-line using an IBM PC. The spectra presentedare an average of two scans, recorded at 20 nrn/min, using a silica quartz cell. Aninstrument sensitivity of ±20 mdeg full scale was routinely used, along with a 4 stime constant (13). Far-UV spectra were recorded over a wavelength range of260-190 nm using 0.1 mm pathlength cells. The secondary structure content ofthe samples was estimated by the CONTIN method (14). The near-UV spectrawere recorded over a wavelength range of 350-250 nm using 10 mm pathlengthcells. Data were converted to units of delta epsilon using -a mean residue weightof 113. The data may be expressed as specific ellipticity by multiplying by 3300.Instrument calibration was regularly checked during the course of the workusing ammonium d-lO-camphorsulphonate (15).

Results

The conductivity of bulk solutions of phosphate buffer, control and irradiatedegg white was measured to ensure that irradiation did not markedly change thesmall ion content of the samples. The conductivity of the 10 mM sodiumphosphate buffer was 1.293 mS/cm. Phosphate buffer containing 0.5 mg/ml(w/v) of control or irradiated egg white had a slightly increased conductivity of-1.35 mS/cm, apparently independent of radiation dose. The influence ofirradiation on the conductivity properties of the egg white samples was measuredmore accurately in solutions made up in distilled water (conductivity 1 us/ern).Solutions of control egg white (0.5 mg/ml w/v) had a conductivity of 60.38 fLS/em. The conductivity ofthe irradiated samples (2-16 kGy) was 56.32 ± 1.83 fLS/em. Therefore, irradiation had a very minor effect on the conductivity of eggwhite solutions. Thus differences in the conductivity of foams of egg whiteprotein in 10 mM phosphate buffer could be confidently attributed to irradiationinduced changes in functional properties.

The stability of foams formed from solutions of spray-dried egg white powderwas examined as a function of dosage with ionizing radiation by a micro­conductimetric method. The decay curves obtained from control and irradiatedegg white protein are plotted in Figure 1. The conductivity at time zero when thegas flow was stopped showed considerable dependence on the irradiation dose.The magnitude of the conductivity signal at time zero is related to thefoamability of the samples (8) and this property increased with the radiation

543

D.C.Clark et al.

8' 4.0~(I)

on

'8~

3.0>-t:~

t3t>A 2.0Z

81.0

0 100 200 300

TIME (SECONDS)

Fig. 1. Foam stability measured by conductivity as a function of time for egg white samples that hadreceived different radiation doses. (e) 0 kGy control sample; (0),2 kGy; (+), 4 kGy; (_),8 kGyand (0), 16 kGy. Each curve was composed from 100 data points. The symbols are to assist in curveidentification.

dose. The initial drop in the level of conductivity (0-20 seconds) was due torapid drainage of the wet foam. The amplitude of conductivity at extended times(e.g. 200-300 s) contains information about the late stages of drainage and thestability of the foam. It is evident that the amplitude of conductivity from foamsof these samples at extended times shows a marked dependence on radiationdose. Thus a marked increase in foamability and foam stability was observed inall the irradiated samples examined (2-16 kGy) when compared with the non­irradiated control sample of egg white protein.

A more detailed study of foam drainage was performed using air-suspendedthin liquid films formed from solutions of control or irradiated samples in aspecially constructed apparatus (11). Visual observations of drainage using amicroscope confirmed that the films drained in a manner characteristic ofprotein stabilization (10). The minimum thickness and rate of drainage of thefilms was measured interferometrically. A comparison of the results obtainedfrom the control sample and the 16 kGy irradiated sample are shown in Figure 2.The rate of film thinning was found to decrease with increased radiation dosewith the difference most marked at the highest dose rate examined. In general,samples that were exposed to lower doses showed drainage behaviourintermediate between these extremes. However, the sample exposed to only2 kGy irradiation drained in a manner indistinguishable from the controlsample. The samples showed only minor differences in the equilibriumthicknesses of their films. The films shown in Figure 2 drained to thicknesses of29.1 nm for the control and 32.1 nm for the irradiated (16 kGy) sample. Theseare not significantly different. This would suggest that irradiation reduces thedrainage rate by increasing the viscosity of the interlamellar phase rather thansignificantly increasing the thickness of the adsorbed protein layer.

544

Effect of irradiation on egg white protein

1400

1200-'"""'Sc 1000'-'Cf.lCf.llI)

.Ei 800o....~.....S 600-....~

400

200

00 10 20 30 40 50 60

Time (minutes)

Fig. 2. A plot of the drainage rate of air-suspended thin liquid films of (.) egg white protein controlsample and (e) 16 kGy irradiated egg white.

The structure of the egg protein samples in solution was examinedspectrophotometrically to determine whether irradiation resulted in a con­formational change. Fluorescence spectroscopy was used to examine the tertiarystructure of the proteins since the fluorescence of the intrinsic tyrosine andtryptophan residues of proteins is environmentally sensitive. Only the 16 kGysample showed a detectable change in its emission spectrum. Fluorescencequenching studies using potassium iodide were attempted, but abandonedbecause only small changes were detected. Near and far-UV CD spectroscopywas used to examine the tertiary and secondary structure of the protein samples,respectively. The results are shown in Figures 3 and 4. Some changes in the CDproperties of the samples were detected. Analysis of the far-UV CD spectra bythe CaNTIN method revealed that irradiation caused a small reduction in the (X­

helix content of the protein solution. The results are summarized in Table I.Some minor changes were also observed in the near-UV CD spectra. However itis not possible to analyse these spectra in a quantitative manner.

Discussion

Measurements of the conductivity of bulk solutions of control and irradiated eggwhite in phosphate buffer showed very minor changes in ionic strength due toirradiation. This validated the use of conductivity in the assessment of foamingbehaviour of the samples.

545

D.C.Clark et al,

5.0

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- 2.5Curve ID

o 3048X 3047

190 200 210 220 230 240 250 260

Wavelength (nm)

Fig. 3. Far-UV circular dichroism spectra of a control sample of egg white ( x) and a sample that hadreceived a 16 kGy radiat ion dose (<:9). Each spectrum contained 351 data points. The symbols are toassist in curve identification.

O.O -t------------.".,.-r"':J?:r"d1"-'~

-0.25

~ - 0.5'm0-w

.2 -0.75a;o

-1.0

- 1.25 Curve IDo 3050x 3049

250 275 300Wavelength (nm)

325 350

Fig. 4. Near-UV circula r dichroism spectra of a control sample of egg white (x ) and a sample thathad received a 16 kGy radi ation dose (<!J) . Each spectrum contained 501 data poi nts. The symbolsare to assist in cur ve ide ntification.

546

Effect of irradiation on egg white protein

Table I. Secondary structure content of egg white protein samples

Sample

ControlIrradiated (16 kGy)

IT-helix(%)

38.8 (1.0)35.1 (0.8)

l3-sheet(%)

26.4 (2.0)27.2 (1.6)

Random coil(%)

35.8 (1.7)37.7 (1.4)

The numbers shown in parentheses are standard errors.

Major differences were observed in the foaming properties of irradiated eggwhite compared with a non-irradiated control sample. Measurements of thefoaming properties of the samples were made using a conductimetric technique.This method has proved sufficiently sensitive to detect changes in the foamingproperties of protein solutions resulting from the presence of competingsurfactants (7,9), a naturally occurring cross-linking agent found in beer (12), andminor differences in primary amino acid sequence (16). The magnitude of theconductivity signal is related to the amount of liquid trapped in the foam. Itfollows that if liquid drainage from the foam is slow a high level of conductivitypersists for longer. Also, if the bubble sizes remain small and their sizedistribution remains narrow (i.e. bubble rupture is minimized) the conductivitysignal will remain high.

The foam conductivity curves show two distinct phases of decay. Initially therewas a rapid decrease in conductivity over the first 20 s after the gas flow wasterminated. This was due to rapid drainage of liquid from the wet foam. This wasfollowed by a second more gradual drop in conductivity. This derived fromlonger term drainage and the magnitude of the signal was in part related to thestability of the foam. The results reported here show that even low doseirradiation resulted in a measurable and significant improvement in thefunctional properties of egg white protein.

More detailed information concerning the drainage properties of foams wasobtained by examining isolated air-suspended thin liquid films (i.e. model foamlamellae). There were significant differences in the drainage behaviour ofcontrol and irradiated samples of egg white protein determined by interfero­metry. Although initially the films were of equivalent thickness, the rate ofdrainage of the irradiated samples was slower than the control (with theexception of the 2 kGy sample) and was proportional to the irradiation dose.

Radiation-induced chemical changes in egg white have been reported (6).However, this study was confined to the identification of radiation-inducedcovalent cross-linking of proteins as determined by SDS-PAGE. Major changeswere observed in samples that were irradiated in aqueous solution. However,egg white samples that were irradiated in a freeze-dried powdered form werelargely unaffected. These observations suggest that aqueous solvent wasrequired for significant covalent cross-linking to occur. More recent preliminarymeasurements with spray-dried egg white samples support this hypothesis sinceno evidence for radiation-induced covalent cross-linking was detected. How­ever, there was some evidence of thermally induced aggregation of the protein

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D.C.Clark et al.

sample during spray-drying. The resulting minor changes in the molecularweight distribution of the spray-dried protein are currently being investigated.These changes may induce some alteration in the functional properties of thespray-dried sample compared to that of the freeze-dried protein but will notinfluence the results here since all samples, including the control, were spray­dried.

An alternative explanation for improved functionality was that irradiation­induced improvements in the foaming properties of egg white derive fromstructural changes in the protein sample. CD measurements of proteinconformation were performed to examine this hypothesis. Some small differ­ences were observed but it is difficult to interpret these structural changes insuch a complex multi-protein system. The far-UV CD spectra showed anapparent conversion of a small quantity of a-helix into primarily randomstructure. Although small, this change could be significant since it is widelyrecognised that denatured or disordered proteins do have enhanced functionalproperties (17). The near-UV CD measurements would support the concept of aradiation-induced decrease in protein structure as the spectrum of the irradiatedsample appeared to be weaker in the 295 and 280 nm regions. This is consistentwith the transfer of aromatic amino acid residues into a less hydrophobicenvironment i.e. partial denaturation.

References

1. Anon. (1991) Regulations in the FieldofFood Irradiation. IAEA-TECDOC-585. lAEA, Vienna,February 1991.

2. UNEC Standard No. 63: Standard for certain hens egg-products for use in industry. UN, 1987.3. Kiss,!., Beczner,J., Kovacs,E. and Kovacs,S. (1985) Final report to IAEA, Res. Agric. No.

3033/CF, time period covered 1 Dec. 1981-1 September 1985, pp. 15.4. Kiss.lF,, Beczner,J. and Kovacs.S, (1988) Radioisotope Application and Radiation Processing in

Industry, 19-23 September, Leipzig, DDR, p. 21.5. Kiss.L, Halmagyi.A. and Kiraly.A. (1987) 18th Annual Meeting of ESNA, 31 August-4

September 1987. Stara Zagora, Bulgaria, Abstracts, p. 105.6. Hajos.G., Kiss.I. and Halasz.A, (1990) Radial. Phys. Chem., 36, 639-643.7. Coke,M., Wilde,P.l., Russell,E.l. and Clark,D.C. (1990) J. Colloid Interface Sci., 138,489­

504.8 Wright,D.l. and Hernmant.J. (1987) J. Sci. Food Agric., 41, 361.9. Clark,D.C., Wilde,P.l. and Wilson,D.R. (1991) Colloids and Surfaces, 59, 209-223.

10. Clark,D.C., Coke,M., Mackie,A.R., Pinder,A.C. and Wilson,D.W. (1990) J. Colloid InterfaceSci., 138, 207-219.

11. Clark,D.C., Dann,R., Mackie,A.R., Mingins.J., Pinder,A.C., Purdy,P.W., Russell,E.l.,Smith,L.l. and Wilson,D.R. (1990) J. Colloid Interface Sci., 138, 195-206.

12. Clark,D.C., Wilde,P.l. and Wilson,D.R. (1991) J. Institute Brewing, 97, 169-172.13. Clark,D.C. and Smith,L.l. (1989) J. Agric. Food Chem., 37, 627-633.14. Provencher,S.W. and Glockner,J. (1981) Biochemistry, 20, 33-37.15. Takakuwa,T., Konno,T. and Meguro,H. (1985) Anal. Sci., 1,215-218.16. Bacon,l.R., Hemmant,l.W., Lambert,N., Moore,R. and Wright,D.l. (1988) Food Hydrocoll.,

2,225.17. Kato,A., Fujimoto,K., Matsudomi,N. and Kobayashi,K. (1986) Agric. Bioi. Chem., 50, 417­

420.

Received on September 17, 1991; accepted on November 22, 1991

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