Effect of Electron Beam Irradiation on Physical, Physicochemical, and ...

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Jun 9, 1997 - (Key words: electron beam irradiation, egg yolk, physicochemical characteristics, ... Salmonella, have increased interest in food irradiation,.
Effect of Electron Beam Irradiation on Physical, Physicochemical, and Functional Properties of Liquid Egg Yolk During Frozen Storage S. HUANG, T. J. HERALD,*,1 D. D. MUELLER† *Department of Foods and Nutrition, †Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506 ABSTRACT Raw yolk of 1-d-old eggs was either subjected to linear electron beam irradiation at ∼2.5 kGy dosage or not processed. Both irradiated and nonprocessed egg yolk samples were stored at –15 C after irradiation. Testing was conducted on 0, 1, 7, 15, 30, and 60 d of storage. Development of storage modulus (G′) was delayed in irradiated samples after 7 d, which suggests that less structure was developed in irradiated egg yolk than in nonprocessed egg yolk during storage. Irradiated samples retained more soluble protein within the first 7 d and showed slightly improved emulsion capacity over that from nonprocessed samples. However, irradiated egg yolk was less bright than nonprocessed samples. No differences were observed in

SDS-PAGE patterns of soluble proteins and delipidized low density lipoprotein (LDL). The LDL isolated from irradiated liquid egg yolk showed no difference in Nterminal amino acids compared to that of nonprocessed egg yolk, indicating no detectable cleavage of LDL. However, the denaturation temperature of irradiated samples at Day 0 shifted about 1 C lower than that of the nonprocessed sample. Results indicated that electron beam irradiation did not cause significant physical, chemical or functional changes of egg yolk, or cleavage of egg yolk protein. Therefore, electron beam irradiation could serve as a preservation method for liquid egg yolk.

(Key words: electron beam irradiation, egg yolk, physicochemical characteristics, functional properties) 1997 Poultry Science 76:1607–1615

INTRODUCTION The increasing number of foodborne bacteria and the worldwide outbreaks of bacteria, particularly of Salmonella, have increased interest in food irradiation, which is a promising technique for the elimination of these bacteria. Irradiation of egg products has been used experimentally as an alternative to heat pasteurization to eliminate Salmonella, which is a naturally occurring pathogen in eggs and a cause of infection. Most research has tried to determine the proper radiation dosage to pasteurize egg products and the effects of irradiation on functional properties of egg products (Brooks et al., 1959; Katusin-Razem et al., 1989; Ma et al., 1990; Matic et al., 1990; Clark et al., 1992; Narvaiz et al., 1992). Extensive research showed that irradiating egg products to a dosage of about 2 to 3 kGy was adequate to inactivate Salmonella without significant changes in sensory properties (Katusin-Razem et al., 1989; Ma et al., 1990; Matic et al., 1990; Narvaiz et al., 1992). The investigation of radiation-induced chemical changes could provide important information on the potential risk factors of radiation for food safety.

Received for publication November 13, 1996. Accepted for publication June 9, 1997. 1To whom correspondence should be addressed.

Katusin-Razem et al., (1992) studied g-radiation-induced buildup of hydroperoxides and destruction of carotenoids as functions of radiation dose. Lipid hydroperoxide was proposed to be the indicator of irradiated egg powder based on a linear relationship between the formation of hydroperoxides and radiation dose below 4 kGy, a sufficient dose to inactivate Salmonella in egg powder (Katusin-Razem et al., 1990). The radiationinduced loss of amino acids was selective and amino acids in egg yolk were less sensitive to g-irradiation than those in egg white (Katusin-Razem et al., 1989). However, another study indicated that g-radiationinduced change in the amino acid composition of frozen liquid egg was not significant (Ma et al., 1993). In most experiments, no significant change in amino acid composition was found when radiation dose was below 50 kGy (Diehl, 1995). g-Radiation-induced aggregation was found in 1% of whole egg white protein at a radiation dose as low as 1.5 kGy (Hajos et al., 1990), and a small amount of conversion of a a-helix to random coil was reported in spray-dried whole egg white protein at a dosage of 16 kGy (Clark et al., 1992); however, studies on electron beam irradiation effects on egg yolk are still limited. The electron beam accelerator is a relatively new, flexible, and more effective source for food irradiation than g-rays. The electron beam is easy to adapt to

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different radiation process requirements, such as operating at different beam energy levels. No radioactivity is present when the accelerator is off, and, therefore, no radioactive waste accumulates, whereas usually 16 to 21 yr are required to dispose of Co-60. The application of electron beam irradiation is limited to thin foods because of its low penetration (about 3 cm at beam power of 8.1 kW). However, studies indicated that electron beam irradiation is more effective in decontamination or disinfestation than g-radiation (Blank and Corrigan, 1995). Previous work by Wong et al. (1996) showed that electron beam irradiation at a dosage of ∼2.5 kGy was effective in controlling Salmonella in egg white without significantly decreasing its functionality. Food irradiation offers low operation cost and is a safer alternate not only to heat pasteurization but also to other preservation methods such as chemical treatment. The objectives of this study were to determine 1) the effects of electron beam irradiation on the functional properties of liquid egg yolk during frozen storage, and 2) whether electron beam irradiation caused any chemical change to egg yolk protein.

MATERIALS AND METHODS

Egg Yolk Preparation One-day-old White Leghorn eggs were collected from the University Poultry Farm2 and either irradiated or not processed. Egg yolk was separated from egg white using a household hand separator. The egg yolk was rolled gently on soft facial tissue to remove adhering albumen. The vitelline membrane was ruptured, and the yolk liquid was collected, mixed well by hand, bagged, and sealed in Ziploc3 snack bags. Each snack bag containing about 120 g liquid egg yolk was then put inside a pint-size Ziploc freezer bag so that the total thickness was about 3 cm.

Irradiation Treatment One-day-old shell eggs were transported by a van at ambient temperature to the linear accelerator facility4 at Iowa State University. Egg yolk was prepared as above before it was irradiated. A linear electron beam accelerator with an energy level of 10 Mev and beam power of 8.1 kW was used to irradiate liquid egg yolk at a dosage ranging from 2.3 to 3.0 kGy. The alanine dosimetry system5 was used for absorbed dose measurement. The dosimeters

2Kansas State University Poultry Farm, Manhattan, KS 66506. 3Ziploc freezer bags, Dowbrands, Indianapolis, IN 46268. 4Iowa State University, Ames, IA 50011. 5Bruker Instruments, Inc., Billerica, MA 01821. 6Kool Mate 36 thermoelectric cooler and warmer, Igloo Product

Corp., Houston, TX 77052. 7Hunter Associates Laboratory, Inc., Reston, VA 22090. 8Bohlin Rheologic, Inc., Cranberry, NJ 08512. 9Model Series 874, John Oster Mfg. Co., Milwaukee, WI 53201. 10Beckman Instruments, Inc., Palo Alto, CA 94304.

were calibrated according to standards determined by the National Institute of Standards and Technology. Dosimeters were placed in the center of the top and bottom of each bag. After irradiation, the samples were transported back in an electric cooler6 at 4 C, and then stored at –15 C within 24 h. Tests were conducted on 0, 1, 7, 15, 30, and 60 d of storage. Three bags were chosen randomly on each day and thawed for about 4 h at room temperature prior to testing.

Color Measurement Color was determined using a Hunter Ultrascan Sphere spectrocolorimeter.7 About 20 g of egg yolk sample was put into a testing cup and scanned at three different locations. Values of lightness (L), redness (a), and yellowness (b) were determined using illuminant C and a 10° viewing angle. Hue angle was calculated by the formula tan–1 (b/a) and saturation index by the formula (a2 + b2)Ø.

Dynamic Properties Gelation of liquid egg yolk during frozen storage was studied. The storage modulus (G′) of egg yolk as a function of frequency (1 to 10 Hz) was measured by a Bohlin VOR Rheometer8 at 25 C. A Cone-Plate 5/30 geometry (cone angle 5°, 30 mm) and a 89-gcm torque bar were used. Strain was set at 2% based on a linear viscoelastic region obtained from a preliminary strain sweep test, and the measurement interval was set at 10 s.

Emulsion Capacity A procedure described by Harrison and Cunningham (1986) was used to determine emulsion capacity of egg yolk. Fifteen grams of egg yolk and 20 mL of vinegar (5% acetic acid) were mixed in an Osterizer blender9 for 10 s at the “mix” setting (output ∼167 W). Then 20 mL of soybean oil was added, and the mixture was blended for 20 s. Additional oil then was added dropwise from a 50-mL burette during continuous mixing until a sudden change from a viscous gel to liquid occurred, indicating a “broken” emulsion. The total amount of oil (including the first 20 mL oil) divided by the grams of egg yolk was taken as the emulsion capacity.

Protein Solubility and pH Soluble proteins were isolated using a method described by Morr et al. (1985). Approximately 5 g of egg yolk was weighed and mixed while 0.1 M NaCl was added to a total volume of 40 mL. The pH of the dispersion was monitored and adjusted to pH 7.0 with 0.1 N NaOH or 0.1 N HCl. The dispersion was mixed for 1 h, and the pH was maintained at 7.0. Then the dispersion was diluted to 50 mL with 0.1 M NaCl and centrifuged at 20,000 × g for 30 min by a Beckman L5-75B Ultracentrifuge.10 The superna-

ELECTRON BEAM IRRADIATION OF LIQUID EGG YOLK

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tant was filtered through Whatman No. 1 filter paper. The soluble protein content of the filtrate was determined by a colorimetric method using Coomassie Protein Assay Reagent 23200.11 Absorbance at 595 nm was measured by a UV-VIS spectrophotometer (UV 1201).12 The standard assay procedure provided with the reagent was used. The pH was measured with a digital pH meter.13

Gel Electrophoresis The soluble protein obtained from the protein solubility step was analyzed by SDS-PAGE without 2mercaptoethanol on a BIO-RAD Mini-PROTEIN II Dual Slab Cell system.14 The SDS was used to minimize the effect of lipids on the size of egg yolk protein, because egg yolk proteins usually are associated with lipids, and mercaptoethanol was not used so that the natural disulfide linkage of the proteins could be maintained as much as possible. A 7.5% separating gel and a 4.0% stacking gel were used. The gels were run at a constant 200 V and a 15 to 20 mA current with a Bio-Rad Model 1000/ 500 power supply.14 Protein in the gel was stained using Coomassie brilliant blue.

Differential Scanning Calorimetry Thermal characteristics of egg yolk were analyzed by a differential scanning calorimeter.15 The machine was calibrated using an indium standard. The peak or denaturation temperature (Td) and the heat of transition or enthalpy (DH) were computed by the DARES-DSC Data collection system.16 About 13 ± 2 mg liquid egg yolk was sealed in an aluminum sample pan (Perkin-Elmer kit No. 0219-0062).15 Samples were heated from 30 to 100 C at a rate of 10 C/min. Pieces of aluminum were sealed in a sample pan and served as the reference having approximately the same weight as the sample pan.

Isolation of Low Density Lipoprotein Low density lipoprotein (LDL) was isolated either from fresh unprocessed or fresh irradiated egg yolks by a flocculation procedure developed by Turner and Cook (1958). Egg yolk was diluted 1:1 (wt/wt) with 0.16 M NaCl, mixed for 30 min at 4 C, then centrifuged (Beckman L5-75) at 45,000 × g for 30 min. The plasma was collected, adjusted to 1 M NaCl, and then centrifuged at 106,000 × g for 16 h at 4 C. The floating gel (crude LDL) was dissolved

11PIERE, Rockford, IL 61105. 12Shimadzu Scientific Instruments, Inc. Columbia, MD 21045. 13Corning model 220, Corning Science Products, Corning, NY 14831. 14Bio-Rad Laboratory, Richmond, CA 94804. 15Perkin-Elmer DSC-4 system, Perkin-Elmer Corp., Norwalk, CT

06856. 16DARES-DSC Data Collection System (Version 2.04), Pico Technology Ltd., Hardwick, Cambridge, UK. 17Bio-Sil SEC 250 gel filtration HPLC column, 300 mm × 7.8 mm, BioRad, Hercules, CA 94572. 18Perkin-Elmer, Model 473A, Applied Biosystems Divisions, Foster City, CA 94402.

FIGURE 1. Purity of fresh isolated low density lipoprotein from egg yolk determined by native-PAGE. A) from nonprocessed egg yolk; B) from irradiated egg yolk.

in 1 M NaCl and centrifuged at 106,000 × g for another 16 h at 4 C. The LDL was collected and dialyzed against 0.05 M Tris-HCl buffer, pH 8.2 (containing 10–3 M ethylenediaminetetraacetic acid (EDTA) and 0.02% NaN3). The purity of LDL was tested by HPLC17 and 7.5% nativePAGE gel electrophoresis (Figure 1).

Determination of N-Terminal Amino Acids of LDL Lipids were removed from purified LDL by chloroform and methanol (1:1) extraction five times before the protein sequence was determined for apoLDL. N-Terminal amino acids were determined on a Protein Sequence System,18 which uses Edman degradation. During sequencing, the sample is retained on a solid support in a temperaturecontrolled glass reaction cartridge. At the end of each cycle, the terminal amino acid is removed as an anilinothiazolinone derivative (ATZ). The ATZ is converted automatically to a more stable phenylthiohydantoin-amino acid (PTH-AA) for the next cycle. The PTH-AA was separated by a dual-syringe, gradientprogrammable microbore system based on the unique relative affinities of different PTH-AA for the reversephase analytical HPLC column in the separation system. The data were collected and analyzed using the AppliedBiosystems A/D Converter and the Model 610A Data Analysis Module.

Statistical Analysis The experiment was analyzed as completely randomized design of two treatments (nonprocessed vs irradiated) and 6 d (Days 0, 1, 7, 15, 30, 60). Three replications of the treatments were used with two subsamples per replication. Mean comparisons were made when main effects were significant (P < 0.05). Data were analyzed using the General Linear models procedure (SAS Institute, 1985).

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RESULTS AND DISCUSSION

pH The pH of irradiated samples was significantly higher than that of nonprocessed sample within the first 15 d, then remained at 5.94 through d 60 in both treatments (Table 1). Irradiation caused differences in the pH of egg products were also observed in other research (Ma et al., 1990, 1993). In both treatments, pH increased significantly (P < 0.05) throughout storage. Increasing pH is a natural trend of food during extended storage. Irradiation can inactivate bacteria in food but does not affect enzymatic activity, which could lead to spoilage. Usually, the start of food spoilage is indicated by the increased pH.

Color Measurement Table 2 summarizes the color characteristics (lightness, redness, and yellowness) of egg yolk during 60 d of frozen storage. The hue of irradiated samples (80.2° at Day 0) was more yellow than that of nonprocessed samples (74.7° at Day 0) throughout 60 d of storage, which means that nonprocessed samples had a more reddish yellow color. The saturation index of irradiated samples was lower than that of nonprocessed samples throughout 60 d of storage, indicating that the irradiated samples were not as vivid as nonprocessed samples. This radiation-induced discoloration of the egg yolk was also observed in previous studies (Brooks et al., 1959; Ma et al., 1990). The color change could have been caused by the destruction of carotenoid in egg yolk, whose decay was proportional to the radiation dose (Katusin-Razem et al., 1992). But this bleached effect was much less than the natural variation of pigment concentration among different samples (Brooks et al., 1959). Storage effects in redness lightness, and hue angle also were observed. Hue angle increased from 74.7° (Day 0) to 78.0° (Day 60) for nonprocessed samples and from 80.2° to 83.7° for irradiated samples. This result indicated that the color of egg yolk became slightly less reddish yellow during storage, or the egg yolk lost pigment during

TABLE 1. The pH of irradiated and nonprocessed liquid egg yolk during 60 d storage at –15 C1 pH

Storage day

Nonprocessed

Irradiated

LSD2

0 SE 1 SE 7 SE 15 SE 30 SE 60 SE x P value

5.79b 0.01 5.74cd 0.02 5.71d 0.01 5.77bc 0.01 5.94a 0.03 5.94a 0.06 5.81 0.00013

5.86b 0.03 5.97a 0.03 5.86b 0.04 5.86b 0.03 5.94a 0.02 5.94a 0.03 5.90 0.00224

0.05 0.06 0.06 0.06 0.06 0.10 0.02

a–dMeans within a column with no common superscript differ significantly (P < 0.05). 1Values represent means of three replications. 2LSD is listed for comparing treatment difference. 3For nonprocessed samples, the pH among different day levels are significantly different (P value = 0.0001). 4For irradiated samples, the pH among different day levels are significantly different (P value = 0.0022).

storage. Freezing brightened the color of egg yolk as shown by the increase in lightness after 1 d of storage (Table 2). However, at Day 60, lightness of nonprocessed samples decreased, which could be a sign of spoilage starting in the sample. Lightness of irradiated samples did not decrease, suggesting that radiation delayed the beginning of spoilage in egg yolk.

Protein Solubility Irradiation caused some loss in soluble protein content of egg yolk as shown in Table 3 (5.63% for nonprocessed samples and 5.09% for irradiated samples at Day 0), but the rate of loss of protein solubility in irradiated samples

FIGURE 2. SDS-PAGE (without b-mercaptoethanol) gel electrophoretic patterns of soluble proteins from nonprocessed (N) and irradiated (I) egg yolk at Days (d) 0, 1, 7, 15, 30, and 60. Unlabeled lanes represent standard protein number.

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ELECTRON BEAM IRRADIATION OF LIQUID EGG YOLK TABLE 2. A summary of color characteristics of nonprocessed (N) and irradiated (I) egg yolk during 60 d storage at –15 C1 L2

Hue angle3

Saturation index4

Day

N

I

LSD5

N

I

LSD

N

I

LSD

0 SE 1 SE 7 SE 15 SE 30 SE 60 SE x P value

51.54b 0.14 55.31a 0.23 54.98a 0.33 54.99a 0.18 54.79a 0.31 51.79b 1.46 53.90 0.0001

50.15c 0.58 51.01c 1.30 55.18b 0.32 56.10b 0.45 56.12b 0.39 57.77a 0.49 54.39 0.0001

0.96

74.66d 0.21 76.42bc 0.55 76.92b 0.08 76.45bc 0.12 76.10c 0.25 78.03a 0.27 76.45 0.0001

80.20c 0.20 80.54c 0.72 82.91b 0.17 83.09ab 0.26 82.90b 0.23 83.67a 0.36 82.22 0.0001

0.46

30.68ab 0.15 30.82a 0.28 29.67bc 0.32 30.29ab 0.01 30.57ab 0.03 28.72c 1.42 30.12 0.0089

26.96ab 0.44 26.01a 0.79 26.59bc 0.51 26.79ab 0.44 27.06ab 0.43 27.50a 0.19 26.82 0.0558

0.74

2.11 0.74 0.78 0.8 2.46 0.45

1.45 0.31 0.46 0.55 0.73 0.23

1.35 0.97 0.70 0.69 2.29 0.38

a–dMeans

within a column with no common superscript differ significantly (P < 0.05). represent means of three replications. 2L, a, b were measured with illuminant C and the 10 degree observation mode. 3Hue angle = tan–1 (b/a). 4Saturation index = (a2 + b2)Ø. 5LSD is listed for comparing treatment difference. 1Values

was much slower than that in nonprocessed samples. The initial loss of soluble protein content could have been caused by radiation-induced changes resulting in less soluble aggregates in egg yolk. Soluble protein content was decreased significantly (P < 0.05) throughout storage in both treatments. Most soluble protein loss (significant) occurred within the first 7 d for nonprocessed samples and within 15 d for irradiated samples. After 15 d of frozen storage, a limited decrease in protein solubility was observed for either treatment. Overall, irradiated samples showed a delayed loss of soluble protein. The loss of soluble protein during frozen storage was mainly due to freezing-induced aggregation or gelation (Table 3). Low density lipid protein, a major protein in egg yolk, is thought to play the main role in freezing-induced gelation. Further, water in LDL is a critical factor in gelation. Ice-crystal formation dehydrates the protein and causes the aggregation of yolk lipoprotein (Powrie et al., 1963).

Gel Electrophoretic Pattern of Soluble Proteins The SDS-PAGE (without 2-mercaptoethanol) patterns of soluble proteins were similar from irradiated and nonprocessed egg yolk throughout the entire storage period (Figure 2). The results showed that electron beam irradiation did not cause a substantial change in the electrophoretic pattern of soluble proteins in liquid egg yolk. Irradiation usually causes degradation of fibrous proteins to smaller proteins, which probably involves C-N bonds in the backbone of polypeptide chain or splitting of

disulfide bonds, whereas globular proteins undergo aggregation (Diehl, 1995). g-Radiation at a pasteurizing dosage did not cause significant changes to egg proteins. But at higher doses, some changes, such as aggregation (Hajos et al., 1990) or breakdown (Ma et al., 1990, 1993), of egg white protein were suggested. Radiation-induced TABLE 3. Protein solubility of irradiated and nonprocessed liquid egg yolk during 60 d storage at –15 C1 Soluble protein content

Storage day

Nonprocessed

Irradiated

0 SE 1 SE 7 SE 15 SE 30 SE 60 SE x P value

5.63a 0.81 3.95b 1.02 2.73c 0.20 2.87c 0.37 2.73c 0.38 2.66c 0.27 3.43 0.00023

(%) 5.09a 0.41 4.47b 0.33 3.60c 0.27 2.85de 0.15 3.06cd 0.26 2.45e 0.47 3.57 0.00014

LSD2 1.46 1.72 0.54 0.63 0.74 0.87 0.33

a–eMeans within a column with no common superscript differ significantly (P < 0.05). 1Values represent means of three replications. 2LSD is listed for comparing treatment difference. 3For nonprocessed samples, the soluble protein content among different day levels are significantly different (P value = 0.0002). 4For irradiated samples, the soluble protein content among different day levels are significantly different (P value = 0.0001).

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HUANG ET AL. TABLE 4. Emulsion capacity of irradiated and nonprocessed liquid egg yolk during 60 d storage at –15 C1 Emulsion capacity

Storage day

Nonprocessed

0 SE 1 SE 7 SE 15 SE 30 SE 60 SE x P value

3.93 0.66 4.49 0.47 4.18 0.21 4.21 0.72 4.04 0.36 4.59 0.45 4.24 0.603

Irradiated (g oil/yolk) 4.89 0.69 5.49 0.39 4.47 0.38 4.54 0.38 4.52 0.19 4.89 0.10 4.79 0.074

LSD2 1.53 0.98 0.70 1.30 0.65 0.74 0.314

1Values

represent means of three replications. is listed for comparing treatment difference. 3For nonprocessed samples, the emulsion capacity among different day levels are not significantly different (P value = 0.60). 4For irradiated samples, the emulsion capacity among different day levels are not significantly different (P value = 0.07). 2LSD

FIGURE 3. A) Storage modulus (G′) of nonprocessed egg yolk during 60 d of storage at –15 C. B) Storage modulus (G′) of irradiated egg yolk during 60 d of storage at –15 C.

covalent cross-linking of egg white protein in aqueous solution was found using SDS-PAGE (Hajos et al., 1990).

Rheological Properties The storage modulus (G′) of the egg yolk as a function of frequency increased during frozen storage for both nonprocessed and irradiated samples (Figure 3). This might have been due to the freezing-induced aggregation and gelation of lipoprotein in egg yolk. The storage modulus (G′) of irradiated samples was a little higher than that of nonprocessed samples before storage. This result might suggest some coagulation of lipoproteins in irradiated egg yolk, thus causing the loss of soluble protein observed at d 0. However, G′ of irradiated egg yolk was delayed after Day 1, then almost stopped increasing after Day 7 (G′ ∼6 kPa). Even at Day 7, irradiated samples had substantially lower G′ than nonprocessed samples. This effect could explain the early stage of soluble protein loss. The G′ of nonprocessed samples reached about 80 kPa at Day 7, then climbed to approximately 1,100 kPa (10 Hz) at Day 15, which indicated that less structure or aggregation was developed during storage in irradiated samples. Soluble protein loss after Day 30 might have been due to the

enzyme activity within egg yolk. A big decrease in G′ of nonprocessed samples occurred from Day 30 (300 kPa, 1 Hz) to 60 (2.62 kPa, 1 Hz), which indicated some structure breakdown in the proteins and other polymers by enzyme or microbial activities. A corresponding decrease in irradiated samples was delayed and very small. The slope of logG′ vs logv (frequency) decreased during frozen storage for both treatments. This showed that G′ became less dependent on frequency, which is typical rubber-like behavior of a highly cross-linked polymer.

Differential Scanning Calorimetry Figure 4 represents the typical differential scanning calorimetry thermogram of nonprocessed and irradiated

FIGURE 4. Differential scanning calorimetry thermograms of nonprocessed and irradiated egg yolk at Day 0.

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egg yolk at Day 0. The peak temperature of irradiated samples shifted about 1 C lower than that of nonprocessed samples, which indicated an alteration in the structure of the egg yolk proteins by radiation as also suggested by the higher G′ and the loss of soluble protein at Day 0. gRadiation-induced aggregation of 1% whole egg white protein solution was found at a radiation dose as low as 1.5 kGy (Hajos et al., 1990). Proteins of egg yolk were the major contributors to heat transition during differential scanning calorimetry from 30 to 90 C. Although radiationinduced lipid oxidation has been reported, lipid structure transition doesn’t contribute to heat transition in this case (30 to 90 C). The differential scanning calorimetry thermograms of LDL from nonprocessed and irradiated samples did not exhibit any endotherms. The lack of endotherms might have been due to the random coil protein moieties of LDL.

Emulsion Capacity Table 4 shows the emulsion capacity of both treatments during storage. Irradiated samples had a significantly higher emulsion capacity than nonprocessed samples, and differences were significant within the first 7 d of frozen storage. Irradiation at pasteurizing dosage has slightly improved the functional properties of both egg white and egg yolk (Ma et al., 1990, 1993; Clark et al., 1992; Wong et al., 1996). The radiation-induced improvement in the functional properties of egg products may have been due to structural changes of the proteins. The far-UV circular dichroism (CD) spectra of Clark et al. (1992) showed conversion of a small quantity of egg white protein a-helix into primarily random structure at a dose of 16 kGy. This partial denaturation of proteins is a critical contributor to the functional properties of egg. Either foaming of egg white or emulsifying of egg yolk has been found to be highly related to partial protein denaturation and exposed hydrophobicity of proteins. Emulsion capacity increased slightly from Day 0 to 1, then declined through Day 60 for both treatments. Slight freezing-induced gelation could help to separate and entrap the oil droplets in the continuous phase of emulsion and provide some steric hindrance.

TABLE 5. N-terminal amino acids determination of low density lipoproteins (LDL) from nonprocessed (N) and irradiated (I) egg yolk1 Cycle no.

N-terminal amino acids of LDL N

I

1

Lys2 Ala Arg? Asn?

2

Ser2 Val? Glu?

3

Ile2 Phe? Thr? Gly?

Lys2 Arg?3 Ala His? Asn? Ser2 Glu? Ile? Phe? Ile2 Thr? Phe?

1All observed amino acids were recorded and listed in order of signal intensities for each cycle. 2Those were major signals in each cycle. 3Amino acids with question marks were not 100% certain because of weak signals.

N-Terminal Amino Acid of LDL Determination Breakdown of protein or cleavage of egg protein by radiation has been suggested (Katusin-Razem et al., 1989; Ma et al., 1990, 1993). Therefore, if electron beam irradiation causes cleavage of egg yolk protein, new Nterminal amino acids should appear in the spectra of the protein sequence. The N-terminal amino acids were similar. Lysine was found to be the predominant N-terminal amino acid of LDL in both treatments (Table 5). Small portions of arginine, alanine, and asparagine also appeared in the spectra. The first three amino acids of apoLDL were LysSer-Ile-. . .. Lysine was reported (Vadehra and Nath, 1973) as one of the N-terminal amino acids along with arginine, alanine, serine, and valine. The complete amino acid sequence of the major apoprotein from White Leghorn hen plasma very low density lipoproteins (VLDL) was reported by Jackson et al. (1977). Chan et al. (1980) reported the amino acid sequence of apoVLDL-II, the major apoprotein in avian VLDL (York LDL is also called VLDL because of its similarity to plasma VLDL) as follows:

Lys-Ser-Ile-Ile-Asp-Arg-Glu-Arg-Arg-Asp-Trp-Leu-Val-Ile-Pro-Asp-Ala-Ala-Ala-Ala-Tyr-Ile-Tyr-Glu-Ala-Val-. . . 1 5 10 15 20 25 They also found that the apoVLDL-II contains two identical polypeptide chains of 82 amino acids residues each, which are linked by a single disulfide bond at residue 76. The amino acid sequences of apoVLDL-II from plasma and apoLDL (apovitellenin I) from egg yolk (White Leghorn hen) are identical (Dugaiczyk et al., 1981).

The small portion of Ala found in N-terminal of LDL may have been contributed to the presence of yolk vitronectin in chick yolk LDL. The partial amino acid sequence of yolk vitronectin was reported (Nagano et al., 1992) as follows:

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Ala-Glu-Asp-Ser-?-Glu-Gly-Arg-?-Asp-Glu-Gly-Phe-Asn-Ala-Met-Lys-Lys-?-Gln-Gly-Asp. . . 1 5 10 15 20 Further analysis showed no fragments that possibly could match any of complete sequence of apoVLDL-II, indicating no detectable cleavage of the apoVLDL-II (apoLDL) polypeptide bond. The SDS-PAGE patterns of delipidized LDL of nonprocessed and irradiated samples (Figure 5) were nearly identical. N-terminal determination of LDL results also indicated no or little cleavage of LDL in irradiated egg yolk. Amino acids in egg yolk have been reported to be less sensitive to irradiation than those in egg white. Therefore, the amino acid composition of egg yolk was not affected by g-irradiation (Katusin-Razem et al., 1989; Ma et al., 1993). The CD measurement of egg white protein indicated that, even at 16 kGy, only limited changes in protein secondary structure were observed (Clark et al., 1992). On the other hand, the lipid content in egg yolk is high, and lipid, especially unsaturated lipid, is more reactive than protein, especially in radical induced chain reactions. The lipid in egg yolk could act as a buffer for protein against radiationinduced reactions.

Summary A rheological test, N-terminal amino acid analysis, and gel electrophoresis appear to be better choices than DSC to study changes in a complex system like egg yolk. For liquid egg yolk products, electron beam irradiation at pasteurizing dosages did not affect physicochemical properties and actually improved the functional properties slightly. No breakdown of egg yolk proteins was found, although some possible aggregation was observed.

FIGURE 5. SDS-PAGE gel electrophoretic patterns of delipidized low density lipoprotein (LPL) from egg yolk. N = delipidized LDL from nonprocessed egg yolk; I = delipidized LDL from irradiated egg yolk.

These results indicated that electron beam irradiation could be an attractive alternative to other preservation method for liquid egg products.

ACKNOWLEDGMENT This work was supported by Regional Projects NC183 (KAN945). Contribution No. 97-139-J from Kansas Agricultural Experiment Station.

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