Irradiation of Food and Packaging - American Chemical Society

Chapter 11

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Application of Electron Beam to Surimi Seafood 1

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J. J a c z y n s k i a n d J. W. Park 1

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Division of Animal and Veterinary Sciences, West Virginia University, Morgantown, WV 26506 Seafood Laboratory and Department of Food Science and Technology, Oregon State University, Astoria, O R 97103

Color, texture, microbial inactivation, and protein-protein interactions of surimi seafood subjected to electron beam (e— beam) were investigated. Color whitening and stronger gel of surimi seafood was measured. The D value for Staphylococcus aureus was 0.34 kGy. Modeling of microbial inactivation demonstrated that two-sided e-beam may control S. aureus if the surimi seafood package is thinner than 82 mm. SDS-PAGE showed gradual degradation of myosin heavy chain (MHC) as e-beam dose increased. Degradation rate was slower when frozen samples were treated. The integrity of actin (AC) was slightly affected by e-beam. 10

© 2004 American Chemical Society Komolprasert and Morehouse; Irradiation of Food and Packaging ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

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166 The Centers for Disease Control and Prevention (CDC) estimated that foodborne diseases in the U S A annually cause 81 million illnesses, 616,377 hospitalizations, and 5000 deaths. Food-borne disease outbreaks implicating seafood are 5, 19, 4, and 12 times as frequent as outbreaks linked to beef, pork, chicken, and turkey, respectively (7). The Food and Drug Administration (FDA) established a "zero tolerance" for Listeria monocyotogenes, Salmonella, Vibrio cholerae, Vibrio vulnificus, and for the presence of toxin, viable spores, or vegetative cells of Clostridium botulinum in ready-to-eat fishery products. If enterotoxigenic Escherichia coli are present at 1 x 10 /g or Vibrio parahaemolyticus at 1 x 10 /g, the F D A will consider regulatory action. The product may be recalled i f it tests positive for staphylococcal toxins or i f 1 x !0 /g of Staphylococcus aureus are present. Fishery products, like other muscle foods, require good pasteurization practices to maintain microbial safety (2,3). Psychrotrophic microflora, inherent to seafood, grows well under refrigeration conditions. Therefore, fishery products are particularly susceptible to microbial deterioration. The surimi seafood industry traditionally uses hot water or steam as a pasteurizing medium (4). However, various pasteurization regimes are used (5, 6). Consequently, products may be overcooked, resulting in undesirable changes of quality (7). It is feasible though to determine a point at which minimum loss of physicochemical quality and desired microbial inactivation occur simultaneously (8). However, some quality deterioration is still inevitable (8, 9). Current thermal pasteurization methods, therefore, may be inadequate to simultaneously maintain microbial safety and product quality. Consequently, in the wake of the September 11 incident, it is increasingly important that the food industry take appropriate action to assure a continuous risk-free food supply (10, 3

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ID. Electron beam (e-beam), in contrast to thermal pasteurization, utilizes highenergy electrons for pasteurization or sterilization. Electrons are accelerated to the speed of light by a linear accelerator. Then, electrons are passed through the food product, inactivating bacteria. The electron source is electricity and, unlike gamma radiation, e-beam does not use radioisotopes (72). E-beam enables the application of high dose rates (e-beam, 10 -10 Gy/sec; gamma, 0.01-1 Gy/sec), resulting in a short exposure time (75). E-beam processing does not affect the temperature of processed food. Therefore, e-beam is likely to minimize the degradation of food quality (14). E-beam, unlike gamma rays, has limited penetration depth (75), which may affect microbial inactivation depending on the package size. The overall antimicrobial effects, though, of gamma rays and e-beam are comparable (75, 16). Joint Expert Committee on Food Irradiation representing F A O / I A E A / W H O (Food and Agriculture Organization/International Atomic Energy Agency/World 3

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Komolprasert and Morehouse; Irradiation of Food and Packaging ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

167


Health Organization) concluded that irradiation of any food up to 10 kGy caused no toxicological hazards and introduced no nutritional or microbiological problems (/ 7). Application of e-beam to surimi seafood has not been reported. Our objectives were to determine color and texture, microbial inactivation, and protein-protein interactions of surimi seafood subjected to e-beam.

Materials and Methods

Color Un-pasteurized surimi seafood crabsticks were obtained from Louis Kemp/Bumble Bee Seafoods (Motley, M N ) . Sticks were tightly placed on plastic trays and sealed in plastic pouches made of a 3-mil (76 microns) standard barrier nylon/PE film (Koch, Kansas City, M O ) under either aerobic (nonvacuum pack) or anaerobic (vacuum pack) conditions. Before e-beam treatment (Ion Beam Applications, San Diego, C A ) , the temperature of the sticks was equilibrated to either -18, 5, or 23°C. The sticks were exposed to four doses of e-beam (0, 1, 2, and 4 kGy) with energy fixed at 10 M e V . Tristimulus color values L * a* b* were measured using a Minolta chroma meter CR-300 (Minolta Camera Co. Ltd., Osaka, Japan) (75). The external (redcolored) layer of the crabstick was peeled off and the remaining stick was ground to a paste. The paste was transferred onto a Petri dish and packed compactly inside for color measurement. To eliminate the effect of compactness on color values, the same amount of paste was applied for each measurement. Color measurement of at least three sticks was taken with at least five measurements per crabstick. A paired t-test based on the pooled standard deviation was used to determine differences between means of various treatments (19).

Texture Frozen Alaska pollock surimi was tempered and cut into small chunks. Surimi chunks were chopped in a silent cutter (Model U M C 5 , Stephan Machinery Corp., Columbus, OH) at low speed for 1 min. Salt (2 %) was added and the surimi paste was chopped at low speed for 0.5 min. Final moisture content was adjusted to 78% by adding ice to the paste, followed by chopping at



168 low speed for 1 min. High speed chopping under vacuum (0.5 bar) was applied for the last 3 min. During chopping, the temperature was kept below 5°C. The paste was then stuffed into torsion gel molds and cooked at 90°C for 15 min, which resulted in hourglass-shaped surimi gels (length = 2.9 cm, end diameter = 1.9 cm, and minimum diameter =1.0 cm). Surimi gels were subjected to one-sided e-beam (0, 1, 2, 4, 6, 8, 10, and 25 kGy) with energy fixed at 10 M e V (Ion Beam Applications, San Diego, C A ) . Surimi gels were kept at room temperature for 2 h prior to measurement. Hourglass-shaped gels were glued to plastic discs and subjected to torsional shear using a Hamman Gelometer (Gel Consultant, Raleigh, N C ) set at 2.5 rpm. Shear stress and shear strain were measured at mechanical fracture to determine gel strength and gel cohesiveness, respectively (20). A t least five measurements per e-beam dose were taken.

Microbial Inactivation

Electron Penetration Surimi gels were prepared as described in "Texture" except that the paste after chopping was not stuffed into torsion gel molds but the paste was placed in a waxed cardboard box (4 cm x 4 cm x 20 cm) and the air gaps were carefully removed. The boxes were vacuum packed and cooked in a water bath at 90°C for 45 min. Immediately following cooking, the surimi gels were cooled in ice slush. Surimi gels with two different dimensions were prepared: (1) 3 cm x 3 cm x 7 cm, and (2) 3 cm x 3 cm x 9 cm. Surimi gels were subjected to two doses (3 and 20 kGy) using one-sided ebeam with energy fixed at 10 M e V (Ion Beam Applications, San Diego, C A ) . The experiments were performed in duplicate. Dosimeters (calibrated radiochromatic dye films) were distributed every 1 cm from the top surface to the bottom of the surimi gels. Exposed dosimeters were read by a spectrophotometer at 605 nm and the doses absorbed at their respective locations were calculated. Absorbed doses were plotted against distance between dosimeters and the surimi gel surface, creating a dose map. The dose map allowed determination of Ro (depth of surimi gel at which the absorbed dose equaled the dose at the surface of the surimi gel), R (depth of surimi gel at which the absorbed dose has decreased 50 % of the absorbed dose at the surface of surimi gel), R (depth of surimi gel at which the absorbed dose pt

5 0 e

m a x


169 reached its maximum value), and Rsomax (depth of surimi gel at which the absorbed dose decreased 50 % of its maximum value). A polynomial regression equation was fitted to the experimental data. Microsoft Excel was used for the calculations. A paired t-test based on the pooled standard deviation was used to determine differences between means of various treatments (19).


Microbial Inactivation Surimi seafood crabsticks (hereinafter surimi seafood) were obtained from a commercial factory (Louis Kemp/Bumble Bee Seafoods, Motley, M N ) . The sticks were ground into a paste. The paste was placed on plastic trays and inoculated (5%) with a cocktail of six strains of Staphylococcus aureus, followed by incubation at 37°C for 72 h, resulting in a final concentration of 10 CFU/g. Following incubation, the inoculated paste was packed in a plastic pouch made of 3 mil (76 microns) standard barrier nylon/PE film (Koch, Kansas City, MO). A half of the packages was anaerobically packed (vacuum); the other half was aerobically packed (non-vacuum). The samples (23°C) were subjected to four doses (0, 1, 2, and 4 kGy) of one-sided e-beam with energy fixed at 10 M e V (Ion Beam Applications, San Diego, C A ) . Following the e-beam treatment, the survivors were enumerated. The six strains of S. aureus were from Dr. M . A . DaeschePs collection (Oregon State University, Corvallis, OR) and identified as 138-cps, 146-cps, 153-cps, 648-gf, 649-gf, and 657-gf. The strains were stored at -70°C. Before inoculation in the paste, the strains were grown in staphylococcus broth (Difco Laboratories, Detroit, MI) at 37°C for 24 hr in an incubator shaker set at 200 rpm. In our preliminary experiments, it was determined that under these conditions each strain reached a stationary phase of growth and concentration of 10 CFU/g (data not shown). Enumeration of S. aureus survivors was performed on staphylococcus 110 agar (Difco Laboratories, Detroit, MI) by a serial 10-fold dilution using the spread plating method (21). Following e-beam treatment, before the survivors were enumerated, the samples were thoroughly mixed in order to obtain uniform distribution of survivors. Bacterial enumeration was performed in triplicate. The presence of S. aureus was confirmed by gram staining, catalase, and coagulase tests. The tests were performed according to the manufacturer (Difco Laboratories, Detroit, MI). A paired t-test based on the pooled standard deviation was used to determine the differences between means of various treatments (19). 9

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170 Predictive Model of Microbial Inactivation by E-Beam Survivors were plotted on a logarithmic scale as a function of dose, resulting in a survivor curve (22). D value defined as the dose in kGy necessary to reduce the microbial population by 90 % (1 log) (22), was calculated as a negative reciprocal of the slope of the survivor curve (23,22) (eq 1).


1 0

log N N D t

0

-

D

W

number of survivors at e-beam dose, initial microbial concentration, Dio value, decimal reduction dose, e-beam dose.

Dose absorbed, as a function of surimi seafood thickness, was simulated by the polynomial equation obtained from the dose map. The dose absorbed was related to the D value, resulting in a total log reduction of S. aureus in the surimi seafood. The number of S. aureus (CFU/g) that survived e-beam treatment was calculated based on the total log reduction and the initial number of S. aureus (CFU/g). Microsoft Excel was used for the calculations. J 0

Protein-Protein Interactions Alaska pollock surimi (hereinafter called surimi) and surimi gels were used in this experiment. Surimi gels were prepared as described in "Texture". Half of the surimi gels and surimi was at room temperature (23°C) when subjected to e-beam. The other half was frozen (-18°C) when subjected to ebeam. Surimi gels and surimi were subjected to 0, 1, 2, 4, 6, 8, 10, and 25 kGy of one-sided e-beam with energy fixed at 10 M e V (Ion Beam Applications, San Diego, C A ) . Surimi (3 g) or surimi gel (3 g) was solubilized in 27 m L of 5 % sodium dodecyl sulfate (SDS) solution (24). Collected supernatant was analyzed for protein concentration by the Lowry assay (25). Samples were diluted by 50-fold so that residual SDS did not interfere with the Lowry assay. Protein concentration was adjusted to 2 mg/mL, and then mixed with 5x sample buffer (1 M Tris-HCl (pH 6.8), 50 % glycerol, 10 % SDS, 14.4 m M pmercaptoethanol (p-ME), 1 % bromophenol blue, and distilled deionized (dd) H 0 ) , followed by heating at 90°C for 5 min (26). Aliquots o f 12.5 p L (25 pg) of proteins per well were used for SDS-PAGE. Discontinuous (12 % 2


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polyacrylamide separating and 5 % polyacrylamide stacking gel) SDS-PAGE under denaturing conditions at 200 m A of constant current were performed (26). The electrophoretic patterns of proteins were stained with Coomassie brilliant blue R-250 (Bio-Rad, Richmond, C A ) , followed by destaining with solution containing 25 % ethanol and 10 % acetic acid.

Results and Discussion

Color and Texture The L * and a* values were not affected (P>0.05) by e-beam. The b* value of the crabsticks decreased (P0.05).

0

1

2

4

E-beam dose (kGy)

Figure 1. The b* value (yellowness) as affected by e-beam (different letters on the bars indicate a significant difference at P0.05). Based on the dose map, the Ro , R , R , and Rsomax were calculated as 33, 21, 41, and 39 mm, respectively. According to the R , two-sided e-beam can efficiently penetrate surimi seafood up to 82 mm thick, resulting in the dose 4

3

2

pt

m a x

50e

50e


174 absorbed across the entire thickness as being equal to or higher than the dose applied. This finding is in accordance with literature, which suggests that ebeam could be applied to food up to 8-10 cm thick that has a specific density of 1 g/cm (27). E-beam at 1, 2, and 4 k G y resulted in 2.9-log reduction, 6.1-log reduction, and no detectable colonies of S. aureus in surimi seafood, respectively (Table I). The Dio value was 0.34 kGy. Effects of radiation are linear with dose, up to 15 kGy (36). Therefore, application of 4 kGy in our tests may have resulted in a 12-log reduction, as verified by no colonies at 4 kGy (Table I). Our Dio value for S. aureus in surimi seafood was similar to the Dio value of 0.29 kGy reported for shrimp (27). Oxygen unavailability under vacuum packing did not affect microbial inactivation (P>0.05).


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Table I. Effect of E-Beam on Inactivation of £ aureus in Surimi Seafood E-beam dose (kGy) 0 (control) 1 2 4

S. aureus count (CFU/g) 1.2 x 10" 2.7 x 10 2.7 x 10 Not Detectable 6

3

Log reduction 0 2.9 6.1 =12

Figure 4 shows simulations of dose absorbed (Figure 4, left) and inactivation of S. aureus (Figure 4, right) in surimi seafood subjected to e-beam. The dose absorbed in surimi seafood was simulated using the polynomial function obtained from the dose map. B y applying the Dio value for S. aureus in surimi seafood to the simulated dose absorbed, total log reduction was estimated. Final concentration of S. aureus (CFU/g) was obtained by applying the total log reduction to the initial concentration of S. aureus (CFU/g). The simulations in Figure 4 are based on 90 mm sample thickness, 2 kGy applied dose, 0.34 kGy Dio value for S. aureus in surimi seafood, and 2.3 x 10 CFU/g initial concentration of S. aureus. Based on Ro and R o equaling 33 and 41 mm, respectively, one-sided and two-sided e-beam can efficiently penetrate 33 and 82 mm of surimi seafood, respectively. Efficient penetration is defined as a penetration that results in the dose absorbed in entire surimi seafood equal to or greater level than the dose applied. Therefore, two-sided e-beam represents better utilization of the dose applied. The dose absorbed below 33 mm for one-sided e-beam would be lower than the dose applied, thereby, the desired antimicrobial effect would not be obtained. 5

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Distance from surface (mm)

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TWO SIDED E-BEAM

D-value for S. aureus in surimi seafood =|

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Figure 4. Predictive models for dose absorbed (left) and inactivation ofS. aureus (right) in surimi seafood by two-sided ebeanu This figure represents model predictions under given conditions: product thickness 90 mm, dose applied 2 kGy, D value for S. aureus in surimi seafood 0.34 kGy, initial population ofS. aureus 2.3 x 10 CFU/g.

(

•

•

TWO SIDED E-BEAM

Initial microbial cone = 2.3E+05 CFU/g Final average micro cone = 1.4E-01 CFU/g


Product thickness = 90 mm Dose applied = 2 kGy

Dose abs orbed


176 Simulation of dose absorbed and microbial inactivation by two-sided e-beam for surimi seafood thicker than 82 mm demonstrates under-processing starting at a depth of 33 mm from the top and bottom surfaces (Figure 4). If the thickness of surimi seafood processed with two-sided e-beam exceeds 82 mm, then the maximum under-processing occurs at the geometrical center of the package. This geometrical center may be referred to as the "cold spot". The term "cold spot" is commonly used to describe under-processed areas of food processed with non-uniform heating (i.e., microwave or radio frequency). However, it is obvious that e-beam processing does not generate heat. The analogy refers only to under-processing, which may result in elevated microbial survival in the under-processed areas of the product. Consequently, one-sided and two-sided e-beam processing are not recommended for surimi seafood thicker than 33 and 82 mm, respectively.

Protein Degradation The SDS-PAGE in 12 % polyacrylamide gel (Figure 5) showed gradual degradation of myosin heavy chain (MHC). The degradation was proportional to the increase of e-beam dose. Gradual disappearance of M H C resulted in a subsequent increase of smaller molecular weight proteins (200 to 50 kDa) that appeared in each lane below M H C . The complete disappearance of the M H C band was observed at 25 kGy for raw surimi and surimi gels subjected to e-beam while at 23°C. However, raw surimi and surimi gels subjected to 25 kGy while at - 1 8 ° C showed a very thin M H C band, suggesting slower degradation at the lower temperature. Actin (AC) and other fractions of myofibrillar proteins were not affected by doses from 0 - 1 0 kGy and marginally affected at 25 kGy. Similar observations have been reported in literature (32,37,38). A gradual disappearance of a band associated with the main chain (210 kDa) of myosin subjected to gamma radiation was observed using SDS-PAGE (37). A slower rate of myosin degradation when frozen samples were subjected to radiation was also observed (37). A cross-linking of mackerel actomyosin induced by U V radiation was observed using S D S - P A G E (32). The U V radiation caused gradual disappearance of M H C in flying fish (38). In our experiments, electrophoresis was conducted under denaturing conditions of P-ME and SDS. If e-beam had induced cross-linking by disulfide bonds or hydrophobic interactions, they would have not been seen due to cleavage of those bonds by P - M E and SDS, respectively. However, crosslinking that involves bonds other than disulfide bonds or hydrophobic interactions would have been detected. Therefore, it is suggested that e-beam did not induce cross-linking other than disulfide bonds or hydrophobic interactions.



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Figure 5. SDS-PAGE of Alaska polock surimi (left) and surimi gels (right) applied to 12 % polyacrylamide gel at 25 fig ofproteins/welL Sample temperature was -18°C (top) and 23°C (bottom) during e-beam treatment MHC - myosin heavy chain, AC - actin.


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Irradiation of Food and Packaging - American Chemical Society

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