Effect of salmon plasma protein on Pacific whiting surimi gelation under various ohmic heating conditions

Fowler, M. R., & Park, J. W. (2015). Effect of salmon plasma protein on Pacific whiting surimi gelation under various ohmic heating conditions. LWT-Food Science and Technology, 61(2), 309-315. doi:10.1016/j.lwt.2014.12.049

10.1016/j.lwt.2014.12.049

Elsevier

Accepted Manuscript

http://cdss.library.oregonstate.edu/sa-termsofuse

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Effect of Salmon Plasma Protein on Pacific Whiting Surimi Gelation under Various 1

Ohmic Heating Conditions 2

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Matthew R Fowler, Jae W Park 4

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Oregon State University Seafood Research and Education Center 7

2001 Marine Dr Rm 253, Astoria, OR 97103, USA 8

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Corresponding Author: 10

Jae W Park 11

(503) 325-4531 Ext #3 12

Jae.park@oregonstate.edu 13

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To be submitted to LWT- Food Sci and Tech 16

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ABSTRACT: 18

The effect of salmon blood plasma (SPP) on the gelation of Pacific whiting surimi under 19

different ohmic heating conditions was investigated. SPP was found to significantly increase gel 20

strength in gels heated ohmically to and held at 60° for 30 min followed by heating ohmically to 21

90°C. SPP at a level of 1 g/100g was also found to increase gel strength in gels held at 25°C for 2 22

h prior to ohmic heating. This increase was not seen in gels where EDTA was added to inhibit 23

the activity of endogenous transglutaminase (ETG). SPP also created a more pronounced setting 24

effect as measured by dynamic rheology and SDS-PAGE. SPP was found to effectively inhibit 25

protease activity through TCA-soluble peptide analysis. Scanning electron microcopy revealed a 26

loosely arranged gel network caused by protease enzymes. It was reversed by the addition of 27

SPP as well as setting at 25oC due to ETG. 28

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Keywords: salmon plasma, surimi gelation, ohmic heating, transglutaminase, protease 30

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Highlights: 32

• Various ohmic heating rates effectively isolated enzyme activity in surimi 33

• Salmon plasma protein effectively inhibited protease in Pacific whiting surimi 34

• Salmon plasma protein increased gel strength during setting 35

• Salmon plasma protein is an effective inhibitor at low levels (0.5 g/100g or less) 36

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4

Introduction 38

In the United States, surimi is made from two types of fish: Alaska pollock (AP) and Pacific 39

whiting (PW). Unlike AP, PW contains a high amount of protease enzymes that degrade the 40

quality of the surimi gel when heated slowly (Klesk, Yongsawatdigul, Park, Viratchakul, & 41

Virulhakul, 2000). The major protease enzymes found in PW are cathepsins B, H, and L. After 42

PW has gone through the rinsing step of surimi manufacturing, most of cathepsin B and almost 43

all of cathepsin H proteases are removed. However, cathepsin L, a protease associated with 44

myofibrillar proteins, is not removed during the washing process and was found to be the main 45

protease responsible for degradation of the surimi gel (An, Weerasinghe, Seymour, & 46

Morrissey, 1994). Cathepsin L is a heat activated cysteine protease, having an optimum 47

temperature of around 55-60°C (Seymour, Morrissey, Peters, & An, 1994; Visessanguan, 48

Benjakul, & An, 2003). Incubating PW surimi around this temperature range for 30 min before 49

heating to 90oC will result in a complete disappearance of the myosin heavy chain as well as an 50

inability to form a gel network (Morrissey, Wu, Lin, & An, 1993; Rawdkuen, Benjakul, 51

Visessanguan, & Lanier, 2007a). Surimi that is heated at a slow rate (such as in a water bath) 52

also suffers protoeolytic degradation (Yongsawatdigul, Park, Kolbe, Dagga, & Morrissey, 1995). 53

In the past, bovine blood plasma (BPP) was added to PW surimi as a protease inhibitor. This 54

practice was discontinued however due to public fear of Bovine Spongiform Encephalopathy 55

(BSE). Since then, BPP has been replaced by dried egg white (DEW), which contains mainly 56

serine protease inhibitors (Yongswatdigul, Hemung, & Choi, 2014). Since DEW is not as effective 57

as BPP at inhibiting cysteine proteases, such as cathepsin L (Yongswatdigul et al., 2014), finding 58

an alternative inhibitor that can be used at small concentrations would be beneficial. Blood 59

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plasma from other sources, such as pork (Benjakul, Srivilai, & Visessanguan, 2001; 60

Visessanguan, Benjakul, & An, 2000) and chicken (Rawdkuen et al., 2007a; Rawdkuen, Lanier, 61

Visessanguan, & Benjakul, 2004, 2007b), have been found to be effective inhibitors of protease 62

enzymes found in surimi. 63

In addition to protease inhibitors, blood plasma also contains other proteins that may enhance 64

the gelation of surimi. Blood plasma proteins such as fibrinogen exhibit their own gelling ability 65

upon heating (Davila, Pares, Cuvelier, & Relkin, 2007). Also, blood plasma has been shown to 66

contain endogenous transglutaminase (ETG) enzymes (Lorand, 2007). ETG is a naturally 67

occurring enzyme in PW and other species of fish. ETG is a calcium dependent enzyme that 68

mediates covalent cross linking of myofibrillar proteins, resulting in a higher gel strength (An, 69

Peters, & Seymour, 1996). Since endogenous PW and AP ETG has an optimum temperature of 70

around 25°C, leaving surimi paste at room temperature for 1 or 2 hr before heating results in 71

stronger gels. This ETG mediated formation of non-disulfide covalent cross links before heating 72

is known as “setting”. Addition of calcium and calcium containing compounds to surimi has 73

been shown to increase the effect of setting (Lee & Park, 1998) and the addition of calcium 74

chelating compounds, such as EDTA, has been shown to completely inhibit setting (Kumazawa, 75

Numazawa, Seguro, & Motoki, 1995). Since blood plasma contains ETG, it may also contribute 76

to the setting phenomenon when added to surimi in sufficient amounts. 77

The activity of these two different types of enzymes (proteases and ETG), pose a problem when 78

evaluating the quality of PW surimi gel. Traditionally, surimi is heated in a water bath to 90°C 79

before conducting gel texture measurement. This slow heating allows for the activity of both 80

ETG (enhances gel strength) and proteases (lowers gel strength). Surimi crabstick, however, is 81

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manufactured in a thin sheet under gas and/or steam heating, which quickly deactivates both 82

types of enzymes and does not allow for any activity beyond 75oC. Therefore, these testing 83

methods do not accurately assess the quality of the surimi seafood being produced in a thin 84

sheet under fast heating. Rapid heating methods, such as ohmic heating, allow for a better 85

assessment of surimi containing protease enzymes (Yongsawatdigul et al., 1995). The objective 86

of this study was to isolate the activities of both proteases and ETG at various heating rates 87

under ohmic heating, and evaluate the effect of salmon plasma protein (SPP) on the gelation of 88

PW surimi in combination with these enzymes. 89

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2. Materials and Methods 91

2.1. Materials 92

Pacific whiting surimi (FA grade) without the addition of egg white, 2 months old, was obtained 93

from American Seafoods (Seattle, WA, USA) and kept at -30°C until used. Protein markers and 94

other electrophoresis chemicals were purchased from Bio-Rad Laboratories (Hercules, CA, 95

USA). All other chemicals used were of reagent grade. 96

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2.2. Collection of salmon blood and preparation of plasma 98

Whole blood was collected at the Klaskanine Fish Hatchery (Astoria, OR, USA) from female 99

Chinook salmon immediately before roe collection. Blood was collected from bleeding fish 100

into bottles containing 3.8g/100mL sodium citrate (as an anti-coagulant), and gently mixed at a 101

ratio of 9:1 (mL:mL) blood to sodium citrate (Li, Lin, & Kim, 2008; Rawdkuen et al., 2007b). 102

Blood was kept on ice and transported back to the Oregon State Seafood Laboratory (Astoria, 103

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OR, USA) where it was centrifuged for 15 min at 1,500 × g at 4°C using a Beckman J6-MI 104

centrifuge (Beckman Coulter, Fullerton, CA, USA). The supernatant was then lyophilized in a 105

Labconco freeze drier (Kansas City, MO, USA) and regarded as salmon plasma protein (SPP). SPP 106

was stored at -80°C until used. Samples were not stored longer than 3 months. 107

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2.3. Surimi gel preparation 109

Paste and gels were prepared according the method of Poowakanjana, Mayer, and Park (2012) 110

with various heating methods. Partially thawed surimi was chopped at 1,800 rpm for 1 min 111

using a silent cutter (UM 5 universal, Stephan Machinery Corp, Columbus, OH, USA). After a 112

2g/100g addition of salt, surimi was chopped for an additional 1 min at 1,800 rpm. Moisture 113

content was then adjusted to 78g/100g using ice. At this time SPP (0, 0.5 or 1 g/100g) as well as 114

EDTA (0 or 0.1 g/100g) was added. A preliminary study conducted in our laboratory indicated 115

that 0.1 g/100g EDTA was sufficient to completely inhibit ETG activity in PW surimi (data not 116

shown). Following the addition of ice and other dry ingredients, surimi was chopped again for 1 117

min at 1,800 rpm. Chopping was then continued at 3,600 rpm under vacuum (40-60 kPa) for an 118

additional 3 min and a total chopping time of 6 min. Care was taken so that the final 119

temperature of the surimi paste was less than 15°C. Paste was packed in a polyethylene bag 120

and subjected to vacuum to remove any air pockets developed during packing. The paste was 121

then stuffed into a 15 cm x 3 cm nylon tube. 3 different heating methods were used: 1. Ohmic 122

(rapid) heating to 90°C at a voltage gradient of 12.62 V/cm with settings of 250 V and 10 kHz to 123

prevent the activity of both ETG and proteases (OH); 2. Ohmic heating to 60°C followed by 124

ohmically holding at 60°C for 30 min before ohmically heating to 90°C to prevent the activity of 125

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ETG and maximize the activity of proteases (60/OH); and 3. Holding in a 25°C water bath for 2 h 126

(to maximize the activity of ETG) followed by ohmic heating to 90°C to prevent the activity of 127

proteases (25/OH). Two sausages were made per heating method. Following heating, gels were 128

placed in a plastic bag, submerged in ice water for 15 min, and stored overnight at 4°C. 129

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2.4. Oscillatory dynamic measurement 131

Surimi paste was subjected to a temperature sweep using a CVO rheometer (Malvern 132

Instruments Ltd., Worcestershire, UK) using a cone (4° and 40 mm diameter) and plate 133

geometry with a gap of 150 μm. Surimi gels prepared as described above were thinly cut to a 134

thickness of 2 mm and subjected to a frequency sweep using parallel plate geometry (20 mm 135

diameter) and a gap of 1 mm. Surimi gel (3 cm diameter) was trimmed to 2 cm using a small 136

knife and moisture trap containing a moistened sponge was used to minimize drying of sample. 137

Temperature sweeps were conducted from 10 °C-90 °C at a heating rate of 2 °C/min at a fixed 138

frequency of 0.1 Hz. Frequency sweeps were conducted from 0.1 to 10 Hz at a fixed 139

temperature of 25 °C. A shear stress of 50 Pa, determined by stress sweep to be in the linear 140

viscoelastic region, was used. 141

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2.5. Fracture gel evaluation 143

The day after heating, gels were removed from refrigerated storage and held at room 144

temperature for 2 h prior to testing. Gel samples were cut into 30 mm lengths and the breaking 145

force (g) and penetration distance (mm) were determined using a texture analyzer (TA-XT plus, 146

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Texture Technologies Corp, NY, USA). Gels were punctured with a spherical probe (5 mm 147

diameter) at 1 mm/sec. 148

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2.6. Color analysis 150

L*, a*, and b* values of surimi gels were determined from 30 mm samples using a Minolta 151

colorimeter (CR-310; Minolta Camera Co. Ltd., Osaka, Japan). The instrument was standardized 152

using a Minolta calibration plate and a Hunter Lab standard hitching file according to the 153

method of Park (1994). Whiteness was calculated using the equation L*-3b*. 154

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2.7. Determination of TCA soluble peptides 156

Eighteen mL of 5 g/100mL trichloroacetic acid (TCA) was added to 2 g of sample followed by 157

homogenization for 2 min at 15,000 rpm using a Tissue Tearor homogenizer (Biospec Products 158

Inc., Bartlesville, OK, USA). Homogenate was then held at 4 °C for 1 h before centrifugation at 159

8,000 × g for 5 min using a Sorvall RC-5B centrifuge (DuPont Instruments, Newton, CT, USA). 160

The TCA-soluble peptide content of the supernatant was measured by the method of Lowry et 161

al. (1951) using tyrosine as a standard and expressed as μmol tyrosine/ g sample. Samples were 162

measured in triplicate. 163

164

2.8. SDS PAGE 165

Surimi gels were examined for protein patterns based on their molecular weight according to 166

the method of Laemmli (1970). Gels were solubilized in 5g/100mL sodium dodecyl sulfate at 90 167

°C according to the method of Morrissey et al. (1993). A 4g/100mL acrylamide stacking gel and 168

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10g/100mL acrylamide separating gel were used. Gels were fixed and stained in 0.125g/100mL 169

Coomassie R-250 (Bio-Rad, Richmond, CA, USA), and de-stained in a 50mL/100mL methanol, 170

10mL/100mL acetic acid solution. Molecular weights of bands were determined by comparison 171

to a molecular weight standard (Protein Plus All Blue, Bio-Rad Laboratories, Hercules, CA, USA). 172

173

2.9. Scanning electron microscopy 174

Gels were cut into 2 mm x 2 mm sections and rinsed two times in distilled water for 30 min 175

prior to fixing for 2 hr in a 0.1 mol/L cacodylate buffer containing 2.5g/100mL glutaraldehyde 176

and 1g/100mL paraformaldehyde. Samples were then dried through serial acetone dilutions 177

(10, 30, 50, 70, 90 and twice in 100%) followed by critical point drying. Samples were then 178

coated with gold and palladium (40:60 ratio) and examined in a Quanta 600 FEG field emission 179

scanning electron microscope (FEI Inc., Hillsboro, OR). This microscopy work was done at the 180

Oregon State University Electron Microscope Facility (Corvallis, OR, USA). 181

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2.10. Statistical analysis 183

Data were subjected to analysis of variance (ANOVA). Comparison of means was carried out by 184

Tukey test (Ramsey & Schafer, 2012). Statistical analysis was done by Sigma Plot software 185

package (Sigma Plot 12.5, Systat Software Inc, San Jose, CA, USA). Two batches were made for 186

each treatment and all experiments were repeated. 187

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3. Results and Discussion 191

3.1. Oscillatory rheology 192

The maximum elastic modulus (G’) of surimi heated at 2°C/min decreased as more SPP was 193

added (Fig 1A-1C). Visessanguan et al. (2000) found that the addition of pork plasma protein to 194

Pacific whiting actomyosin also decreased G’. It was postulated that this was due to plasma 195

proteins having different thermal stabilities and gelation properties than fish muscle proteins. 196

The formation of two different gel networks (plasma protein and fish protein) with different 197

properties may lead to the observed decrease in G’. However, for pastes with 0.5g/100g SPP 198

(Fig 1B) and 1g/100g SPP (Fig 1C), the maximum G’ was higher for samples containing no EDTA 199

than samples containing EDTA. This difference was not observed in the control containing no 200

SPP. The difference between samples with and without EDTA is attributed to the setting effect 201

due to the action of ETG. This activity, however, may be offset by proteases active in the control 202

paste that are inhibited in the samples containing SPP. Therefore, the influence of setting 203

during heating at 2°C/min is more pronounced when SPP is added. In addition, ETG and calcium 204

present in SPP may serve to enhance the setting effect. Yin & Park (2014) found that adding 205

calcium containing nano scale fish bone to surimi also increased gel strength. 206

Rheological properties of the final gel were also evaluated by frequency sweep (Fig 1D). The G’ 207

of the control sample held at 60°C before ohmic heating showed a significantly higher 208

frequency dependence than the control sample ohmically heated directly to 90°C. Samples 209

containing both 0.5g/100g SPP and 1g/100g SPP and held at 60°C before ohmic heating showed 210

similar frequency dependence as the control heated to 90°C. G’ values of less cohesive gels 211

exhibit higher frequency dependency. Therefore, G’ increases at a higher rate as frequency 212

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increases than is observed in a more cohesive gel (Picout & Ross-Murphy, 2003). These results 213

indicate that adding SPP to surimi to inhibit proteases lead to a less frequency dependent, more 214

cohesive gel network in the finished product. 215

216

3.2. Fracture gel evaluation 217

Both breaking force (Fig 2A) and penetration distance (Fig 2B) of 60/OH gels were greatly 218

increased by the addition of 0.5g/100g and 1g/100g SPP with or without the addition of EDTA 219

(P<0.05). There was no difference for this heating method between 0.5g/100g and 1g/100g 220

SPP. This heating condition maximized the action of endogenous proteases while minimizing 221

the activity of ETG to less than 10% (Park, Ooizumi, & Hunt, 2014). This indicates SPP at a level 222

of 0.5g/100g is sufficient for inhibiting protease activity and no added benefit in this regard is 223

seen from increasing concentration. 224

Compared to OH gels, 25/OH gels without EDTA showed a greater breaking force and 225

penetration distance (P<0.05). This heating method was favored by ETG and the activity of 226

protease enzymes was not significantly noted. Therefore, this increase of gel strength is due to 227

the setting phenomenon. In addition 25/OH gels without EDTA and containing 1g/100g SPP 228

showed the highest breaking force and penetration distance of all samples (P<0.05). It may be 229

postulated that transglutaminase present in SPP contributed additionally to enzyme-mediated 230

covalent cross linking during settings. Both OH and 25/OH gels containing no SPP decreased 231

moderately and significantly, respectively, in breaking force and penetration distance when 232

EDTA was added (P<0.05). EDTA chelates calcium, which in addition to being a cofactor for ETG 233

may also play other roles in gelation (Lee & Park, 1998). However, both OH and 25/OH gels 234

13

containing EDTA showed a higher breaking force and penetration distance with the addition of 235

SPP as compared to the control (P<0.05), indicating a significant role of SPP as a gel enhancer. 236

In addition to transglutaminase being present in SPP, it may also be a source of additional 237

calcium (Heaton & Pomare, 1974; Maye, Keaton, Hurst, & Habener, 1979), leading to greater 238

gel strength. 239

240

3.3 Color gel analysis 241

Whiteness decreased markedly as SPP concentration increased (P<0.05) (Fig 3D). Increase in 242

SPP was also associated with a lower L* value (less lightness) (Fig 3A), more negative a* value 243

(more redness) (Fig 3B) and a higher b* value (more yellowness) (Fig 3C). The decrease in color 244

quality is due to hemolysis in the blood plasma. When blood is collected and plasma is 245

processed, damage to the red blood cells can result in plasma containing a pinkish hue as 246

opposed to a straw yellow color (Field, Elvehjem, & Juday, 1943; Li et al., 2008). In addition, as 247

salmon is a cold water species, blood plasma may exhibit lower thermal stability and therefore 248

be more susceptible to hemolysis than blood plasma from mammals. This issue may be partly 249

intervened by the fact that SPP is needed only at very low levels (maximum of 0.5g/100g as 250

shown by the results in Fig 2) in order to prevent proteolysis in PW surimi. It is suggested 251

however that further studies be conducted to determine collection and processing methods 252

that will reduce hemolysis. In addition, 60/OH gels showed greater L* values, less negative a* 253

values, and higher b* values than other gels (P<0.05). However, overall whiteness was not 254

affected by heating conditions. 255

256

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257

3.4 TCA soluble peptide content 258

At a TCA concentration of 5g/100mL, all proteins except for small oligopeptides are insoluble 259

(Yvon, Chabanet, & Pelissier, 1989). Since small peptides are the result of the action of 260

endogenous proteases on PW muscle, the protein content in the 5g/100mL TCA supernatant 261

after centrifugation is related to total protease activity in the surimi. Control 60/OH gels with or 262

without EDTA had significantly higher TCA soluble peptide content compared to other gels 263

(P<0.05) (Fig 4). The addition of SPP to these gels showed a significant decrease in TCA soluble 264

peptide content (P<0.05). There was no difference between 0.5g/100g and 1g/100g SPP. 265

Besides 60/OH gels, no difference was seen in TCA soluble peptide content between other 266

treatments and 60/OH gels containing SPP. This confirms the fact that an SPP concentration of 267

0.5g/100g is sufficient to inhibit protease activity and no additional inhibition is seen from 268

increasing concentrations. In addition, this confirms that the fast heating treatments (OH and 269

25/OH) effectively eliminate protease activity. 270

271

3.5 SDS PAGE 272

For OH gels, there was no discernible difference in protein pattern between gels prepared 273

without EDTA (Fig 5A) and gels with EDTA (Fig 5B). Among this group, SPP also had no 274

noticeable effect on protein pattern. This is because the OH treatment eliminated the activity of 275

both protease enzymes and ETG, therefore there was no setting effect or degradation of the 276

myosin heavy chain. For the 25/OH samples, however, a protein band is visible in the high 277

molecular weight range (>250 kDa) for gels treated without EDTA that is not present for gels 278

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treated with EDTA (dotted line in Fig 5A). Yin and Park (2014) found that ETG-mediated cross 279

linking in surimi led to the appearance of high molecular weight bands. This band is due to the 280

crosslinking of myosin heavy chain proteins, leading to higher molecular weight proteins 281

(Kamath, Lanier, Foegeding, & Hamann, 1992). The disappearance of this band in the EDTA 282

samples confirms the inhibition of ETG by EDTA. The high molecular weight band is also darker 283

for 1g/100g SPP than the control, indicating that SPP might have played a role in the setting 284

phenomenon as transglutaminase is one of various proteins present in blood plasma (Folk, 285

1980). 286

The 60/OH group showed no difference between samples with and without EDTA. In this 287

treatment, only protease enzymes are active and the action of ETG was eliminated. This heating 288

treatment completely destroyed the myosin heavy chain band in the control samples due to the 289

activity of proteases. When SPP was added at 0.5g/100g however, the myosin heavy chain band 290

remained intact. Increasing SPP concentration did not affect the intensity of the myosin heavy 291

chain band. This confirms that 0.5g/100g SPP is sufficient to inhibit proteases and prevent 292

myosin heavy chain degradation. 293

294

3.6 Scanning electron microscopy 295

The 60/OH gel with no SPP added showed the greatest number of voids and the least compact 296

structure among the samples tested (Fig 6B). This is consistent with the results from the 297

puncture test as well as the TCA-soluble peptides. This confirms that protease enzymes active 298

at this temperature serve to break up and weaken the gel structure. 60/OH gel with 1g/100g 299

SPP (Fig 6E) showed a significantly more orderly and continuous gel structure with less voids 300

16

than the 60/OH gel with no SPP present, indicating effective inhibition of protease enzymes. 301

However, there were a greater number of voids present in this gel than in the OH gels (Fig 6A 302

and 6D), indicating residual protease activity. 25/OH gels without and with 1g/100g SPP (Fig 6C 303

and 6F, respectively) had a more compact and continuous structure compared to OH gels 304

without and with 1g/100g SPP (Fig 6A and 6D, respectively). This is due to the addition of extra 305

covalent cross linking in the gel structure due to the action of ETG. In addition, 25/OH gel with 306

1g/100g SPP had a more compact structure with less voids than the 25/OH gel without SPP. 307

This may be due to transglutaminase present in SPP in addition to ETG of surimi, leading to 308

additional cross linking during setting. 25/OH sample containing no SPP and 0.1g/100g EDTA 309

(Fig 6G), had a greater number of voids than the OH samples. The purpose of EDTA addition 310

was to chelate calcium in order to prevent the activity of ETG. However, these results indicate 311

that calcium may play other roles in gelation in addition to being a cofactor for ETG. 312

313

4. Conclusion 314

SPP was found to effectively inhibit endogenous proteases in PW surimi at levels as low as 315

0.5g/100g. Higher concentrations of SPP may also aid in transglutaminase-mediated gel setting, 316

leading to an increase in gel strength. However, increasing SPP concentrations also led to a 317

decrease in the elastic modulus as well as a decrease in whiteness. This balance must be kept in 318

mind when formulating surimi with SPP. Holding at 25°C before ohmic heating was found to 319

optimize ETG activity while controlling protease activity. Rapidly heating to and holding at 60°C 320

before ohmic heating to 90°C was found to optimize protease activity while minimizing ETG 321

activity. 322

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323

5. Acknowledgment 324

This research was supported by a scholarship (2013 NPRD Graduate Research Award) from the 325

North Pacific Research Board (Anchorage, AK). We are thankful for Teresa Sawyer of the 326

Oregon State University Electron Microscope Facility (Corvallis, OR) for her assistance with the 327

scanning electron microscopy. 328

329

REFERENCES 330

An, H., Peters, M. Y., & Seymour, T. A. (1996). Roles of endogenous enzymes in surimi gelation. 331 Trends in Food Science & Technology, 7(10), 321-327. 332

An, H., Weerasinghe, V., Seymour, T. A., & Morrissey, M. T. (1994). Cathepsin Degradation of 333 Pacific Whiting Surimi Proteins. Journal of Food Science, 59(5), 1013-1017. 334

Benjakul, S., Srivilai, C., & Visessanguan, W. (2001). Porcine plasma protein as proteinase 335 inhibitor in bigeye snapper (Priacanthus tayenus) muscle and surimi. Journal of the 336 Science of Food and Agriculture, 81(10), 1039-1046. 337

Davila, E., Pares, D., Cuvelier, G. r., & Relkin, P. (2007). Heat-induced gelation of porcine blood 338 plasma proteins as affected by pH. Meat Science, 76(2), 216-225. 339

Field, J. B., Elvehjem, C. A., & Juday, C. (1943). A study of the blood constituents of carp and 340 trout. Journal of Biological Chemistry, 148(2), 261-269. 341

Folk, J. E. (1980). Transglutaminases. Annual Review of Biochemistry, 49(1), 517-531. 342 Heaton, K. W., & Pomare, E. W. (1974). Effect of bran on blood lipids and calcium. The Lancet, 343

303(7846), 49-50. 344 Kamath, G. G., Lanier, T. C., Foegeding, E. A., & Hamann, D. D. (1992). Nondisulfide covalent 345

cross linking of myosin heavy chain in setting of Alaska Pollock and Atlantic Croaker 346 surimi. Journal of Food Biochemistry, 16(3), 151-172. 347

Klesk, K., Yongsawatdigul, J., Park, J. W., Viratchakul, S., & Virulhakul, P. (2000). Gel Forming 348 Ability of Tropical Tilapia Surimi as Compared with Alaska Pollock and Pacific Whiting 349 Surimi. Journal of Aquatic Food Product Technology, 9(3), 91-104. 350

Kumazawa, Y., Numazawa, T., Seguro, K., & Motoki, M. (1995). Suppression of surimi gel setting 351 by transglutaminase inhibitors. Journal of Food Science, 60(4), 715-717. 352

Laemmli, U. K. (1970). Cleavage of Structural Proteins during the Assembly of the Head of 353 Bacteriophage T4. Nature, 227(5259), 680-685. 354

Lee, N., & Park, J. W. (1998). Calcium compounds to improve gel functionality of Pacific whiting 355 and Alaska pollock surimi. Journal of Food Science, 63(6), 969-974. 356

18

Li, D. K., Lin, H., & Kim, S. M. (2008). Effect of rainbow trout (Oncorhynchus mykiss) plasma 357 protein on the gelation of Alaska pollock (Theragra chalcogramma) Surimi. Journal of 358 Food Science, 73(4), C227-C234. 359

Lorand, L. (2007). Crosslinks in blood: transglutaminase and beyond. The FASEB Journal, 21(8), 360 1627-1632. 361

Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with 362 the Folin phenol reagent. The Journal Of Biological Chemistry, 193(1), 265-275. 363

Maye, G. P., Keaton, J. A., Hurst, J. G., & Habener, J. F. (1979). Effects of plasma calcium 364 concentration on the relative proportion of hormone and carboxyl fragments in 365 parathyroid venous blood. Endocrinology, 104(6), 1778-1784. 366

Morrissey, M. T., Wu, J. W., Lin, D., & An, H. (1993). Protease Inhibitor Effects on Torsion 367 Measurements and Autolysis of Pacific Whiting Surimi. Journal of Food Science, 58(5), 368 1050-1054. 369

Park, J. W. (1994). Functional protein additives in surimi gels. Journal of Food Science, 59(3), 370 525-527. 371

Park, J. W., Ooizumi, T., & Hunt, A. L. (2014). Ingredient technology for surimi and surimi 372 seafood. In J. W. Park (Ed.), Surimi and Surimi Seafood (3 ed.). Boca Raton, FL: CRC Press. 373

Picout, D. R., & Ross-Murphy, S. B. (2003). Rheology of biopolymer solutions and gels. 374 TheScientificWorld Journal, 3, 105-121. 375

Poowakanjana, S., Mayer, S. G., & Park, J. W. (2012). Optimum chopping conditions for Alaska 376 pollock, Pacific whiting, and threadfin bream surimi paste and gel based on rheological 377 and Raman spectroscopic analysis. Journal of Food Science, 77(4), E88-E97. 378

Ramsey, F., & Schafer, D. (2012). The Statistical Sleuth: A Course in Methods of Data Analysis (3 379 ed.). Stamford: Cengage Learning. 380

Rawdkuen, S., Benjakul, S., Visessanguan, W., & Lanier, T. C. (2007a). Effect of cysteine 381 proteinase inhibitor containing fraction from chicken plasma on autolysis and gelation of 382 Pacific whiting surimi. Food Hydrocolloids, 21(7), 1209-1216. 383

Rawdkuen, S., Lanier, T. C., Visessanguan, W., & Benjakul, S. (2004). Chicken plasma protein: 384 Proteinase inhibitory activity and its effect on surimi gel properties. Food Research 385 International, 37(2), 156-165. 386

Rawdkuen, S., Lanier, T. C., Visessanguan, W., & Benjakul, S. (2007b). Cysteine proteinase 387 inhibitor from chicken plasma: Fractionation, characterization and autolysis inhibition of 388 fish myofibrillar proteins Food Chemistry, 101(4), 1647-1657. 389

Seymour, T. A., Morrissey, M. T., Peters, M. Y., & An, H. (1994). Purification and characterization 390 of Pacific whiting proteases. Journal of Agricultural and Food Chemistry, 42(11), 2421-391 2427. 392

Visessanguan, W., Benjakul, S., & An, H. (2000). Porcine plasma proteins as a surimi protease 393 inhibitor: effects on actomyosin gelation. Journal of Food Science, 65(4), 607-611. 394

Visessanguan, W., Benjakul, S., & An, H. (2003). Purification and characterization of cathepsin L 395 in arrowtooth flounder (Atheresthes stomias) muscle. Comparative Biochemistry and 396 Physiology Part B: Biochemistry and Molecular Biology, 134(3), 477-487. 397

Yin, T., & Park, J. W. (2014). Effects of nano-scaled fish bone on the gelation properties of 398 Alaska pollock surimi. Food Chemistry, 150, 463-468. 399

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Yongsawatdigul, J., Park, J. W., Kolbe, E., Dagga, Y., & Morrissey, M. T. (1995). Ohmic heating 400 maximizes gel functionality of Pacific whiting surimi. Journal of Food Science, 60(1), 10-401 14. 402

Yongswatdigul, J., Hemung, B. O., & Choi, Y. J. (2014). Proteolytic Enzymes and Control in 403 Surimi. In J. W. Park (Ed.), Surimi and Surimi Seafood (3 ed., pp. 141-167). Baco Raton, 404 FL: Taylor and Francis. 405

Yvon, M., Chabanet, C., & Pelissier, J. P. (1989). Solubility of peptides in trichloroacetic acid 406 (TCA) solutions hypothesis on the precipitation mechanism. International Journal of 407 Peptide and Protein Research, 34(3), 166-176. 408

409 410 411

412

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FIGURES 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 Figure 1 – Elastic (Storage) modulus of Pacific whiting surimi paste as affected by temperature 445 sweep and 0, 0.5 and 1g/100g SPP additions (A, B, and C, respectively) and gel as affected by 446 frequency sweep (D) after various treatments. without EDTA with EDTA 447 Control OH Control 60/OH 0.5g/100g SPP 60/OH 1g/100g SPP 60/OH 448

A B

C D

21

0100200300400500600

Brea

king

For

ce (G

)

02468

101214161820

Pene

trat

ion

Dist

ance

(mm

)

449

450

451

452

453

454

455

456 457 Figure 2 - Breaking force (A) and penetration distance (B) of Pacific whiting surimi gel as 458 affected by SPP concentration, EDTA, and heating method. Error bars represent the standard 459 deviation of 6 determinations. control 0.5g/100g SPP 1g/100g SPP 460 461

462

463

464

465

466

467

468

469

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B A

22

60

65

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85L*

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b*

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ss (L

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*)

-4

-3

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474

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476

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479 480 481 482

483 484 485 486 487 488 489 490 491 492 493 494 Figure 3 - L* (A), a* (B), b* (C), and whiteness (D) values of Pacific whiting surimi gels as 495 affected by SPP concentration, EDTA, and heating methods. Error bars represent the standard 496 deviation of 6 determinations. 497 498 499 500 501 502 503 504 505 506 507 508 509 510

A B

C D

23

511 Figure 4 - TCA soluble peptide contents in Pacific whiting surimi gel as affected by SPP 512 concentration, EDTA, and heating method. Error bars represent the standard deviation of 3 513 determinations. 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540

0

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ol ty

rosi

ne/g

sam

ple

24

541 542 543

544 545 Figure 5 - SDS PAGE pattern of surimi gels without (A) and with (B) 0.1g EDTA/100g as affected 546 by SPP concentration and heating methods. MW=molecular weight marker, C=control, 547 0.5=0.5g/100g SPP, 1=1g/100g SPP, MHC=myosin heavy chain, Ac=Actin, 20-250=molecular 548 weight in kDa. 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574

25

575 576 OH 60/OH 25/OH 577 578 579 580 581 582 Control 583 584 585 586 587 588 589 1g/100g 590 SPP 591 592 593 594 595 596 Control 597 EDTA 598 599 600 601 602 603 Figure 6 – Scanning electron microscope image of surimi gels. A = Ohmic heating with no SPP 604 added; B = 60°C holding for 30 min followed by ohmic heating with no SPP added; C = 25°C 605 holding for 2 hr followed by ohmic heating with no SPP added; D = Ohmic heating with 1g/100g 606 SPP added; E = 60°C holding for 30 min followed by ohmic heating with 1g/100g SPP added; F = 607 25°C holding for 2 hr followed by ohmic heating with 1g/100g SPP added; G = 25°C holding for 2 608 hr followed by ohmic heating with no SPP added and 0.1g EDTA/100g. Magnification = 10,000x. 609 610 611 612 613 614 615 616

A B C

D E F

G