Nutritional Composition Changes in Alaska Pollock (Gadus Chalcogrammus) During and Between Bering Sea a and B Seasons

Home , Chikuwa, Kamaboko, Milt, Pollock roe

AN ABSTRACT OF THE THESIS OF

Clara Hintermeister for the degree of Master of Science in Food Science and Technology presented on October 10, 2017.

Title: Nutritional Composition Changes in Alaska Pollock (Gadus chalcogrammus) During and Between Bering Sea A and B Seasons.

Abstract approved: ______Christina A. Mireles DeWitt

Alaska pollock (Gadus chalcogrammus) is a schooling whitefish native to the

Bering Sea that is prized for its fillets, surimi, roe, and milt. Fillets are frequently used for popular products such as fish and chips. If collected, roe and milt are commonly exported to South Korea and Japan. However, no markets currently exist for roe and milt in the United States.

The Alaska pollock fishery is one of the largest and most sustainably managed in the world. The fish are caught during two seasons in the year, season A and season B, which correspond to pre- and post-spawning periods in the fish reproductive cycle. To date, there has been no in-depth published data on seasonal changes in the composition of Alaska pollock fillets, roe and milt. In fact, there has been little data published on milt composition at all.

This study found that the nutritional composition of Alaska pollock changes significantly from season A to season B, with only small changes within seasons.

Fillets were higher quality in season B, with significantly higher protein, fat, vitamin

D, omega-3 fatty acids, and essential amino acid index (EAAI) scores than in season A

(p<0.05). Remarkably, the 1% increase in fat in season B correlated to a 20% increase in vitamin A, 99% increase in vitamin D and a 34 % increase in omega-3 fatty acids. The fat content in roe increased 71% from season A to season B, which correlated to significant increase in omega-3 and omega-6 fatty acids (p<0.05).

However, vitamin D in roe decreased from the start to end of season A, with significant differences between Feb 3 and Mar 31 catch dates (p<0.05). Vitamin D content was significantly lower (64%) in season B than in season A (p<0.05). Milt composition remained remarkably consistent throughout season A, with only sporadic changes in fat, moisture and ash content (p<0.05). No other compositional changes were observed. Mineral content in fillets, roe and milt did not change significantly by catch date or season. On a dry basis, roe and milt have high protein, vitamin, mineral and omega-3 content, which could allow for the development of new nutritional supplements or functional ingredients.

Nutritional Composition Changes in Alaska Pollock (Gadus chalcogrammus) During and Between Bering Sea A and B Seasons.

by Clara Hintermeister

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Master of Science

Presented October 10, 2017 Commencement June 2018

Master of Science thesis of Clara Hintermeister presented on October 10, 2017

APPROVED:

Major Professor, representing Food Science and Technology

Head of the Department of Food Science and Technology

Dean of the Graduate School

I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request.

Clara Hintermeister, Author

ACKNOWLEDGEMENTS

From family and friends to the faculty and staff of Oregon State University, thank you to everyone who has supported me during this journey. In particular, I would like to acknowledge the following people:

Dr. Christina DeWitt, for her endless support, encouragement, and knowledge. I couldn’t have asked for a better mentor. Give Rocket some belly rubs for me!

Dr. Jae Park, Dr. Jung Kwon, and Dr. Claudia Hase for taking time out of their busy schedules to be on my committee.

Sue Hansell and Craig Holt for always being there to help.

Silvana Harikedua, Ryan Smith, Megan Ooi, and Peter Scruggs for helping me with lab work and always putting a smile on my face.

Everyone who has been at the seafood lab over the past few years for making my time in Astoria enjoyable and memorable.

Michael Haupt for always going above and beyond to support me, make me laugh, and keep me on track.

CONTRIBUTION OF AUTHORS

Dr. Christina DeWitt was involved with project design, results interpretation, and editing of each chapter. Dr. Quentin Fong was involved with the design of the project presented in Chapter 3. Silvana Harikedua, Ryan Smith, Megan Ooi, and

Peter Scruggs were involved with data collection for Chapter 3.

TABLE OF CONTENTS

Page

1 Introduction ..………………………………………………………………………………………………….. 1

2 Literature Review.……………………………………………………………………………………………. 3

2.1 Alaska Pollock……………………………………………………….…………………………… 3

2.1.1 Basic Information…………………………………………………………………………. 3

2.1.2 Alaska Pollock Fishery…………………………………………………………….……. 4

2.1.3 Economic Impacts……………………………………………………………………….. 5

2.2 Utilization of Alaska Pollock……...………………………………………………………. 6

2.2.1 Current Commercially Available Products…………………...………………. 6

2.2.1.1 Fillets………………………….……………………………………………………. 6

2.2.1.2 Surimi and Surimi Seafood……………………………………………….. 8

2.2.1.3 Roe………………………………………………………………………………….. 9

2.2.1.4 Milt…………………………………………………………………………………. 11

2.2.1.5 Fish Meal and Fish Oil…………………………………………….……….. 11

2.2.2 Products Being Developed or Researched…………………………………… 13

2.3 Nutritional Composition of Alaska Pollock……..…..……………………………. 15

2.3.1 Whole Fish………………………………………………………………………………….. 15

2.3.2 Fillets………………………………………………………………………………………….. 16

2.3.3 Roe…………………………………………………………………………………………….. 17

2.3.4 Milt…………………………………………………………………………………………..… 17

2.4 Analytical Methods………………………………………………….………………………. 18

TABLE OF CONTENTS (Continued) Page

2.4.1 Fat……………………………………………...... 18

2.4.2 Protein………………………………………………………………………………….……… 21

2.4.3 Moisture……………………………………………………………………………………... 25

2.4.4 Ash……………………………………………………………………………………..………. 25

2.4.5 Fat-Soluble Vitamins……………………………………………………………..……. 27

2.4.6 Fatty Acids………………………………………………………………………………..… 29

2.4.7 Amino Acids………………………………………………………………………..…..…. 30

2.4.8 Minerals……………………………………………………………………………………… 32

2.5 Dietary Importance………………………………………...…………………………..….. 32

2.5.1 Roles in the Body…………………………………………………………………….…. 32

2.5.1.1 Protein and Amino Acids……………………………………………..…. 32

2.5.1.2 Fat and Fatty Acids..………………………………………….………..….. 34

2.5.1.3 Fat Soluble Vitamins…………………………………………………..….. 36

2.5.1.4 Minerals…………………………………………………………………..……. 37

2.5.2 Recommended Consumption……………………………………..…………..… 39

3 Nutritional Composition Changes in Alaska Pollock (Gadus chalcogrammus) During and Between Bering Sea A and B Seasons……………………………………..…….. 41

3.1 Abstract……………………………………………………………………………..…..……… 42

3.2 Practical Application….....…………………………………………………………....… 42

3.3 Introduction………………………………………………………………………………..... 43

3.4 Materials and Methods……………………………………………………………….… 45

TABLE OF CONTENTS (Continued) Page

3.5 Results and Discussion………………………………………………………..…………..... 51

3.6 Conclusion…………………………………………………………………………………………. 61

3.7 References…………………………………………………………………………………………. 63

4 General Conclusion……………………………………………………………………………………..…… 79

Bibliography ……………………………………………………………………………………..……………….. 81

Appendices …………………………………………………………………………………………………..…… 92

LIST OF FIGURES

Figure Page

1. Figure 3.1 - HPLC chromatograms from Alaska pollock fillets, roe, and milt. The different wavelengths were overlaid. Vitamin A was viewed at 325 nm, vitamins D2 and D3 were viewed at 265 nm, and vitamin E was viewed at 296 nm ……………………………………………………………………………………………………………..… 77

2. Figure 3.2 - Principle component analysis component scores comparing the amino acid composition of fillets, roe, and milt. Samples labeled 1 are from Feb 3, 2 are from Feb 17, 3 are from Mar 3, 4 are from Mar 16, 5 are from Mar 31, 6 are from Jul 15, and 7 are from Aug 15.………………………………………….....………….…. 78

LIST OF TABLES

Table Page

2.1 Recommended Daily Intake (RDI) of vitamins and minerals relevant to Alaska Pollock for adults and children ages 4 and older...... 39

2.2 Daily Reference Value (DRV) of food macronutrients for adults and children ages 4 and older……………………………………………………………………………………………. 40

3.1a Mean values of Alaska Pollock fillet, roe, and milt proximate composition on a wet basis throughout Seasons A and B in 2015……………………………………….………. 68

3.1b Mean values of Alaska Pollock fillet, roe, and milt proximate composition on a dry basis throughout Seasons A and B in 2015…………………………………………….…… 69

3.2 Mean values of Alaska Pollock fillet, roe, and milt vitamin content per serving on a wet basis and % daily values (% DV) throughout the A and B Seasons in 2015. The serving size for fillets was 85 g, and the serving size for both roe and milt was 15 g…………………………………………………………………………………………………………………..… 70

3.3 Total saturated fatty acids (SFA), omega-3 fatty acids, omega-6 fatty acids, omega-9 fatty acids, and undifferentiated monounsaturated fatty acids (MUFA) on a mg/ serving basis of Alaska pollock fillets, roe and milt during and between seasons A and B in the Bering Sea. Servings sizes were 85 g for fillets and 15 g for roe and milt………………………………………………………………………………………………………… 71

3.4 Amino acid scores for essential amino acids and the essential amino acid index (EAAI) for Alaska pollock fillets, milt and roe by catch date and season. Amino acid scores and EAAI values were calculated using amino acid requirements for preschool-aged children as the reference protein………………….. 72

3.5 A comparison of amino acid requirements for adults as determined by the WHO (2002) and the average seasonal amino acid content of Alaska pollock on a mg/g protein basis………………………………………………………………………………….………. 73

3.6 Mean amino acid content for fillets, milt and roe from all catch dates…….….. 74

3.7 Iron (Fe), sodium (Na), calcium (Ca), magnesium (Mg), and copper (cu) content per serving for Alaska pollock fillets, roe and milt on a wet basis. The serving size for fillets was 85 g, and the serving size for both roe and milt was 15 g. % Daily values (% DV) were rounded based on FDA rules………………….………. 75

LIST OF TABLES (Continued)

Table Page

3.8 Alaska pollock fillets, roe and milt macronutrients and micronutrients based on serving size for nutritional supplements (25 g), calculated on a dry basis…….76

1. Introduction

Alaska pollock (Gadus chalcogrammus) is an abundant whitefish found in the northern parts of the Pacific Ocean. Although there are other designated catch regions, most are caught in the Bering Sea. As the world’s largest and most sustainably-managed whitefish fishery, its economic impact and consumption are global. Products from Alaska pollock are consumed widely in Asia, Europe, and North

America. Alaska pollock is roughly 45% of the catch volume of Alaska fisheries, and averages a wholesale value over one billion dollars per year (Strong and Criddle

2013). The value of the Alaska pollock fishery is not just company profits, but also the creation of jobs in all levels of production. People come from all over the United

States and other countries to work in the Alaska fishing industry.

The Alaska pollock fishery is managed by allowing fish to be caught during two periods throughout the year. The first period, referred to as season A, occurs from late January until the total allowable catch (TAC) for the season is harvested, usually in early April. The second period, referred to as season B, occurs from early June until that TAC for that season is harvested, typically in early October. Season A and season B roughly correspond to pre-and post-spawning periods in the fish life cycle.

Thus, season A is known for roe production in addition to fillet and surimi production. Season B is primarily for fillet and surimi production. Byproducts are generated in both seasons.

Alaska pollock is processed primarily into either fillets, surimi, or mince. Fillets are frequently breaded, frozen, and fried. Fish mince is used to make fish sticks or

patties, which are also breaded and fried. Despite widespread consumption, there has not been any published data on fillet compositional data over the course of the two catch seasons. Alaska pollock roe and milt are the reproductive organs and are exported to Japan and South Korea for consumption. Popular products are salted and seasoned roe, called tarako or mentaiko in Japan and myongran in Korea, or milt prepared in a variety of ways, called shirako in Japan and goni in Korea. However, in recent years the market for American-caught Alaska pollock roe and milt has been less competitive with the growth of Russian markets. Therefore, American processors are interested in finding new uses for roe and milt to appeal to different consumers.

Thus, the main goal of this project was to determine how the nutritional composition of Alaska pollock fillets, roe, and milt changed within and between seasons by looking at proximate composition, fat-soluble vitamins, fatty acids, amino acids, and minerals. Implications of this research vary by sample type. Regarding fillets, changes in proximate composition could affect processing yields. Changes in fatty acids, vitamins, amino acids and minerals could also affect nutritional value and be used as marketing tools. For roe and milt, nutritional data from throughout the season could be used to develop alternative markets and create higher value products such as nutritional supplements and functional ingredients.

2. Literature Review

2.1 Alaska Pollock

2.1.1 Basic Information

Alaska pollock (Gadus chalcogrammus, formerly Theragra chalcogramma) is a species of schooling whitefish native to the North Pacific, ranging from central

California to the Bering Sea, and into the Sea of Japan (Rodger 2006). Only those fish caught in Alaskan waters may be called Alaska pollock. However, fish of the same species may be referred to as walleye pollock, Pacific pollock, Pacific tomcod, pollock, or pollack regardless of where they were caught. It should be noted that despite the similar common names, Alaska Pollock should not be confused with fish of the Pollachius genus, which are also referred to as pollock. In fact, Alaska Pollock is more closely related to cod, Gadus morhua, than Pollachius spp.

Alaska Pollock are lean, fast-growing fish that can reach up to 105 cm in length and to 6.5 kg in weight, and can live 22 years (NOAA Fisheries Service 2010).

However, most fish caught are smaller and younger. Alaska Pollock have three dorsal and two ventral fins in addition to their pelvic and pectoral fins. Their lower jaw slightly protrudes. In contrast to their pale bellies, their sides and backs are darker and speckled with dark green and brown patches.

The diet of Alaska Pollock is variable based on size, season and region.

Juveniles eat primarily zooplankton including copepods and various euphausiids

(NOAA Fisheries Service 2010). As Alaska Pollock grow and enter adulthood, they begin to include smaller fish and sometimes juvenile Alaska Pollock into their diet.

Regardless of age, Alaska Pollock tend to feed more heavily in warmer months to build up a lipid reserve to sustain them through the winter and spring.

Alaska Pollock can be found in both shallow and deep water, but are mostly found at 100-300 m deep. They are regarded as semi-demersal fish because their tendency to increasingly swim closer to the ocean floor as they age (NOAA Fisheries

Service 2010).

For the majority of Alaska Pollock, sexual maturity is reached around 4 years old (NOAA Fisheries Service 2010). In the Bering Sea, spawning occurs in early spring.

Batches of eggs are released multiple times during the spawning period as they mature. This is called batch spawning. At any given time there will be a “reserve fund” of yolkless, immature eggs in the ovary regardless of how developed the other eggs in the ovary are. These immature eggs become the next batch to mature (Stahl

2004). The eggs are fertilized by the males once released by the female. Eggs will hatch 1-3 weeks after fertilization, and the young are referred to as larvae.

2.1.2 Alaska Pollock Fishery

The Alaska Pollock fishery is the largest single species fishery in the world intended for food. It is divided into separately-managed stocks, including the Bering

Sea and Aleutian Islands (BSAI) stock and the Gulf of Alaska (GOA) stock. The Bering

Sea stock will be the focus of this thesis. 90% of Alaska Pollock caught in the U.S. is caught in the Bering Sea (Fissel et al. 2016)

Fishing in the Bering Sea stock is divided into two seasons, called the A season and the B season. The A season begins January 20th and continues through early-mid

April. It is associated with roe production, as spawning takes place in early spring.

The B season, which is officially from June 10th – November 1st, occurs post- spawning (Ianelli and others 2015). Alaska pollock are caught by trawling, which is done by dragging a large conical net behind a boat.

Each year the total allowable catch (TAC) for the Alaska Pollock fishery is determined by the North Pacific Fishery Management Council and the National

Marine Fisheries Service. It is usually between 1.1 and 1.5 million metric tons per year (“Harvest Specification Tables | NOAA Fisheries Alaska Regional Office” 2017).

A portion of the TAC is set aside for the Western Alaska Community Development

Quota. The remaining TAC is divided among the inshore processors, catcher/processors, and vessels harvesting for processing by motherships, while also factoring in incidental bycatch from other fisheries (Strong and Criddle 2013).

2.1.3 Economic Impacts

In 2015, the Alaska Pollock fishery comprised 67% of the total Alaska groundfish catch, equating to a harvest of 1.5 million metric tons and a value of $480 million. Prices for fillets dropped to $1.40/pound, but prices rose to $1.12/pound for surimi. Roe, which is primarily shipped overseas to Asia, saw a 9% decrease in production and 23% decrease in price to $2.15/pound (Fissel et al. 2016).

Alaska Pollock products are popular in the North America, Asia, and Europe.

The total wholesale revenue of Alaska Pollock products from fish harvested in the

United States from 1999 to 2010 averaged to $1.1 billion per year. This included fillets, roe, surimi, fish meal, etc (Strong and Criddle 2013).

2.2 Utilization of Alaska Pollock

Alaska Pollock contributes a wide variety of products from both primary and secondary processing to the seafood market in the United States and abroad.

Approximately half of the products produced from Alaska Pollock are fillets, one quarter surimi and one fifth roe (“Alaska Pollock | FishWatch” 2016). Though there is a large and established market for Alaska Pollock fillets and roe, the majority of the fish - the frames, viscera, skin, fillet trimmings, and heads - is leftover. Because discarding this processing waste at sea can have negative environmental impacts, and because discarding wastes at land-based processing facilities can be costly, there is increasing interest in the utilization of the remainder of the Alaska Pollock for other purposes. Likewise, there is also legislation regulating the amount of waste that can be discarded at sea. In this section, both the current commercially available

Alaska Pollock products will be discussed, along with by-products being researched and developed.

2.2.1 Current Commercially Available Products

2.2.1.1 Fillets and Fish Mince

Alaska Pollock may be caught, frozen, and then brought to land-based processing facilities where they are thawed, processed and refrozen. It is also common for them to be both caught and processed at sea. These catcher-processor vessels have the capability to quickly and automatically process Alaska Pollock into frozen blocks of fillets, individually quick frozen (IQF) fillets, mince or surimi. The rapid turnover from catch to processing on the vessels allows for a higher quality product, as the fillets are only frozen once. Repeatedly freezing and thawing can lead to quality defects in a variety of sensory attributes, including flavor, texture and aroma (“Genuine Alaska Pollock Producers” 2016). However, in value-added products, where twice-frozen fillets are the norm due to the extra processing requirements, these quality changes are not significant enough to be detectable by consumers (Ovissipour, Rasco, and Bledsoe 2014).

Fish mince is a versatile product created from the by-products of fillet production. The process of filleting Alaska Pollock leaves behind a large amount of edible fish flesh from the fillet trim, frames, tongue and cheeks. These parts can be processed into a mince, and may also include scraps from other species of fish. In the case of fish mince sold directly to consumers, it may be necessary to modify color, texture or a variety of other properties to increase acceptability. (Marsh and

Bechtel 2012). Likewise, special care must be taken during processing steps to prevent lipid oxidation, enzymatic activity, or bacterial contamination due to the mixing of multiple fish parts.

The majority of Alaska Pollock fillets sold to consumers are processed into value added products. Particularly in American and European markets, fillets are minced, reshaped, seasoned in some manner, and sold frozen. Typical products found in grocery stores include fish burgers, breaded fillet patties and fish sticks, and battered and fried fish as part of “fish and chips.” Alaska Pollock is also commonly served in fast food restaurants and by food service companies, particularly as breaded patties. These fillet products are popular due to their mild flavor, flaky texture, and nutritional value as a low-carbohydrate, low-fat source of protein

(“Alaska Pollock | FishWatch” 2016). Additionally, the Moscow Plekhanov Institute of the National Economy has prepared food products by adding vegetable fillers and bone tissue. This contributes phosphorus and calcium to fish mince (Marsh and

Bechtel, 2012). Thus, there is potential to use fish mince to create alternative burger patties or other innovative products, creating more revenue for companies while simultaneously reducing processing waste.

2.2.1.2 Surimi and Surimi Seafood

Surimi seafood is the largest product category created from Alaska Pollock fillets or headed and gutted fish. The first step of creating surimi seafood products is creating surimi, a paste made from isolated and stabilized myofibrillar protein. The steps for surimi production are as follows: 1) heading and gutting the fish to remove sources of proteolytic enzymes, 2) mincing and deboning, 3) washing and dewatering to remove water-soluble proteins, impurities, and fat, 4) refining to remove remaining impurities such as skin, pin bones, scales, and connective tissue,

5) passing the meat through a screw press to remove excess water from washing process, 6) adding cryoprotectants such as sucrose, sorbitol, sodium tripolyphosphate, and tetrasodium pyrophosphate to impart stability during frozen storage, 7) freezing (typically in a 10 kg block) with a plate freezer, and 8) metal detection, which is the only critical control point during surimi production (Park et al. 2014).

Surimi is often made by a primary fish processor, though sometimes mince can be purchased and then processed into surimi. Surimi is processed into surimi seafood, which are the cooked, ready-to-eat products of the gelled myofibrillar protein (Park 2015; Park and Lin 2005). In the United States, the most popular example of surimi seafood is imitation crab meat. However, in Asia, other types of surimi seafood are popular, such as fish balls (Southeast Asia), ah-mook (South

Korea), and kamaboko, chikuwa, and hanpen (Japan) (Park 2005). Although surimi is made from a number of fish species, the Alaska Pollock fishery is the largest contributor to the industry (Guenneugues and Morissey, n.d.). Much of the surimi industry is centered around Asian consumers and a large portion is exported, but interest in surimi-based products in the United States may increase with development of innovative applications and marketing.

2.2.1.3 Roe

Roe, sometimes referred to as hard roe, is the group of eggs in the ovaries of a female fish. As with most fish species, female Alaska Pollock have two ovaries, and

roe is obtained from each fish as a pair of connected egg skeins. The eggs are small, and it can be hard to distinguish individual eggs while they are still in the skeins. On average, eggs in harvested roe are 1.3-1.5 mm in diameter (Bledsoe, Bledsoe, and

Rasco 2003). Although there is variation from fish to fish, Alaska Pollock roe is generally reddish in color, ranging from pink to orange. This color may be a reflection of maturity level of the ovary; ovaries with more yolked eggs tend to be more consistently orange than ovaries with fewer or no yolked eggs, which display much more color variability. During spawning, eggs are more transparent due to increased hydration and the ovarian wall is very thin and stretched out. However, diet may also affect the color of the ovary, so color is not the sole indicator of maturity level (Stahl 2004).

Alaska Pollock roe is frequently consumed in South Korea and Japan, so exporting roe as tarako or mentaiko can be a lucrative business for Alaska Pollock processors. Both tarako and mentaiko are salted roe; however, mentaiko may or may not also contain other seasonings. Both are served as intact pairs of skeins and graded based on texture and appearance, i.e. firmness, color, size and shape.

Bruising, separation of skeins, or large veins all decrease the quality and lower the grade of the roe (Bledsoe, Bledsoe, and Rasco 2003; Hui 2006; Ovissipour, Rasco, and Bledsoe 2014). Another popular use of mentaiko is in sushi or rice balls. In this case, the membranes surrounding the eggs would be removed. Roe is harvested with quality in mind, as the price is affected by the grade. Alaska Pollock roe may also be used as an ingredient in salad dressing or dried and used as seasoning for a

variety of products, including sea vegetable snacks and salads. It is also commonly substituted for cod roe in a variety of products, such as “Norwegian Peanut Butter,” a blended paste of roe and cream cheese. Eggs may also be removed from their skeins and served as a caviar product. There is growing interest in caviar-style Alaska

Pollock roe, particularly in the European market (Bledsoe, 2003). Alaska Pollock roe is not yet a popular product among American consumers, though it can be found in the freezer section of large Asian grocery stores in the United States.

2.2.1.4 Milt

Milt, the male reproductive organ, is also referred to soft roe (Medina 2012).

However, in this thesis only the term milt will be used. Milt is filled with pale, creamy seminal fluid and resembles mammalian small intestines in shape.

In Japan, milt is called shirako and is considered a delicacy. Traditionally,

Atlantic or Pacific cod milt (Gadus morhua or Gadus microcephalus, respectively) is served as shirako; however, given the similarities between Alaska Pollock and cod,

Alaska Pollock would be an easy substitution. Appearance-wise, cod and Alaska

Pollock milt are very similar. According to Medina (2012), milt has a creamy texture and can be eaten raw or cooked, fried, pickled or smoked. In the American market, milt is often discarded or processed with the viscera.

2.2.1.5 Fish Meal and Fish Oil

Fish meal and fish oil are well-established by-products from the fishing industry and traditionally used in feed for aquaculture, pets and farm animals. Fish meal and fish oil are produced concurrently from the remaining portions of the fish after being processed for human consumption. The first step of production is cooking the fish, often by steaming. The solid portion that forms is referred to as a presscake and, as the name suggests, the solids are pressed to remove as much liquid as possible. The presscake contains mainly bone and coagulated protein, whereas the liquid section, called the press liquor, contains water, oil, and suspended solids such as proteins, vitamins and minerals. Press liquor can be separated into oil, stickwater, and sludge by centrifugation or natural separation after allowing it to sit undisturbed (Fisheries and Aquaculture Department 2016).

To produce fishmeal, the presscake is dried and either ground into a powder or pelletized. Fishmeal yield can be increased by adding sludge from the press liquor.

Sometimes the stickwater is concentrated by evaporation and added to the fishmeal, but the stickwater concentrate may also be sold on its own as condensed fish solubles (Fisheries and Aquaculture Department 2016).

Fish oil is produced from the press liquor. After separation from sludge and stickwater components of the press liquor, the oil is refined and used in animal feeds or human supplements (Fisheries and Aquaculture Department 2016). Fish oil is often consumed as a source of n-3 fatty acids, particularly eicosapentaenoic acid

(EPA) and decosahexaenoic acid (DHA). Fish oil can also be used as a fuel source.

2.2.2 Products Being Developed or Researched

The majority of fish products being developed or researched are related to by-products and maximal utilization of the fish.

Fish Protein Isolate (FPI) is a product created by solubilizing and extracting fish protein using pH shifts. The fish is heat treated under alkali or acidic (less common) conditions to alter the muscle pH from 7 to 10 (3 to 10 for acidic solubilization conditions) under dilute conditions, thus solubilizing the myofibrillar protein. The proteins are only kept at high temperature and pH for a short time period (approximately 5 minutes) to avoid damaging the proteins. The solubilized proteins are separated from insoluble matter such as bones and scales by centrifugation, and the pH is then shifted down to 5.5, the typical pI of muscle foods, to facilitate water removal (Hultin and Kelleher 1999). Although FPI has been around for nearly 20 years, it has not found a steady place in the market. Researchers are still working to learn more about its functional properties and to develop products that will be accepted by consumers.

FPI can be used to create surimi seafood products, though gelation of FPI occurs through a different mechanism than conventional surimi. There has been research looking into making nutraceuticals by fortifying surimi products created from FPI with n-3 fatty acids and dietary fiber, as well as using salt substitutes to reduce sodium content (Tahergorabi, Matak, and Jaczynski 2015).

Protein hydrolysates are another product created from fish by-products. Unlike FPI, fish protein hydrolysate (FPH) is made from breaking down proteins into small

peptides and amino acids. Because of this, protein hydrolysates may vary in functionality. FPH is not capable of forming a gel, and may or may not interact with other molecules. Factors affecting functionality include the size of the peptides, the amino acids present, and the pH of the solution. Protein hydrolysates can be used as emulsifiers, fat binders, foaming agents, and water binders to increase water holding capacity of a product. Despite the many possible uses, protein hydrolysates from fish add a bitter off-flavor to products (Pires & Batista, 2013). However, bioactive properties of FPH have been widely studied. It has been found that consumption of

FPH can decrease serum and liver cholesterol as well as acyl-CoA: cholesterol acyltransferase activity, which in turn increases the proportion of high density lipoprotein (HDL) cholesterol compared to low density lipoprotein (LDL). This characteristic was observed in both Alaska pollock and salmon FPH (Hosomi et al.

2011; Wergedahl et al. 2004). This is particularly desirable because high LDL cholesterol can cause heart attacks and strokes. It has also been found that FPH from Pacific hake (Merluccius productus), a gadoid fish similar to Alaska pollock, exhibited in vitro angiotensin converting enzyme (ACE)-inhibitory activity

(Samaranayaka, Kitts, and Li-Chan 2010). Such activity is important because ACE inhibitors are used for treating hypertension and congestive heart failure. FPH may also be useful as an antioxidant in the body. However, antioxidative capabilities are greatly reduced if the FPH contains larger quantities of oxidized peptides, indicating that production methods may affect this particular health benefit (Halldorsdottir et al. 2014). Therefore, FPH has great potential as a functional or nutraceutical food,

but more research is needed to find effective ways to incorporate FPH into food products that will be accepted by consumers.

2.3 Nutritional Composition of Alaska Pollock

Since Alaska pollock is a widely-consumed food fish, there have been several studies investigating their fillet composition. Alaska pollock roe has not been studied to the same degree as fillets, and published information on Alaska pollock milt was not found. The effect of seasonality on fillets, roe, and milt individually, has not been studied. However, information is available on seasonality of whole fish. Other

Gadoid fish such as cod, haddock and whiting are expected to have similar composition, so they can be used to estimate Alaska Pollock composition.

2.3.1 Whole Fish

There have been several studies on the composition of whole fish. Adult fish contain14.02% protein, 1.58% fat, 2.58% ash, and 82.55% moisture, and juveniles

70-78 mm in length were found to have similar composition (Payne, Johnson, and

Otto 1999). Kitts et al. (2004) studied both seasonality and gender differences of whole fish. There were no significant differences between genders, but the fish did display seasonal differences in crude lipid content. The fish had higher lipid contents in summer and fall, roughly 21-22% dry mass, than in the winter and spring, roughly

15-17.5% dry mass. This is because the fish feed heavily in warmer months to build up a lipid reserve to last through winter, as mentioned in Section 2.1.1.

2.3.2 Fillets

Fish fillets are a widely consumed product from Gadoid fish. The USDA nutrient database (2015) provides data on raw Alaska Pollock fillets, presenting information from fish purchased from 12 different stores based on a probability- based sampling plan. It does not specify when the fish were caught. For proximate analyses and fatty acids, 8 samples were tested. However, only 1-3 samples were tested for vitamin content. Thus, there is likely less confidence in the vitamin results than for the other tests. The proximate composition is listed as 86.75% moisture,

12.19% protein, 0.41% fat, and 1.36 % ash, with 0% carbohydrate. Alaska Pollock fillets contain all essential amino acids. Vitamins A and D are present in insignificant quantities (<2% Daily Value, or DV), though approximately 3% DV of Vitamin E could be obtained in a standard 85 g serving of Alaska Pollock. Most minerals in Alaska

Pollock fillets are also present in insignificant quantities. However, approximately

241 mg of phosphorus, 19% DV, and 13.5 µg of selenium, 24% DV, are present in an

85 g serving. This would allow for the fillets to be labeled as a good source of phosphorus and an excellent source of selenium. Of the fatty acids present, roughly half were polyunsaturated fatty acids, including EPA and DHA.

Because cod is a similar Gadoid fish, it can be used for insight on Alaska

Pollock composition. According to Jangaard, Ackman and Sipos (1967), phospholipids make up approximately 80-86% of all lipids in cod fillets. They found that fatty acid variation was high from fish to fish, that there were no significant

differences between fillets from male and female fish, and that the minimal seasonal changes were mostly found in the triglycerides.

2.3.3 Roe

Chiou, Matsui and Konosu (1988) compared raw and prepared Alaska Pollock roe. In this study, they removed the ovary membranes for analysis. They found that raw Alaska Pollock roe contained 71.8% moisture, and on a dry basis 78.7% protein,

6.7% lipid, and 8.2 % ash. The prepared roe, called tarako, contained only 64.7% moisture, and on a dry basis, 70.3% protein, 6.0% lipid, and 19.6% ash. The salting process caused the significant increase in ash content due to addition of minerals.

For both raw and prepared roe, most amino acids were free rather than in proteins, indicating that the calculated amount of total protein may actually be from amino acids. The major inorganic ions in raw and salted roe were phosphate, chloride, sodium, potassium, calcium and magnesium. In cod, it was found that the fatty acid composition of the roe remained constant during development (Jangaard, Ackman, and Sipos 1967). However, the gonads were found to increase in percent body weight when approaching spawning (Eliasson 1982; Jangaard, Ackman, and Sipos

1967).

2.3.4 Milt

Milt has not been as widely studied as fillets and roe. However, there is still insight into its composition. Alaska pollock milt was studied in the context of

producing a dried meal product. Proximate composition of raw milt was determined to be 12.7% protein, 3.1% lipid, 1.8% ash, and 82.4% moisture. The milt was dried until it only contained 6% moisture before undergoing compositional analysis. The dried product contained 3.10 ppm copper and 33.50 ppm iron. Dried Alaska pollock milt contained relatively low histidine, methionine and phenylalanine when compared to standard fishmeals. The majority of the fats present in the milt were phospholipids (82%), and the dominant fatty acids were C16:0, C18:1n9c, C20:5n3, and C22:6n3 (Plante et al. 2008).

Interestingly, one study about inshore cod from Nova Scotia found that there was no relationship between milt maturity and fatty acid composition. The total lipids accounted for 0.9-2.3% of the milt, with 18:1n9, 20:5n3, 16:0, and 22:6n3 being the major fatty acids present (Jangaard, Ackman, and Sipos 1967). This was similar to what was observed in the study by Plante et al. (2008).

2.4 Analytical Methods

2.4.1 Fat

Commonly used fat extraction methods used for fish and fish products include acid hydrolysis, Folch or Bligh and Dyer methods, and Soxhlet extraction.

Choice of procedure depends on the amount of lipid, type of lipid (i.e. phospholipids or triglycerides), cost, and time constraints.

Acid hydrolysis uses hydrochloric acid (HCl) to digest samples. The samples are refluxed in the acid, which breaks covalent and ionic bonds between fats and other cellular components. This not only releases fats from the food matrix, but also breaks the molecules into smaller, easily extractable pieces. Due to the polar head of the molecule, phospholipids are somewhat water soluble and will tend to form a layer between the aqueous and organic phases during an extraction. Thus, ethanol is often added to the sample after digestion to force phospholipids into the organic layer for maximal extraction (Min and Ellefson 2010). In AOAC method 948.15, after the acid hydrolysis is complete, the fats are extracted using a mixture of diethyl ether and petroleum ether in Mojonnier flasks (Hungerford 1995). The design of the

Mojonnier flask allows for easy separation of the aqueous and organic phases by trapping the aqueous phase in a small compartment at the bottom of the flask. The organic phase containing the fats can easily be poured into collection flasks. Once the extraction steps have been repeated three times, the ether in the collection flasks is boiled off, leaving the fat behind. The fat is then dried and weighed.

The choice of solvents is critical to the success of this extraction procedure following acid hydrolysis. Multiple solvents of different polarities are often used because lipids also vary in polarity. Diethyl ether- petroleum ether is a commonly used solvent combination because diethyl ether is more polar than petroleum ether, allowing for extraction of both polar phospholipids and relatively nonpolar triglycerides, respectively (Min and Ellefson 2010). Regarding lean fish, the ability to extract phospholipids is critical to obtaining accurate fat content data because most

fat in the muscle comes from phospholipids in cell membranes (Jangaard, Ackman, and Sipos 1967).

Chloroform and methanol are used as extraction solvents in the Folch method and Bligh and Dyer method, though in these methods there is no hydrolysis step prior to extraction (Bligh and Dyer 1959; Folch, Lees, and Sloane Stanley 1957).

Both the Folch and Bligh and Dyer methods involve homogenizing sample in chloroform and methanol to extract fat, and then removing the remaining solids via filtration. Phase separation is induced by the addition of salt, and the chloroform layer, which contains the fats, is removed. The chloroform is then evaporated and the amount of fat is determined by weight (Min and Ellefson 2010).

The Bligh and Dyer method is a modified, rapid version of the Folch method.

Whereas the Folch method can be used on smaller samples, the Bligh and Dyer method was designed on larger, high-moisture cod muscle samples. However, according to Bligh and Dyer (1959), their method could be used on any sample containing 80% water. It is now a recommended method for measuring fat content in biological tissues (Iverson, Lang, and Cooper 2001). In a comparative study by

Iverson et al. (2001), it was concluded that the Bligh and Dyer method and the Folch method produce similar results with samples with <2% fat. However, the Bligh and

Dyer method increasingly underestimates true fat values in samples with increasing amounts of fat.

Soxhlet extraction is a semi-continuous batch method of extracting fat from a sample. In the traditional method, solvent, most commonly anhydrous ethyl ether, is

heated and condensed. The condensed solvent drips into an extraction chamber containing the sample. The fat is extracted as the solvent passes through the sample. When the chamber is completely full, the solvent is siphoned off and returned to the boiling flask. This process of condensation, extraction and siphoning continues for approximately 4 hours and the extracted fat collects in the boiling flask. The percent fat of the sample can either be determined by drying and weighing the boiling flask or drying the remaining sample and determining the difference in weight (Min and Ellefson 2010). Automated Soxhlet extraction is now possible, which reduces the amount of solvent and time necessary for analysis. High pressure, ultrasound, and microwave-assisted procedures have also been developed

(Luque de Castro and Priego-Capote 2010). However, even with more advanced

Soxhlet methods, analysis is time consuming and not as sensitive as the acid hydrolysis method.

2.4.2 Protein

In nutritional testing, estimation of crude protein content is done by measuring total nitrogen and multiplying by a nitrogen-to-protein conversion factor. There are two widely-used and accepted methods for determining total nitrogen: Kjeldahl and

Dumas. These methods use different approaches to determine nitrogen content, but ultimately yield similar results with proper calculations.

The Kjeldahl method of nitrogen determination has been widely used since its conception and is considered a standard method for nutritional analysis.

Originally developed by Johan Kjeldahl in 1883, the method was novel because it converted organic nitrogen into ammonia (NH3), an easily quantifiable compound.

The method can be condensed into three steps: digestion with sulfuric acid, neutralization, distillation and titration. Kjeldahl’s method was demonstrated to be fairly simple, accurate, and time efficient in that many samples could be analyzed in one day (Kjeldahl 1883). However, by today’s standards, the method is considered time-consuming even with semi-automation.

Although the method has been modified and improved since its initial development, the basic steps of digestion, neutralization, distillation and titration remain the same. Samples are digested with sulfuric acid, an oxidizing agent. A metallic catalyst, commonly copper, is added to the digestion flasks to speed up digestion. Likewise, potassium sulfate is also added to speed up digestion by increasing the boiling point of the sulfuric acid. The result of the digestion process is formation of water and carbon dioxide from carbon and hydrogen atoms, and most importantly, the formation of ammonium sulfate from the reaction of amine nitrogen and sulfuric acid. Following digestion, the samples are diluted with water and the acid is neutralized with the addition of sodium hydroxide. This neutralization step shifts the pH of the solution and favors formation of ammonia from ammonium. Since ammonia boils at -33°C (Pubchem 2016), it is in the gas state and easily separated from the remainder of the sample through distillation. The ammonia is distilled directly into a trapping solution containing boric acid and indicator solutions. Because the trapping solution is acidic, the ammonia removes a

hydrogen atom from the boric acid, forming ammonium and borate ions. In the final titration step, a standard acid, such as HCl, is used to titrate against the negatively charged borate ions (Chang 2010).

The Dumas method was originally devised in 1831, decades prior to the development of the Kjeldahl method, but it was not an immediate success because of low accuracy and reproducibility of results. However, with modern technology it is beginning to supersede the Kjeldahl method as the preferred standard method for nitrogen determination. In the Dumas method, samples are combusted with pure oxygen at a temperature between 950 and 1050°C (Moore et al. 2010). Nitrogen is converted into N2 and nitrogen oxides, and using a copper column the nitrogen oxides are reduced to N2 as well. Then, using helium as the carrier gas, the nitrogen is quantified using gas chromatography and a thermal conductivity detector (Chang

2010). The entire procedure can be automated and many samples can be run quickly and consecutively without attention. The major disadvantages in comparison to the

Kjeldahl method is the high initial cost of equipment and inclusion of inorganic nitrogen in quantification (Chang 2010; Moore et al. 2010). However, in the long- term, using the Dumas method is cheaper than the Kjeldahl method (Moore et al.

2010).

For either method, a conversion factor is needed to calculate the percent protein from the total nitrogen. The factor historically used for seafood and meat products is 6.25, based on the assumption that protein is 16% nitrogen. Certain other food groups, such as milk and wheat, have slightly different amounts of

protein nitrogen, which is reflected in their respective conversion factors (Chang

2010). Variation in protein nitrogen is due to expected proportions of each amino acid residue, since histidine, lysine, arginine, asparagine and glutamine all contain nitrogen in their side chains. However, some research suggests that in general, these conversion factors may be too high, since organic nitrogen is also found in nucleic acids, free amino acids, and urea. These other sources could contribute up to 20% of the total nitrogen in the sample (Boisen, Bech-Anderson, and Eggum 1987), of which

0.1-9.6% is likely nucleic acid (Sosulski and Imafidon 1990). Diniz et al (2013) calculated the average nitrogen-to-protein conversion factor for nine coastal

Brazilian fish from the ratio of the mass of amino acid residues to the total nitrogen, and concluded 5.71 was a more appropriate factor. Similarly, after reviewing several studies and data sets, Mariotti, Tomé, and Mirand (2008) concluded the 6.25 conversion factor should be reduced to 5.60 to account for the other organic nitrogen sources. However, they acknowledge that changing such a long-standing value across the entire industry would be a difficult task, and to date, 6.25 remains the convention

It should be noted that use of these conversion factors with total nitrogen from the Dumas method would yield slightly higher results than with total nitrogen from the Kjeldahl method. This is because the Dumas method’s nitrogen measurements also include inorganic nitrogen. As a result, many researchers use a coefficient along with the conversion factor to correct for the extra nitrogen from

the sample, resulting in roughly equal results between the two methods (Moore et al. 2010).

2.4.3 Moisture

Understanding the moisture content and total solids of a sample is important not only for determining sample composition, but also for determining proper storage and processing methods to ensure product safety and quality. Water in food samples can be found in three forms: free water, adsorbed water, and water of hydration (Bradley 2010). Adsorbed water, which is the water bound by proteins or included in cells, is particularly important in fish and meat products.

The Association of Analytical Chemists (1995) has published an official method for the determination of moisture in meat products by air drying (Method

950.46 B). In this method, a homogenous sample is weighed, dried in an uncovered dish for 16-18 hours at 100-102°C, cooled in a desiccator, and re-weighed. The difference in initial and final weights is reported as the moisture, and the weight of the dried sample is the total solids.

2.4.4 Ash

Ash is the residue that remains behind after combustion of the organic matter in a food sample, and serves as an approximation of the total mineral content. Ash content is often a slight overestimation of total mineral content because the minerals are in forms such as metal oxides, phosphates, and chlorides, among

others, as opposed to the pure forms (Miller 2008). This causes the weight of the ash to be slightly higher than the sum of the weights of individual minerals. However, the estimate suffices for determination of proximate composition. Ashing can be done with either a dry or wet procedure.

Dry ashing is the preferred choice of the two ashing procedures due to its simplicity and hands-off nature. Traditionally, dry ashing is done in a muffle furnace set between 500-600°C. The specific temperature is chosen based on the minerals expected to be present. Elements such as Cu, Fe, Zn, Hg and others volatilize at high temperatures (Marshall 2010). Because these elements are commonly found in seafood, the recommended ashing temperature for such products is ≤ 550°C

(Hungerford 1995). Ashing is complete when the ash is white, which indicates complete combustion of carbon. Samples higher in carbohydrate tend to require extra steps such ash filtration or addition of nitric acid to facilitate complete ashing

(Marshall 2010). Time needed for complete ashing depends on the composition of the sample and the discretion of the researcher. For example, Kitts et al. (2004) ashed samples of dried and homogenized whole Alaska Pollock for 32 hours at

550°C. In contrast, Bechtel (2003) ashed ground samples of wet or freeze-dried

Alaska Pollock and Pacific Cod whole fish, viscera, frames, skinless fillets, and skins for only 6 hours at 550°C.

Wet ashing is a procedure which involves use of acid and heat to digest samples. Wet ashing is primarily used as a preparatory procedure for specific mineral analysis rather than as a method for determining proximate composition.

However, it can be advantageous in samples where mineral volatilization is a concern (Marshall 2010).

2.4.5 Fat-Soluble Vitamins

High performance liquid chromatography (HPLC) is a common method for determining the presence and quantity of fat-soluble vitamins, vitamins A, D, E and

K. HPLC is a technique that separates compounds based on affinity for the mobile or stationary phase as samples are pumped through a column under high pressure.

Samples are injected via a valve injector, which operates on a loop to incorporate the sample into the mobile phase. The mobile phase and sample are then pumped into the column. As the sample components elute, they pass through a detector and a chromatogram is created. There are a variety of detection methods used in HPLC, including UV-Vis, fluorescence, refractive index and electrochemical (Reuhs and

Rounds 2010). Fat-soluble vitamin procedures primarily use UV-Vis detection, though fluorescence detection may be used for vitamin E.

Sample preparation for fat-soluble vitamins requires saponification and extraction steps. Saponification is the process of using alkali to break down neutral fats, called triglycerides, into their component glycerol and free fatty acids (O’Keefe and Pike 2010). The strongly basic conditions created by the alkali can also break down other large molecules such as proteins and carbohydrates. Thus, the fat- soluble vitamins A, D and E are freed from the matrix. Their non-polar characteristics allow for extraction from the other non-saponifiable materials, i.e. protein,

carbohydrates, sterols, minerals and water, which are much more polar. A non-polar solvent, typically n-hexane, diethyl ether, or a mixture of petroleum ether and diethyl ether is used for extraction. After extraction, the sample is concentrated if needed, filtered, and then injected into the HPLC. It should be noted that vitamin K is destroyed during the alkaline saponification conditions (Nollet 2013); however, because it is not present in significant quantities in fish, it is not relevant to this thesis.

To date, there are no official analytical methods for the simultaneous extraction and quantification of all fat-soluble vitamins at once for fish or meat products. However, there have been several published studies over the past three decades regarding successful extractions and quantifications of multiple fat-soluble vitamins at once. Problems that arose when developing such methods included vitamin oxidation and isomerization during saponification, interference from other compounds during separation, and widely varying vitamin concentrations (Nollet

2013; Qian and Sheng 1998; Zonta, Stancher, and Bielawny 1982).

In general, many fat-soluble vitamin analysis procedures for A, D, and E follow similar principles. A large proportion of vitamin analysis procedures involving vitamin D included a normal-phase clean-up step with reverse-phase quantification

(Brouwer et al. 1998; Mattila et al. 1995; Purchas et al. 2007; Salo-Väänänen et al.

2000). According to Nollet (2013), the clean-up step is used to remove interfering compounds in vitamin D analysis such as sterols and other vitamins. However, there are also successful procedures that did not include a clean-up step. In such cases

quantification was possible using either reverse-phase HPLC (Gomis, Fernandez, and

Guitierrez Alvarez 2000; Lopez-Cervantes, Sanchez-Machado, and Rios-Vazquez

2006; Qian and Sheng 1998; Stancher and Zonta 1983; Zonta, Stancher, and

Bielawny 1982) or normal-phase HPLC (Pozo et al. 1990). Given the low cholesterol quantities found in Alaska Pollock, it may be possible to forego a clean-up step because sterols absorb much less light than vitamin D at the detection wavelength used for vitamin D analysis, 265 nm (Nollet 2013).

2.4.6 Fatty Acids

To determine fatty acid content, fats from samples must be extracted, saponified and converted to methyl esters prior to analysis. A common extraction method is the Folch method which uses 2:1 mixture of chloroform: methanol (Folch, Lees, and

Sloane Stanley 1957; Huynh et al. 2007; Kitts et al. 2004; Ross, Van Nieuwenhove, and Gonzalez 2012). Saponification under heat and alkali, such as methanolic NaOH, is used to break the fats into their fatty acid components (Ross, Van Nieuwenhove, and Gonzalez 2012). Fatty acids are converted to fatty acid methyl esters (FAME) using boron trifluoride in methanol (Huynh et al. 2007; Kitts et al. 2004). Other esterification agents include acidic methanol and iodomethane (Kaspar 2009).

FAMEs can be separated and quantified using gas chromatography equipped with a flame ionization detector (FID) and a capillary column (Huynh et al. 2007; Kitts et al.

2004; Ross, Van Nieuwenhove, and Gonzalez 2012).

2.4.7 Amino Acids

Analysis by HPLC or GC are the most common methods for determining amino acid content in foods. Sample preparation generally consists of hydrolysis, purification and derivatization. Protein is most commonly hydrolyzed using a combination of acid and heat under vacuum or nitrogen, though alkaline hydrolysis with a strong base has also been successfully used. Problems during acid hydrolysis include partial oxidation of cysteine, deamination of glutamine and asparagine, and destruction of tryptophan (Aristoy and Toldra 2013; “EZ:faast User’s Guide,” n.d.). Most purification procedures use precipitation or solid phase extraction (Kaspar 2009).

Derivatization assists in the separation step by altering analytes so they can more easily be detected and ensuring reproducible results. Derivatizing agents can tag analytes or alter the analyte’s physical properties by making them less volatile or increasing thermal stability (Sellers 2010).

For liquid chromatography, derivatization can be done post-column or pre- column. In post-column derivatization, the free amino acids are separated in the column before a derivatization agent, such as ninhydrin or o-phthaldehyde (OPA), is added. The sample and derivatizing agents are mixed and then sent through the detector. This is a disadvantage over pre-column derivatization because mixing require extra equipment, and the addition of the derivatizing agent increases peak broadening by increasing the dead volume behind the column (Aristoy and Toldra

2013). In pre-column derivatization, the derivatization agent is added before the amino acids are separated. This method is more sensitive and selective than post-

column derivatization. For both post- and pre-column derivatization procedures, however, analysis times can be long depending on the compounds to be analyzed

(Kaspar 2009), which is a disadvantage when analyzing large numbers of samples.

Gas chromatography must be done with an FID or couple with mass spectroscopy

(MS), and derivatization must be done prior to injection. Sample preparation is slightly longer than with HPLC, but analysis is much more rapid and GC offers better resolution of analytes (Husek 1991; Kaspar 2009; Silva et al. 2003). Possible derivatization agents include N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) or

N-methyl-trimethylsilyltrifluoroacetamide (MSTFA), fluorinated alcohols, or alkyl chloroformates (Kaspar 2009). Alkyl chloroformates are the quickest derivatization reagents, making them a popular choice (Husek 1991; Kaspar 2009).

There are several commercially available kits for amino acid analysis, including the

Waters PicoTag Method and AccuTag Method (Milford, MA) and Phenomenex

(Torrance, CA). The Waters PicoTag method is based off the Edman degradation reaction which allows amino acids to be derivatized be phenyl isothiocyanate and analyzed by HPLC; the Waters AccuTag method uses 6-aminoquinolyl-N- hydroxysuccinimidyl carbamate as a derivatizing reagent and HPLC equipped with fluorescence detection (“AccQ•Tag Ultra, AccQ•Tag and Pico•Tag Columns” 2017;

Cohen 1989; Kaspar 2009). Phenomonex provides amino acid kits for GC-FID, GC-MS and LC-MS, using alkyl chloroformates as derivatizing reagents(“EZfaast Amino Acid

Information” 2017, “EZ:faast User’s Guide,” n.d.).

2.4.8 Minerals

Minerals are commonly analyzed using inductively coupled plasma in tandem with spectroscopy. Atomic emission spectroscopy (ICP-AES), optical emission spectroscopy (ICP-OES), and mass spectroscopy (ICP-MS) are frequently used.

Regardless of the method, sample preparation is similar. The samples should either be wet ashed or dry ashed and dissolved in dilute acid. Wet and dry ashing procedures were discussed in section 2.4.4.

ICP methods can be used to detect trace elements at the parts-per-billion level. ICP uses an argon torch, which creates an oscillating magnetic field. This environment is ideal for atomization and excitation of samples, which are introduced as an aerosol. From there, the samples are sent through a detector

(Miller and Rutzke 2010).

2.5 Dietary Importance of Nutrients

2.5.1 Roles in the Body

2.5.1.1 Protein and Amino Acids

Protein serves both structural and functional roles in the body. Consumption of protein is necessary in the diet because body proteins are repeatedly broken down and reconstructed, but not with 100% efficiency. Thus, some body proteins are lost in various metabolic processes and must be replenished. It is necessary to consume complete proteins or balance protein sources in the diet to obtain all necessary amino acids.

Amino acids can be divided into essential and non-essential. Non-essential amino acids can be synthesized by the body, while essential amino acids cannot. The following amino acids are essential for humans: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine (Smith 2010).

Protein quality is determined by amino acid content. Most animal proteins contain all essential amino acids at or above reference levels, so fish products are generally high quality protein sources. There are several methods for determining the quality of a protein. The simplest is the amino acid score which can be used to determine the first limiting amino acid of a protein. These scores are calculated by comparing the mg amino acid per g test protein to the mg of amino acid per g reference protein (Damodaran 2008). An extension of this method is the protein digestibility-corrected amino acid score (PDCAAS), which adjusts the score of the first limiting amino acid based on an in-vivo digestibility study. However, this is only required if a % Daily Value (% DV) is printed on the nutrition label (Smith 2010).

Another method is the essential amino acid index (EAAI), which factors in all essential amino acids and is calculated by the formula below:

푬푨푨푰

ퟗ 풎품 풍풚풔풊풏풆 풊풏 풕풆풔풕 풑풓풐풕풆풊풏 풆풕풄. 풇풐풓 ퟖ 풐풕풉풆풓 풆풔풔풆풏풕풊풂풍 = √( ) ∗ ( ) 풎품 풍풚풔풊풏풆 풊풏 ퟏ 품 풓풆풇풆풓풆풏풄풆 풑풓풐풕풆풊풏 풂풎풊풏풐 풂풄풊풅풔

2.5.1.2 Fats and Fatty Acids

In nutrition labeling, the “Fat” is a general term that refers to the lipid component of a food, where lipids are molecules that are insoluble in water. The most common example of fats are triglycerides, which are large, nonpolar, energy- storing molecules consisting of three fatty acids connected to a glycerol backbone via ester bonds. Although primarily characterized by their hydrophobicity, some types of lipids are amphipathic, meaning there is also a polar component to the molecule. These types of lipids are found in cell membranes and include phospholipids and cholesterol (Institute of Medicine Staff and National Research

Council, Subcommittee on Metabolism Staff 1989)

Consumption of fats is important for several reasons. First, fats are a concentrated source of energy, providing approximately 9 kcal per gram. Second, fats are the major structural component of cell membranes. The types of fats consumed in the diet can affect membrane fluidity and functionality. Fats also assist and in the transport and absorption of fat-soluble vitamins. Finally, some fats contain essential fatty acids, which are not synthesized by the body but necessary for several physiological functions and brain health (“Facts on Fats - Dietary Fats and

Health” 2015).

Saturated fatty acids ranging from 8-carbon to 18-carbon chains can be produced by the body and serve in cell membranes. They can also be consumed in the diet, primarily from animal sources. Monounsaturated fatty acids contain one double bond and are also important in myelin. Oleic acid (C18:1n9c) is 92% of

monounsaturated fatty acids consumed (Institute of Medicine, Food and Nutrition

Board 2005).

Although they are required for proper health and critical body functions, the human body cannot produce omega-6 or omega-3 fatty acids (Pigott and Tucker

1990). Linoleic acid (C18:2n6), an omega-6 fatty acid, and α-linolenic acid, an omega-

3 fatty acid, are both considered essential fatty acids and must be obtained through the diet. Arachidonic acid (C20:4n6), which can be produced from linoleic acid, may be considered a third essential fatty acid if there is not enough linoleic acid in the body. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are both important omega-3 fatty acids that can be synthesized from and α-linolenic acid

(Institute of Medicine Staff and National Research Council, Subcommittee on

Metabolism Staff 1989). However, EPA and DHA can also be consumed directly by eating fish and shellfish because they are present in fish phospholipids (Pigott and

Tucker 1990). It should be noted that recommended ratio of consumption for omega-6 and omega-3 fatty acids is 1:1 (McClements and Decker 2008). The average western diet, however, involves consuming 10 times more omega-6 than omega-3, which can cause or make chronic conditions such as heart disease, diabetes and cancer worse. Omega-6 fatty acids compete for uptake with omega-3 fatty acids, so even if adequate intake of omega-3 is met, it may not matter if omega-6 is still consumed at a high ratio (Simopoulos 2004).

Both linoleic and α-linolenic acid are important for maintenance of cell membrane, fluidity, structure and function. Omega-3 fatty acids in particular also

play roles in cell signaling, gene expression and eicosanoid metabolism (McClements and Decker 2008). Eicosanoids are signaling molecules that help regulate immune responses, cell growth, and blood pressure, among other functions. EPA in particular is important for maintenance of inflammatory responses and modification of platelet function (Institute of Medicine Staff and National Research Council,

Subcommittee on Metabolism Staff 1989) In the brain, retinal receptors and sperm,

DHA is the preferred membrane phospholipid. DHA is also thought to play a role in brain development and learning ability, particularly in a developing fetus (Pigott and

Tucker 1990).

2.5.1.3 Fat-Soluble Vitamins

The fat-soluble vitamins consist of vitamins A, D, E and K. When absorbed by the body, they are often stored in the liver. Since they cannot be readily excreted in urine like the water-soluble vitamins, toxicity may occur with overconsumption.

Vitamin A consists of both retinol compounds and carotenoid compounds.

However, in fish and other animal products, retinol is the primary source of vitamin

A activity. In contrast to carotenoids, retinol is easily absorbed by the human body

(Gregory 2008). Vitamin A is essential for vision, helps regulate gene expression, and supports the immune system. It is critical for proper embryonic development.

Vitamin A also serves as an antioxidant and may have an anti-carcinogenic effect

(Ball 2004).

There are two forms of vitamin D, ergocalciferol (D2) and cholecalciferol

(D3).They are considered equally biopotent (Ball 2004). Vitamin D3 is the dominant form in animal sources because it can be synthesized by the body with exposure to sunlight. Vitamin D2 is often produced synthetically and used for fortification

(Gregory 2008). Because exposure to sunlight is dependent on geographic location, lifestyle, and genetics, need for dietary consumption may vary significantly from person to person. Regardless of source, vitamin D serves several important purposes in the body. Vitamin D assists in intestinal absorption of Ca2+, development and mineralization of bone, and maintenance of Ca2+ homeostasis between bone, intestine, and kidneys. Vitamin D also helps regulate insulin secretion and boost the immune system (Ball 2004).

Vitamin E primarily consists of α-tocopherol, but also includes other tocols and tocotrienols that provide vitamin activity (Gregory 2008). However, in the case of fish muscle, α-tocopherol is the only vitamin E compound found in significant quantities. α-tocopherol serves as an antioxidant in cell membranes and also helps stabilize membrane lipoproteins. Particularly in aging populations, consumption of vitamin E can increase cell immune response. Vitamin E is also thought to prevent atherosclerosis from oxidized lipoproteins (Ball 2004).

2.5.1.4 Minerals

Minerals are often divided into micronutrients and macronutrients depending on the recommended daily intake. Micronutrients are generally classified

as minerals with a daily requirement of less than 100 mg. Macronutrients require consumption of greater than 100 mg per day. There are 11 trace elements, or those with daily requirements of <18 mg per day, which are classified under micronutrients (Quintaes & Diez-Garcia, 2015). Of relevance to Alaska pollock are the macronutrients sodium, calcium, iron and magnesium, and the micronutrient copper.

Calcium accounts for approximately 1-1.2 kg of body mass in adult humans

(Miller 2008). The bones contain 99% of calcium in the body. Calcium also plays a structural role in teeth. Small quantities of calcium are needed for nerve impulses and muscle contraction, blood coagulation, and hormone secretion. It can also serve as a cofactor in certain enzymatic reactions (Miller 2008; Zand, Christides, and

Loughrill 2015).

Although toxic in large amounts, copper is an essential trace element in the body. It assists in synthesis of the connective tissues collagen and elastin, serves as an antioxidant, and is involved in hemoglobin formation. Copper is also a part of several metalloenzymes such as lysyl oxidase (Quintaes and Diez-Garcia 2015).

Iron assists in the transportation of oxygen throughout the body due to its presence in both hemoglobin and myoglobin. Iron is also present in cytochrome enzymes and assists in electron transport. Adequate intake of iron is also important for prevention of microcytic hypochromic anemia (Quintaes and Diez-Garcia 2015).

Iron can be stored in the bone marrow, liver and spleen for later hemoglobin or myoglobin synthesis (Miller 2008).

Most magnesium in the human body can be found in the bones and teeth.

Magnesium assists in skeletal development and maintenance by influencing hormone and active vitamin D secretions. It also serves as a cofactor for various enzymes. Magnesium also helps maintain electrical potential across membranes and assists in active transport of calcium and potassium across cell membranes (Zand,

Christides, and Loughrill 2015).

Sodium, in tandem with potassium, is responsible for maintaining a gradient inside and outside of cells to keep them functioning properly. It is also involved in nerve impulse transmission (Quintaes and Diez-Garcia 2015). It is important to note that excess sodium consumption, which has become problematic in developed countries, contributes to high blood pressure and therefore increases the risk of stroke and coronary heart disease (Miller 2008).

2.5.2 Recommended Consumption

There are two important acronyms in nutrition labeling which refer to the recommended quantities of nutrients to be consumed in a day: RDI and DRV. The

RDI, or reference daily intake, is used for vitamins and minerals. The DRV, or daily reference value, is used for larger food components. The term “Daily Value” (DV) can refer to both of these terms. This thesis will use the RDI and DRV amounts for a 2000 calorie diet, which is used on a standard nutrition label and intended for adults and children ages 4 and older. It should be noted, however, that there are different RDI and DRV amounts for pregnant/lactating women, children 1-3 years old, and infants through 12 months.

On a food label, the % DV is used to express the amount of a food component in comparison to the RDI or DRV (Metzger 2010). For example, a serving of food containing 5 µg vitamin D, which has an RDI of 20 µg, would have be 25%

DV. The RDIs from 21 CFR 101.9 (c) (8) (iv) (2016) relevant to Alaska Pollock are shown in Table 2.1. The DRVs relevant to Alaska Pollock from 21 CFR 101.9 (c) (9)

(2016) are shown in Table 2.2. Serving sizes defined by the FDA are provided in 21

CFR 101.12 (b) Table 2. For fish meat, the serving size is 85 g.

Table 2.1. Recommended Daily Intake (RDI) of vitamins and minerals relevant to Alaska Pollock for adults and children ages 4 and older.

Vitamin or Mineral Recommended Daily Units Intake (RDI) A 900 µg or RAE D 20 µg E 15 mg Calcium 1300 mg Iron 18 mg Magnesium 420 mg Copper 0.9 mg Table 2.2. Daily Reference Value (DRV) of food macronutrients for adults and children ages 4 and older. Component Daily Reference Value Units (DRV) Fat 78 g Saturated Fat 20 g Total Carbohydrate 275 g Sodium 2300 mg Protein 50 g

3. Nutritional Composition Changes in Alaska Pollock (Gadus chalcogrammus) During and Between Bering Sea A and B Seasons

Clara A. Hintermeister1, Christina A. Mireles DeWitt1, Quentin Fong2

1 Seafood Research and Education Center, Oregon State University, 2001 Marine Dr,

Astoria, OR 97103, USA

2 University of Alaska Fairbanks, Marine Advisory Program Kodiak Seafood and

Marine Science Center, 118 Trident Way, Kodiak, AK 99615, USA

3.1 Abstract

The Alaska Pollock fishery is the largest whitefish fishery in the world and is harvested from January through April (season A) and June through October (season

B). Effect of time within a season on nutritional content has not been previously investigated in depth, though changes could impact further processing. Literature on macro and micro nutrient composition of pollock roe and milt is incomplete.

Therefore, Alaska Pollock fillets, roe and milt were analyzed for proximate composition, fat-soluble vitamins, fatty acids, amino acids, and mineral content to determine how nutritional composition changes within and between seasons, and if these changes could affect product labeling. Samples were collected

(n=10/collection date/product type) biweekly in Season A. Only fillets and roe were collected in Season B as milt was not sufficiently developed. On both wet and dry basis, within season changes were negligible. On a dry basis, protein was significantly higher in fillets (90.2±4.9%) than roe (82.2±5.0%) and milt (82.5±3.7%).

However, fillets were significantly lower in fat (2.6±0.7%) than roe (6.0±1.6%) and milt (12.5±2.2%). This correlated to higher fat-soluble vitamin content on a dry basis in roe and milt than in fillets. Also noteworthy is that the majority of fatty acids in all three sample types were omega-3 fatty acids, with the highest quantities in roe (dry basis). Roe exceeded protein quality requirements for all essential amino acids.

Fillets and milt had similar amino acid contents and were both deficient in at least one essential amino acid. Finally, mineral content in fillets, roe, and milt did not

change significantly, and all sample types were good sources of copper (10% or more of daily requirements).

3.2 Practical Application

Despite being the largest whitefish fishery in the world, there is little information on how catch date affects nutritional content in Alaska pollock fillets, roe and milt. Roe and milt are exported byproducts, but there is no current market for them in the United States. Having information on their nutritional content could lead to the development of new markets and higher value products.

3.3 Introduction

Alaska pollock, Gadus chalcogrammus, is a species of schooling whitefish caught primarily in the Bering Sea. It is widely consumed across North America,

Europe, and Asia. Alaska pollock is primarily prized for its mild-flavored fillets, which are often consumed as surimi seafood or breaded and battered products such as fish sticks. In 2015, Alaska pollock was 67% of the Alaska groundfish catch, totaling 1.5 million metric tons worth $480 million (Fissel and others 2016). With additional processing, the value only increases. From 1999 to 2010, total wholesale revenue of all Alaska pollock products averaged to $1.1 billion per year (Strong and Criddle

2013). If roe is harvested, it is almost exclusively exported to South Korea or Japan.

In 2014, the wholesale value of roe was $151 million for only 24,100 metric tons.

However, markets for roe have been declining since 2010 due to the increase in production from Russia (Fissel and others 2016). Other Russian pollock products include whole frozen fish, which are hand-processed in China and Vietnam and then

refrozen. These fish products, often called twice-frozen products, are also sold in the

United States and compete with American-caught, once-frozen products.

Regardless, the Alaska pollock fishery is the largest whitefish fishery in the world. The fishery is divided into three stocks, with the Bering Sea stock being the largest (Alaska Fisheries Science Center 2017). The fish are caught from January-April

(Season A) and June-October (Season B) each year as part of a fishery management plan (Strong and Criddle 2013). Season A and Season B correspond to the pre- and post-spawning periods in the fish reproductive cycle. Despite its popularity as a food source, little research has been done on seasonal changes in Alaska pollock composition and no data exists on within season changes. Kitts and others (2004) investigated seasonal changes on whole Alaska pollock and found that % fat content increased in summer months, including omega-3 fatty acids. Seasonal data on Alaska pollock roe is virtually nonexistent. However, studies have been done to determine maturity of roe (Stahl 2004) or investigate the composition of immature roe (Bechtel and others 2007). Data found on the nutritional composition of milt was limited.

Raw Alaska pollock milt proximate composition and composition of dried meal made from the milt was analyzed for amino acids, fatty acids, and mineral content.

However, the effect of catch date was not investigated. Thus, the main goal of this study was to develop nutritional profiles for Alaska pollock fillets, milt and roe based on catch date and season. Regarding fillets, changes in nutritional composition could affect processing yields and efficiency. This data could also serve as a basis of comparison against other whitefish used for similar products, for example cod

(Gadus morhua) in fish sticks, or also against Russian twice-frozen pollock products.

With declining markets for roe from the United States in Asia, alternative markets and uses are needed to appeal to consumers all over the world. Having nutritional data on roe and milt could be used in the development of new products such as protein powders or functional ingredients (Sathivel 2006). Protein powders from a variety of fish species have already been studied for functional properties and nutritional value (Shaviklo 2015). Such products would be valuable to processors and would be a good source of many nutrients.

3.4 Materials and Methods

3.4.1 Sample Preparation

Randomly selected fillet, milt, and roe samples (n = 10/week) were collected biweekly from February through March 2015 (season A). Fillets were also collected once per month in July and August (season B) in the Bering Sea by an at-sea processor and immediately frozen. Due to the yearly reproductive cycle, roe was only present in season A and the July catch date, and milt was only present in season

A. Samples were prepared for analysis by partially thawing, slicing into 1 cm2 pieces, re-freezing in liquid nitrogen, and blending to form a powder. The powdered samples were stored at -80 °C.

3.4.2 Proximate Analysis

Acid hydrolysis was used to determine fat content by using AOAC Official

Method 948.15 (AOAC 2005), modified to use only 3.00 ± 0.10 g of each sample.

Protein content was determined using the Kjeldahl method for nitrogen in meat,

AOAC Official Method 928.08 (AOAC 2005), using 1.350 ± 0.150 g of each sample and copper catalyst in a ratio of 3.5:1 potassium sulfate: copper sulfate pentahydrate. Moisture and ash were determined by modifying and combining

AOAC official methods 938.08, Ash of Seafood, and 950.46, Moisture in Meat using

4.0 ± 0.1 g sample (AOAC 2005). Sample was weighed into a crucible and placed in a drying oven for 16 hours at 105°C, cooled to room temperature in a desiccator, and weighed. The same dried samples were then used for ashing (Kitts and others 2004).

Samples were heated on a hot plate (PC-620D, Corning, Corning, NY, USA) at 550°C until they were charred and had stopped smoking, then transferred to a muffle furnace set to 525°C. Fillets were ashed for 12 hours. Roe and milt were ashed for 48 hours.

3.4.3 Fat-Soluble Vitamin Analysis

To account for small sample amounts, a method was developed to extract and analyze vitamins A, D, and E simultaneously. Sample preparation was based on a study by Podda and others (1996) using 1.00 ± 0.10 g sample. The sample was weighed into a 50 ml centrifuge tube and 5 ml 1% ascorbic acid and 10 ml ethanol were added. The tubes were then vortexed for 5 seconds. Samples were saponified by adding 1.5 ml 50% KOH solution and heating for 30 minutes at 70°C in a water bath. Samples were cooled to room temperature on an ice water bath and 125 µl vitamin D2 internal standard (2 ug/ml), 25 µl 1 mg/ml BHT, 5 ml 1% ascorbic acid, and 10 ml hexane were added. Samples were mixed by inversion for 1 minute, then centrifuged at 3250 g for 5 minutes at 4°C (J6-M1, Beckman Instruments Inc.,

Fullerton, CA, USA). An 8 ml portion of the hexane layer was transferred into a scintillation vial and evaporated under nitrogen in a 55°C water bath. To prepare for analysis, 250 µL 50% ethanol: 50% methanol solution was added and samples were vortexed for 5 seconds. The solution was added to a vial and either stored in a freezer (-20°C) or analyzed using a Shimadzu Prominence UFLC System (Shimadzu

USA MFG INC, Canby, OR, USA). HPLC settings were as follows: 50 µL injection, flow rate 1.5 ml/min, column oven at 44°C. Two 25 cm columns (Phenomenex Synergi™ 4

µm Hydro-RP 80 Å LC Column 250 x 4.6 mm and Phenomenex Kinetex 5 µm XB-C18

80 Å Colum 250 x 4.6 mm; Phenomenex, Torrance, CA, USA) were used in tandem with a guard column (Phenomenex SecurityGuard™) as per Stancher and Zonta

(1983). Results were viewed at 265 nm for vitamins D2 and D3, 296 nm for vitamin

E, and 325 nm for Vitamin A, and peaks were identified based on the following standards, respectively: ergocalciferol, cholecalciferol, α-tocopherol, and retinol

(Supelco, Bellefont, PA, USA). Standards had been prepared as per AOAC methods

992.04 for all-trans-retinol, 992.26 for vitamins D2 and D3, and 971.30 for alpha- tocopherol (2005). Vitamin results were calculated based on serving size and by % daily value (% DV), the portion of the reference daily intake (RDI) present in the food, for adults and children over 4 years old.

3.4.4 Fatty Acid Analysis

The fat extraction procedure was based on the Folch method (Folch and others 1957), using 1.0 ± 0.1 g sample weighed into 50 ml centrifuge tubes.

Tridecanoic acid internal standard (Supelco) at a concentration of 10 mg/ml (200 µl

for fillets, 800 µl for roe and milt) and 15 ml 2:1 chloroform:methanol solution were added to each sample tube and an empty tube to be used as a blank. Samples were mixed for one minute using a Pro 200 homogenizer (Pro Scientific, Oxford, CT, USA) and then filtered through #415 filter paper into clean test tubes. To each tube 2 ml of 0.88% KCl solution were added. Samples were then vortexed for 10 seconds and centrifuged at 1000 x g for 5 min at 20 °C (J6-M1, Beckman Instruments Inc).

Conversion to fatty acid methyl esters was based on a procedure by Morrison and

Smith (1964). A 5 ml aliquot from the bottom layer (chloroform) of each sample was transferred to a clean test tube and evaporated to dryness under nitrogen at 55°C.

To saponify the fats, 0.5 ml of 0.5 M NaOH in methanol was added to each tube.

Tubes were vortexed for 5 seconds and heated for 5 minutes at 100 °C in a water bath. Upon removal, fatty acids were esterified using 0.5 ml boron trifluoride in 14% methanol, then vortexed for 5 seconds and heated for 5 minutes at 100 °C in a water bath. Samples were cooled to room temperature, and 2 ml DI water and 2 ml hexane were added. Again, samples were vortexed for 10 seconds and centrifuged at 1000 x g for 5 min at 20 °C (J6-M1, Beckman Instruments Inc.). A 1.5 ml aliquot of the top layer (hexane) were transferred to a clean test tube and evaporated to dryness under nitrogen at 55 °C. Fillet fatty acids were resuspended in 250 µl hexane. Roe and milt fatty acids were resuspended in 1 ml hexane. Samples were transferred to a spring bottom insert in a GC vial and stored at -80 °C until analysis.

Analysis was performed using a Shimadzu GC-2010 with AOC-20i + s autoinjector and autosampler (Shimadzu Corporation, Kyoto, Japan). The column used was a

Supelco-2560 100m x 0.25mm x 0.25 µm film thickness column (Supelco). The analysis program was a modified version of the method from Lapis and others

(2013). Injections were 1 µl per sample with an inlet temperature of 250 °C and a column flow of 0.44 ml/min, using the linear velocity mode. A split ratio of 50:1 was used. The FID detector temperature was set at 275 °C, with N2 make-up flow at 35.0 ml/min, hydrogen flow at 40.0 ml/min, and air flow at 450.0 ml/min. The column oven program was as follows: 140-180°C at 2°C/min, 180-200°C at 2.5°C/min, 200-

220°C at 0.5°C/min, and 220-235°C at 10°C, hold 5 min. Total run time was 74.50 min/sample. Peaks were identified using a C4:C24 fatty acid methyl ester reference standard (18919-1 AMP, Supelco).

3.4.5 Amino Acid Analysis

The protein hydrolysis procedure was based on a study by Rutherford (2009).

Approximately 0.1 g sample and 800 µl sequencing grade 6N HCl (Pierce, Rockford,

IL, USA) containing 0.1% phenol (Acros Organics, Morris Plains, NJ, USA) were added to vacuum hydrolysis tubes (Chemglass, Vineland, NJ, USA) sealed under vacuum and heated in an oil bath at 110°C for 22 h. Hydrolysates were prepared for gas chromatographic analysis by solid phase extraction (SPE) and derivatization using a

Phenomenex EZ:Faast amino acid kit. Sample preparation was modified by using 125

µl hydrolysate, 25 µl 10% NaOH, and 300 µl Reagent 2 to raise the pH of the hydrolysate into the acceptable range given by the kit. It should be noted that methionine, cysteine, and tryptophan were partially or completely destroyed during hydrolysis. Arginine and glutamine were deaminated during the process and are

reported as aspartic acid and glutamic acid, respectively. For analysis, a Shimadzu

GC-2010 with AOC-20i + s was fitted with a Zebron ZB-AAA Column for Protein

Hydrolysate (Phenomenex). Injections were 1.6 µl, with inlet temperature at 300 °C and a split ratio of 1:15. Helium at 1.5 ml/min was the carrier gas. The oven program was 110 – 320 °C at 32 °C/min for a run time of 6.56 minutes per sample. The FID was set at 320 °C. Hydrogen flow was 40 ml/min, air flow was 350 ml/min, and the makeup gas (N2) flow was 35 ml/min.

3.4.5 Mineral Analysis

One sample per catch date of fillets, roe and milt were randomly selected for mineral testing. Mineral analysis was completed by the Oregon State University

Central Analytical Laboratory (Corvallis, OR, USA). Samples were dried, microwave digested, and analyzed for iron, sodium, calcium, magnesium, and copper using ICP-

OES (PerkinElmer 2100 DV, PerkinElmer, Waltham, MA, USA).

3.4.6 Statistical Analysis

All samples were considered random and representative of the population, with ten replicates per week. One-way ANOVA and T-tests were used to test for differences during and between seasons, respectively, for proximate analysis and vitamins. Fatty acids and amino acids were analyzed using principal component analysis, t-tests, and two-way ANOVA. All statistical analyses were conducted with

SigmaPlot (Version 13, Systat Software, San Jose, CA, USA) at a significance level of

0.05.

3.5 Results and Discussion

3.5.1 Proximate Analysis

The proximate analysis results for fillets, roe, and milt are shown in Table 3.1a.

Composition of fillets was similar to that found in the USDA Nutrient Database for

Alaska pollock (2015). Fillets within season A did not vary significantly for fat, protein and moisture. Although ash content on Feb 3 was significantly different (p < 0.05) from ash content on Mar 16, there were no other significant differences within the season. Within season B, fat, protein, and ash content were significantly higher (p <

0.05) on Jul 15 than on Aug 15, but moisture content was lower. On average, fat increased 0.1%, protein increased 1%, moisture decreased by 0.5%, and ash increased 0.1% in season B. The increased protein in season B could positively impact processing yields for fillet products such as surimi and fish sticks. For roe, there were no significant compositional differences within Season A (p > 0.05).

Season B only had one catch date, Jul 15, which differed significantly in fat and moisture content from the season A average (p < 0.05). Fat increased by 1.2% and moisture decreased by 1.4%. Milt values for fat, moisture and ash fluctuated throughout the season. Although significant differences were found, no trends were noticed and overall no particular week stood out as different compared to all other weeks. Protein content did not change significantly throughout season A.

3.5.2 Fat-Soluble Vitamin Analysis

The necessity for developing a method to extract and analyze fat-soluble vitamins A, D, and E simultaneously was based on limited sample amounts of roe

and milt needed for all the other analyses in this study. Although there are AOAC official methods for analyzing fat-soluble vitamins individually, there are no approved methods for analyzing them together. Thus, the method developed and used in this study was novel. The use of two columns in the developed method allowed for complete separation of vitamins D2 and D3, which eluted at 22.0 and

22.5 minutes, respectively (Figure 3.1). The complete baseline resolution of these two vitamins allowed for the use of vitamin D2 as an internal standard since it is not naturally present in fish.

Vitamin content per serving is shown in Table 3.2 as total amount present and % DV. Fillet results were calculated based off an 85 g serving size as recommended by the FDA (21 CFR §101.12 2017). Although there is some vitamin A present in the fillets, and variation within and between seasons, the quantity was so low at each catch date it would still be regarded as 0% DV. Vitamin D, on the other hand, did not change significantly within season A (p>0.05), but increased significantly between season A and season B (p<0.05). However, the changes within season B were not large enough to affect the % DV. The vitamin E content did not change significantly within season A (p>0.05), but did during the B season (p<0.05).

The season averages were not significantly different (p>0.05). Again, though there were changes noticed, they were not large enough to affect the % DV of vitamin E present in the food. The values obtained for vitamins D and E are higher than those in the USDA Nutrient Database for Alaska pollock (2015), which lists 0.2 µg vitamin D and 0.42 mg vitamin E per serving. However, the samples used in the current study

were stored at -80°C and most likely fresher than those tested by the USDA.

Vitamins D was found to be degraded by both light and oxygen exposure (Renken and Warthesen 1993), and vitamin E by light (Nhan and Hoa 2013).

A serving size of 15 g was estimated for roe based off FDA recommendations for caviar (21 CFR §101.12 2017). Vitamin A content fluctuated significantly throughout Season A (p<0.05). However, the seasonal average was 0% DV. The

Season B average was also 0% DV and not significantly different from Season A

(p<0.05). The vitamin D content significantly decreased from 6% at the start of

Season A to 2% at the end of the season (p<0.05). Although still considered 2% DV, the Season B average (0.32 µg/15 g) was even lower than the lowest point of Season

A (Mar 31; 0.57 µg/15 g). Vitamin E content did not change significantly within or between either season and stayed at 6% DV (p>0.05).

Milt vitamin content was also based off a 15 g serving size due to its similarity to roe. No significant changes for any vitamin were observed in milt

(p>0.05), and at 2% DV, vitamin E was the only vitamin of nutritionally relevant quantity. Vitamin A and vitamin D were found to be 0% DV.

The fat-soluble vitamins are critical for proper health. Vitamin A is well known as a necessity for vision, but it is also necessary for embryonic development

(Ball 2004). This may be why vitamin A is found in significantly higher quantities in roe than in fillets and milt (p<0.05). Vitamin D is responsible for uptake and homeostasis of calcium and mineralization of bones (Ball 2004). Unlike humans, fish cannot synthesize vitamin D and instead obtain it from their diet, though it is used

for similar purposes in the body (Lock and others 2009). Because of this, vitamin D content in pollock fillets is likely largest in summer months because the fish are most actively feeding. Variations in diet may also further explain the discrepancy between the vitamin D content found in this study and that presented in the USDA Nutrient

Database. Vitamin E is a membrane lipoprotein stabilizer and an antioxidant (Ball

2004). The importance of this role may explain why vitamin E content did not exhibit many significant changes in fillets, roe, or milt.

3.5.3 Fatty Acid Analysis

Fatty acid content is summarized in Table 3.3. Using principle component analysis, it was shown that in fillets, higher C14:0, C16:0, C20:5n3 (eicosapentaenoic acid, EPA), and C22:6n3 (docosahexaenoic acid, DHA) were correlated with lower

C16:1. This trend was noticed in Season B, and except for C14:0, also followed in

February of Season A. A significant increase from Season A to Season B was found for DHA, C14:0 and C16:0 (p<0.05). In roe, Season B had significantly higher amounts of C14:0, C18:2n6c, C20:1, C22:1n9, EPA and DHA, and significantly lower C16:0 and

C18:1n9c (p<0.05). Principal component analysis did not reveal strong trends in the data. Significant changes within either season were not found for fillets, roe or milt

(p>0.05).

Of particular interest are omega-3 fatty acids, specifically EPA and DHA, because they are important to gene expression, inflammatory responses, and brain function (McClements and Decker 2008; Pigott and Tucker 1990; Institute of

Medicine Staff and National Research Council, Subcommittee on Metabolism Staff

1989). Both EPA and DHA are the most prominent fatty acids in Alaska pollock fillets, roe and milt. Alaska pollock fillets from both seasons and roe from Season B contain roughly 25-35% of adequate intake of total omega-3 fatty acids for adults, which is

1.6 g per day for males and 1.1 g for females (Institute of Medicine, Food and

Nutrition Board 2005). The ratio of omega-6 to omega-3 fatty acids is also important. In the typical Western diet, people consume 10 times more omega-6 than omega-3 fatty acids, though they should be consumed in roughly equal amounts. In Alaska pollock fillets, the ratio of omega-6 to omega-3 is 1:420 in Season

A, and approximately 1:230 in Season B. Omega-6 fatty acids in roe were only observed in Season B at a ratio of 1:345. There were no omega-6 fatty acids in milt.

These ratios make Alaska pollock a preferably choice over similar cod (Gadus morhua), which has a 1:10 ratio of omega-6 to omega-3 fatty acids (Jangaard and others 1967).

From an organoleptic perspective, higher amounts of palmitic acid (C16:0) increase palatability and improve mouthfeel (Institute of Medicine, Food and

Nutrition Board 2005). Palmitic acid comprises 20% of the fat in roe in Season A, but is only 10% of the fat in Season B. Therefore, roe from Season A may be liked better that roe from Season B regarding mouthfeel. In fillets, regardless of season, C16:0 comprises 17% of the fat. Fat in milt also contained approximately 20% C16:0.

3.5.4 Amino Acid Analysis

Essential amino acids cannot be synthesized by the human body and must be consumed through food sources. These amino acids include histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Amino acid scores and essential amino acid index (EAAI) are two methods of determining protein quality (Table 3.4). In this case amino acid scores were calculated using the recommended values for consumption for preschool-aged children as a reference protein (Smith 2010). By these scores, histidine is the first limiting amino acid for fillets and milt. As shown in Table 3.5, neither met the requirement of 15 mg/g protein for adult daily consumption of histidine, though all other amino acids exceeded their respective requirements. For roe, the sulfur amino acids, methionine and cysteine, combined are the first limiting amino acid. However, all amino acids, including the sulfur amino acids, exceeded requirements (WHO, FAO, and UNU

2002). The differences in amino acid content between fillets, roe and milt can be seen using component scores generated during principal component analysis, as seen in Figure 3.2. The amino acid content of fillets and milt are more closely related to each other than to roe. However, fillets, roe and milt were all significantly different from each other (p<0.05). Differences in amino acid content by sample type can be seen in Table 3.6.

The EAAI and amino acid scores for histidine, isoleucine, leucine, lysine, phenylalanine, tyrosine, and valine all increased significantly in season B for fillets.

This corresponds to the increase in protein in season B noted previously. Because the fish feed more heavily in the summer months, the fish grow the most during this

time. Similar behavior was noted in cod (Gadus morhua), for which increased growth rates were correlated to warmer water temperatures (Pálsson and Thorsteinsson

2003). The only significant difference between seasons for roe was a decrease in lysine content. No significant changes within seasons were observed for fillets, roe or milt.

3.5.5 Mineral Analysis

Mineral content is shown in Table 3.7. There were no significant changes found within or between seasons for any sample type (p<0.05). Fillets, roe and milt differed significantly from each other in mineral content on a per-serving basis

(p<0.05). Fillets and milt had similar mineral content, only significantly differing in sodium content (p<0.05). Roe differed in sodium and calcium content from fillets and milt, as well as in iron and copper content from milt and magnesium content in fillets. Calcium was not present in nutritionally relevant quantities in a serving of fillets, milt or roe.

Although calcium is the mineral best known for creating strong bones, the vast majority of magnesium in the body is also responsible for the health of bones and teeth. Adequate intake of magnesium is important for hormone and vitamin D secretions involved with the skeleton (Zand and others 2015). Due to their proximity to the bones, it is not surprising that Alaska pollock fillets were higher in magnesium than roe and milt.

Iron is primarily associated with hemoglobin and myoglobin, two types of globular proteins responsible for oxygen transport in the blood and muscles,

respectively. Myoglobin is the cause of coloration in muscles, with redder meats being associated with higher levels of myoglobin. Myoglobin occurs in larger quantities in muscles that are frequently used at regular intervals. Since Alaska pollock are not very active fish and tend to move in short bursts, their meat (fillets) are more white than red in color. Therefore, Alaska pollock are relatively low in iron compared to more active fish such as tuna.

Sodium is a necessity for proper health. However, in the United States and other industrialized countries, sodium is commonly consumed in excess of dietary recommendations and physiological requirements (Jackson and others 2016; Elliott and Brown 2006). Major sources of sodium are processed products, including processed or cured meats and dishes created from them (Jackson and others 2016).

Thus, it is important to note that each serving of roe and milt contains less than 2%

DV of sodium and fillets contain only 6% DV. Consuming large quantities of sodium can lead to or exacerbate hypertension, which can lead to strokes and heart disease

(Jackson and others 2016).

On the Jul 15 collection of samples there were two fillets with abnormally high copper levels (0.66 mg/85 g and 256.85 mg/85 g). Additional samples were tested and the fish with high copper levels were determined to be outliers. They were therefore excluded from the remainder of the statistical analysis. Sources of copper in fish may be ocean water, ocean sediment, food sources, or human pollution (Blossom 2015). This is noteworthy because while copper is an essential trace element in the body responsible for assisting with connective tissue and

hemoglobin synthesis, it is toxic in large quantities (Quintaes and Diez-Garcia 2015).

However, the average Alaska pollock product only contains between 10-15% DV of copper. Considering most Alaska pollock products containing muscle are created from many fish (e.g. surimi, fish sticks, fish patties), the occasional fish high in copper may not be a large concern.

3.5.6 Dry Basis

The proximate composition of fillets, roe and milt on a dry basis is shown in

Table 3.1b. Table 3.8 compares some of the important nutritional components of fillets, roe and milt on a 25 g dry basis. The serving size was selected based on serving sizes observed for protein powders. Protein content in milt changed the most from a wet to dry basis. On a wet basis milt was only 12-13% protein, compared to 23% protein in roe. Dried roe contained 82% protein in season A and

79% in season B, while dried milt contained almost 83% protein. Because they are predominantly composed of muscle, dried fillets contained even higher amount of protein than roe or milt with 88% in season A and 92% in season B. Regarding fat, milt contained the highest amounts at approximately 13% compared to roe at 6% in season A and 10% in season B and fillets at 2% in season A and 3% in season B. This is remarkable since on a wet basis both roe and milt were around 2% fat in season A.

However, on a dry basis, in season A roe and milt still contained similar quantities of omega-3 fatty acids. Both exceeded recommended minimum daily consumption for females (1.1 g), but not for males (1.6 g). Omega-3 fatty acid content in roe nearly doubled from season A to season B. In season B, omega-3 fatty acid content

exceeded recommended consumption for males and females. In season A, dried fillets contained roughly half the omega-3 fatty acids of roe and milt. In season B, dried roe contained almost 2.5 times as many omega-3 fatty acids as fillets. While on a dry basis fillets still did not contain nutritionally relevant amounts of vitamin A, roe and milt increased to 6% and 4% DV, respectively. Dried roe contained 25% DV of vitamin D in season A but only 10% in season B. Fillets and milt contained around 8-

10% DV of vitamin D dependent on season. Vitamin E levels, at 35 % DV, were also highest in dried roe. Iron was found to be at 6% DV per serving for dried milt and roe from both seasons, and only 4% in fillets. Sodium, of concern for health reasons, was only present at 6% of the daily value in dried roe, compared to 10% in fillets and milt. Magnesium was highest in dried fillets, at 10% DV in season B and 12% DV in season B. Dried roe contained only 2% DV in season A and 4% DV in season B. Dried milt contained 8% DV. At 15% DV, copper was highest in dried milt and season B roe.

Dried season A roe and fillets both contained 10% DV of copper, and season B fillets contained 12% DV.

By FDA rules, products can be labeled as “high” in a nutrient if that nutrient is present in quantities of greater than or equal to 20% of the RDI. Products can be labeled as “a good source” of a nutrient if that nutrient is present in quantities from

10-19% of the RDI. Thus, dried roe would be considered high in vitamin E and a good source of copper in both seasons. However, with the significant decrease in vitamin

D content, dried roe vitamin D levels could be labeled “high” in season A and “a good source” in season B. Dried milt would be a good source of vitamin E and

copper. In both seasons, dried fillets would be a good source of magnesium and copper. However, they could only be labeled as a good source of vitamin D in season

These results show the value of dried roe and milt as potential supplements or functional ingredients. Roe could be used as a high-quality protein source since it contains adequate amounts of all essential amino acids. Milt contained only 2 mg/g less than the WHO recommendation of 15 mg/g of histidine per protein, so when paired with other foods would also be a possible protein supplement. The presence of omega-3 fatty acids, vitamins, and minerals could be used as an added benefit for nutritional products from roe and milt. Dried products may also be able to serve as functional ingredients. Pollock viscera excluding liver were found to be as highly nitrogen soluble as egg albumin and capable of forming a stable emulsion (Sathivel

2006). However, the viscera were not just limited to roe and milt so more research would need to be done on specific protein functional properties of the reproductive organs.

3.6 Conclusion

The nutritional composition of Alaska pollock changes significantly from season A to season B, with only small changes within seasons. Fillets were higher quality in season B, with significantly higher protein, fat, vitamin D, omega-3 fatty acids, and EAAI scores than in season A (p<0.05). Roe increased in fat content from season A to season B, which correlated to significant increase in omega-3 and

omega-6 fatty acids (p<0.05). These increases in fillet and roe were likely caused by the tendency of Alaska pollock to feed most heavily in summer months. Milt composition remained remarkably consistent throughout season A, with only sporadic changes in fat, moisture and ash content. No other compositional changes were observed.

Implications of this work affect marketability and usage of Alaska pollock products. Data on fillet seasonal changes could influence how fish are processed or affect processing yields. Additionally, having data on fresh, once-frozen Alaska pollock fillets could serve as a basis of comparison against similar whitefish such as cod, or even against foreign-processed twice-frozen Alaska pollock fillets. However, no published data has been found comparing the nutritional content of once- vs. twice-frozen fillets. This is a possible topic for future research.

The value of roe and milt became apparent on a dry basis. Over both seasons dried roe was a good source of copper and high in vitamin E, and in season A dried roe was high in vitamin D. Dried milt was a good source of copper and vitamin E.

Both roe and milt contained 80-82% protein on a dry basis, with over 1.2 g omega-3 fatty acids per serving. Because of this, dried roe and milt have potential for use as supplement or functional ingredients, and could prove to be lucrative for processors.

However, since the dried results presented here were calculated, more work should be done to determine if the drying process would affect the nutritional content or protein functionality. Likewise, further studies should be done into the nutritional content of roe and milt, particularly regarding cholesterol and B vitamins.

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Table 3.1a - Mean values of Alaska Pollock fillet, roe, and milt proximate composition on a wet basis throughout Seasons A and B in 2015

Type Sample % Fat % Protein % Moisture % Ash Date Fillet Feb 3 0.44 (0.20) 14.51 (0.83) 83.07 (0.37) 1.31 (0.02)a Feb 17 0.40 (0.09) 14.87 (0.86) 83.12 (0.59) 1.30 (0.08)ab Mar 3 0.46 (0.08) 15.13 (0.57) 83.07 (0.78) 1.35 (0.04)ab Mar 16 0.37 (0.14) 15.14 (0.88) 83.31 (0.53) 1.36 (0.05)b Mar 31 0.39 (0.08) 15.23 (0.89) 83.77 (1.10) 1.29 (0.06)ab Season A 0.41 (0.13) 14.98 (0.82) 83.27 (0.37) 1.32 (0.02) Avg Jul 15 0.57 (0.10)x 16.18 (0.71)x 82.61 (0.68) 1.45 (0.07)x Aug 15 0.44 (0.08)y 15.35 (0.68)y 83.02 (0.53) 1.36 (0.08)y Season B 0.50 (0.10)1 15.77 (0.80)1 82.82 (0.61)1 1.40 (0.07)1 Avg Roe Feb 3 1.72 (0.16) 23.99 (3.00) 71.45 (2.74) 1.61 (0.16) Feb 17 1.78 (0.28) 23.66 (2.36) 71.13 (2.51) 1.49 (0.11) Mar 3 1.64 (0.34) 22.92 (2.14) 71.56 (2.42) 1.63 (0.30) Mar 16 1.62 (0.23) 23.36 (1.16) 71.70 (1.54) 1.64 (0.18) Mar 31 1.64 (0.17) 22.53 (2.01) 72.41 (5.83) 1.63 (0.21) Season A 1.68 (0.24) 23.17 (1.87) 71.65 (2.28) 1.60 (0.20) Avg Season B 2.87 (0.20)1 23.60 (2.11) 70.22 (5.80)1 1.63 (0.14) Avg Milt Feb 3 1.93 (0.17)ab 12.77 (0.17) 84.60 (0.07)ab 1.71 (0.08)bcd Feb 17 2.13 (0.53)b 12.22 (0.53) 85.30 (0.07)a 1.61 (0.07)ad Mar 3 1.99 (0.20)a 12.44 (0.20) 84.92 (0.04)ab 1.56 (0.07)a Mar 16 1.72 (0.18)a 12.37 (0.18) 84.91 (0.14)ab 1.64 (0.08)abcd Mar 31 1.75 (0.17)ab 13.02 (0.17) 84.10 (0.11)b 1.75 (0.06)bc Season A 1.91 (0.32) 12.57 (0.77) 84.76 (0.85) 1.65 (0.11) Avg ab Denotes significant differences within Season A (p<0.05) xy Denotes significant differences within Season B (p<0.05) 1 Denotes Season B values significantly different from Season A values (p<0.05)

Table 3.1b - Mean values of Alaska Pollock fillet, roe, and milt proximate composition on a dry basis throughout Seasons A and B in 2015

Type Sample % Fat % Protein % Ash Date Fillet Feb 3 2.58 (1.18) a 85.70 (3.94) abcde 7.72 (0.22) abcd Feb 17 2.36 (0.55) a 88.09 (3.44) abcde 7.69 (0.43) abcde Mar 3 2.70 (0.46) a 89.58 (5.09) bcde 7.99 (0.35) abcdef Mar 16 2.23 (0.84) a 90.70 (3.64) de 8.14 (0.26) bcdef Mar 31 2.39 (0.44) a 94.00 (5.65) e 7.98 (0.45) abcdef Season A 2.45 (0.69) 89.61 (4.35) 7.90 (0.34) Avg Jul 15 3.28 (0.57) ab 93.09 (3.83) e 8.32 (0.41) cdef Aug 15 2.58 (0.45) a 90.47 (4.11) cde 7.99 (0.25) abcdef Season B 2.93 (0.51) 1 91.78 (3.97) 8.16 (0.33) 1 Avg Roe Feb 3 6.11±0.93 c 79.56 (8.34) abc 5.71 (0.87) abc Feb 17 6.13±0.60 abc 81.93 (4.28) abcd 5.19 (0.48) abc Mar 3 5.78±1.19 abc 80.66 (5.12) ab 5.81 (1.51) abc Mar 16 2.28±0.89 abc 91.21 (3.11) abcd 8.12 (0.34) abc Mar 31 5.78±1.19 abc 80.66 (5.12) abcd 5.81 (1.51) abc Season A 5.22 (0.96) 82.80 (5.19) 6.13 (0.94) Avg Season B 9.69±0.90 bc 1 79.34 (4.62) a 5.52 (0.81) ab Avg Milt Feb 3 12.58 (1.38) c 82.84 (4.14) abcde 11.08 (0.33) f Feb 17 14.48 (3.31) c 83.23 (2.48) abcde 10.94 (0.32) ef Mar 3 13.22 (1.35) c 82.52 (5.13) abcd 10.34 (0.40) def Mar 16 11.38 (1.10) c 81.95 (2.38) abcd 10.84 (0.66) def Mar 31 11.03 (0.98) c 82.05 (4.27) abcd 10.98 (0.18) f Season A 12.54 (1.64) 82.52 (3.68) 10.84 (0.38) Avg abcdef Denotes significant differences within column Statistical summary: No effect of season on protein dry basis (over all types). Within product type, lowest protein came from roe, highest from fillets. For fat on dry basis, Season B is higher for fillets. For Roe, highest fat was also in season B. The highest levels of fat were found in milt, then roe, then milt. For Ash, highest levels were found in Milt, followed by fillets then roe.

Table 3.2- Mean values of Alaska Pollock fillet, roe, and milt vitamin content per serving on a wet basis and % daily values (% DV) throughout the A and B Seasons in 2015. The serving size for fillets was 85 g, and the serving size for both roe and milt was 15 g. Type Date % % % A (µg) D3 (µg) E (mg) DV DV DV Fillets Feb 3 2.78 (0.68)a 0 0.76 (0.36) 4 0.25 (0.21) 2 Feb 17 3.80 (0.85)b 0 0.74 (0.31) 4 0.23 (0.17) 2 Mar 3 2.29 (0.64)b 0 0.57 (0.28) 2 0.35 (0.19) 2 Mar 16 2.89 (0.32)ab 0 0.77 (0.25) 4 0.25 (0.21) 2 Mar 31 3.63 (0.97)ab 0 1.25 (0.88) 6 0.47 (0.17) 2 Season A 3.38 (0.07) 0 0.87 (0.50) 4 0.31 (0.20) 2 Average Jul 15 3.79 (0.67) 0 1.32 (0.41) 6 0.21 (0.11)x 2 Aug 15 4.52 (1.20) 0 1.33 (0.59) 6 0.42 (0.22)y 2 Season B 4.14 (1.00)1 0 1.33 (0.49)1 6 0.31 (0.12) 2 Average Roe Feb 3 7.61 (4.61)ab 0 1.10 (0.45)ac 6 0.76 (0.37) 6 Feb 17 6.45 (3.19)ab 0 1.38 (0.82)c 6 0.78 (0.28) 6 Mar 3 15.16 (9.74)b 2 0.74 (0.69)a 4 1.03 (0.45) 6 Mar 16 6.28 (3.45)b 0 0.72 (0.27)ac 4 1.01 (0.44) 6 Mar 31 11.99 (7.93)ab 2 0.57 (0.27)b 2 0.93 (0.55) 6 Season A 9.38 (6.92) 0 0.90 (0.60) 4 0.91 (0.43) 6 Average Season B 10.87 (4.70) 2 0.32 (0.15)1 2 0.90 (0.35) 6 Average Milt Feb 3 2.94 (1.64) 0 0.13 (0.06) 0 0.25 (0.12) 2 Feb 17 3.70 (1.41) 0 0.19 (0.19) 0 0.25 (0.12) 2 Mar 3 4.86 (1.99) 0 0.15 (0.19) 0 0.29 (0.21) 2 Mar 16 3.48 (1.13) 0 0.12 (0.15) 0 0.19 (0.13) 2 Mar 31 3.51 (1.80) 0 0.21 (0.18) 0 0.28 (0.16) 2 Season A 3.70 (1.70) 0 0.16 (0.16) 0 0.25 (0.15) 2 Average abc Denotes significant differences within the Season A (p<0.05) xy Denotes significant differences within Season B (p<0.05) 1 Denotes Season B values significantly different from Season A values (p<0.05)

Table 3.3 - Total saturated fatty acids (SFA), omega-3 fatty acids, omega-6 fatty acids, omega-9 fatty acids, and undifferentiated monounsaturated fatty acids (MUFA) on a mg/ serving basis of Alaska pollock fillets, roe and milt during and between seasons A and B in the Bering Sea. Servings sizes were 85 g for fillets and 15 g for roe and milt. Type Date Undiff. SFA Omega-3 Omega-6 Omega-9 MUFA (mg) (mg) (mg) (mg) (mg) Fillet Feb 3 104.62 463.23 0.21 20.79 6.81 Feb 17 126.07 457.38 0.00 27.05 7.56 Mar 3 121.96 404.40 2.32 24.88 7.82 Mar 16 110.99 353.44 0.76 23.47 11.89 Mar 31 102.91 307.39 1.40 21.84 9.07 Season 113.31 397.17 0.94 23.61 8.63 A avg Jul 15 148.06 579.79 3.61 19.26 6.16 Aug 15 141.48 484.20 0.96 23.99 9.43 Season 144.771 531.991 2.29 21.62 7.79 B avg Roe Feb 3 63.26 191.83 0.00 17.08 17.29 Feb 17 74.90 219.18 0.00 18.26 15.58 Mar 3 60.78 224.79 0.00 17.45 12.51 Mar 16 59.21 207.47 0.00 13.90 11.28 Mar 31 74.93 196.77 0.00 13.86 13.05 Season 66.61 208.01 0.00 16.11 13.94 A avg Season 53.48z 412.841 2.031 9.08 17.91z B avg Milt Feb 3 33.65 114.79 0.00 14.05 2.78 Feb 17 35.39 122.58 0.00 20.62 8.00 Mar 3 38.82 102.82 0.00 17.88 2.75 Mar 16 35.56 112.72 0.00 21.63 3.69 Mar 31 43.56 106.13 0.00 19.99 5.83 Season 37.40 111.81 0.00 18.83 4.61 A avg 1 Denotes Season B averages significantly different from Season A averages (p<0.05) z Denotes that at least one of the fatty acids included under this category was significantly different in Season B than in Season A (p<0.05), but the difference was not large enough to change the significance of the whole category

Table 3.4 - Amino acid scores for essential amino acids and the essential amino acid index (EAAI) for Alaska pollock fillets, milt and roe by catch date and season. Amino acid scores and EAAI values were calculated using amino acid requirements for preschool-aged children as the reference protein. Type Date MET PHE HIS ILE LEU LYS + + THR VAL EAAI CYS TYR Fillet Feb 3 7 24 23 25 13 28 19 21 0.22 Feb 17 4 17 18 17 13 26 18 16 0.18 Mar 3 6 19 19 25 13 20 24 18 0.20 Mar 16 3 21 19 24 15 24 18 17 0.19 Mar 31 8 16 16 15 11 22 15 15 0.18 Season 6L 19 19 21 13 24 19 17 0.20 A Avg. Jul 15 15 32 31 41 23 31 22 28 0.31 Aug 15 15 39 34 41 29 31 36 31 0.35 Season 151 L 351 321 411 26 311 29 291 0.331 B Avg. Roe Feb 3 20 47 41 34 23 39 35 41 0.38 Feb 17 32 53 42 39 24 39 37 46 0.42 Mar 3 36 58 45 43 26 39 39 47 0.45 Mar 16 21 38 37 34 19 31 32 33 0.34 Mar 31 30 42 45 36 21 34 30 36 0.38 Season 28 47 42 37 22L 37 35 41 0.39 A Avg. Season 27 45 43 301 24L 36 31 39 0.38 B Avg. Milt Feb 3 13 32 31 31 17 36 31 39 0.31 Feb 17 14 27 28 20 13 34 28 34 0.27 Mar 3 13 24 24 28 13 39 24 28 0.27 Mar 16 18 31 31 31 17 41 29 34 0.32 Mar 31 13 26 26 24 15 44 24 30 0.28 Season 14L 28 28 27 15 39 27 33 0.29 A Ave. HIS histidine, ILE isoleucine, LEU leucine, LYS lysine, MET + CYS methionine and cysteine, PHE + TYR phenylalanine and tyrosine, THR threonine, VAL valine 1 Denotes Season B values significantly different from Season A values (p<0.05) L Denotes first limiting amino acid of the season

Table 3.5 - A comparison of amino acid requirements for adults as determined by the WHO (2002) and the average seasonal amino acid content of Alaska pollock on a mg/g protein basis

MET PHE Type Date HIS ILE LEU LYS + + THR VAL CYS TYR WHO -- 15 30 59 45 15 25 15 26 Fillet Season A 6 31 61 56 17 41 26 37 Season B 14 56 105 109 35 68 40 63 Roe Season A 26 75 136 96 30 95 47 87 Season B 25 72 136 81 31 39 43 83 Milt Season A 13 45 90 71 20 52 38 71

Table 3.6 – Mean amino acid content for fillets, milt and roe from all catch dates on a 25 g dry basis.

Amino Acid Fillets Roe Milt ALA 115.21a 204.05c 142.95b GLY 113.65a 130.99a 169.13b VAL 150.70a 265.61b 221.65b LEU 247.89a 415.02b 278.40a ILE 128.36a 228.92b 138.89a THR 100.44a 144.88b 117.42ab SER 81.03a 169.475b 102.01a PRO 73.49a 201.26c 112.84b ASP 180.62a 210.91b 147.43a MET 75.87a 92.14b 61.46a GLU 226.42a 283.29b 192.34a PHE 88.07a 139.87b 89.82a LYS 241.04ab 294.69b 220.37a HIS 28.67a 78.80b 41.37a TYR 76.26a 149.22b 70.89a abc Denotes significant differences between fillets, roe and milt

Noted statistical observations: The milt appears to have a very similar composition of amino acids as fillets. Notable amino acids milt is relatively high in comparison to fillets is GLY, VAL (essential), and PRO. Roe is significantly higher in almost every amino acid than fillets. The only amino acids it is not significantly higher in is GLY (highest in milt), VAL (same as milt), and THR (same as milt). In all cases Roe is significantly higher than fillets in all amino acids.

Table 3.7 – Iron (Fe), sodium (Na), calcium (Ca), magnesium (Mg), and copper (cu) content per serving for Alaska pollock fillets, roe and milt on a wet basis. The serving size for fillets was 85 g, and the serving size for both roe and milt was 15 g. % Daily values (% DV) were rounded based on FDA rules.

Type Date Fe %DV Na %DV Ca %DV Mg %DV Cu %DV (mg) (mg) (mg) (mg) (mg) Fillet Feb 3 0.28 2 121.01 6 8.11 0 26.08 6 0.04 4 Feb 17 0.41 2 116.97 6 9.16 0 29.98 8 0.04 4 Mar 3 0.35 2 126.65 6 9.10 0 29.06 6 0.05 6 Mar 16 0.24 2 122.12 6 7.51 0 27.00 6 0.03 4 Mar 31 1.12 6 163.99 8 9.98 0 22.17 6 0.08 10 Season 0.48 2 130.15 6 8.77 0 26.86 6 0.05 6 A avg Jul 15 1.10 6 162.36 8 8.93 0 29.85 6 0.06 6 Aug 15 0.35 2 133.56 6 8.09 0 28.72 8 0.06 6 Season 0.73 4 147.96 6 8.51 0 29.28 6 0.06 6 B avg Roe Feb 3 0.26 2 20.74 0 2.54 0 2.12 0 0.02 2 Feb 17 0.15 0 18.34 0 1.71 0 1.19 0 0.02 2 Mar 3 0.11 0 19.12 0 1.90 0 1.40 0 0.01 2 Mar 16 0.30 2 16.74 0 1.29 0 1.35 0 0.01 2 Mar 31 0.18 2 30.94 2 3.09 0 1.45 0 0.01 2 Season 0.20 2 21.18 0 2.11 0 1.50 0 0.02 2 A avg Season 0.16 2 18.17 0 5.96 0 2.18 0 0.02 2 B avg Milt Feb 3 0.09 0 19.34 0 0.98 0 2.89 0 0.01 2 Feb 17 0.11 0 21.50 0 1.31 0 3.10 0 0.02 2 Mar 3 0.11 0 21.54 0 0.92 0 2.85 0 0.01 2 Mar 16 0.08 0 23.26 2 0.95 0 3.11 0 0.01 2 Mar 31 0.19 2 27.90 2 1.70 0 3.77 0 0.01 2 Season 0.12 0 22.71 0 1.17 0 3.14 0 0.01 2 A avg

Table 3.8 - Alaska pollock fillets, roe and milt macronutrients and micronutrients based on serving size for nutritional supplements (25 g), calculated on a dry basis. Type Season Total Total Fat Total Vitamin Vitamin Vitamin Fe Na Mg Cu Protein (g) (g) Omega-3 A (% DV) D (% DV) E (% DV) (% DV) (% DV) (% DV) (% DV) (mg) Fillet A 22.02 0.61 687.14 0 8 4 4 10 10 10 Fillet B 22.95 0.73 920.40 0 10 4 4 10 10 10 Roe A 21.50 1.49 1222.86 6 25 35 6 6 2 10 Roe B 19.84 2.42 2293.57 6 10 35 6 6 4 15 Milt A 20.63 3.14 1242.31 4 8 20 6 10 8 15

Figure 3.1 – HPLC chromatograms from Alaska pollock fillets, roe, and milt. The different wavelengths were overlaid. Vitamin A was viewed at 325 nm, vitamins D2 and D3 were viewed at 265 nm, and vitamin E was viewed at 296 nm.

Component Scores

Roe 5 Roe 6Roe Roe1 5 Fillet 2 Roe 4 Fillet 1 Roe 4 Roe 2 1 Roe 4 FilletFillet 4 3 Roe 5 FilletFillet 5 2Fillet 5 Roe 6 Fillet 1 Roe 6 Roe 3 Milt 5 Roe 2 Milt 3 Roe 1RoeRoe 2 3 Roe 3 Fillet 1 0 Milt 2 Roe 1 Fillet 6 Milt 3 Fillet 6 Milt 3

PC(7.31%) 2 -1 Milt 4 Mit 1 Fillet 7 Milt 4 Fillet 7 Milt 1 Fillet 7 Milt 5 -2 Milt 4

Milt 2 -3

-4 -10 -8 -6 -4 -2 0 2 4 6 8 10

PC 1 (75.28%) Figure 3.2 – Principle component analysis component scores comparing the amino acid composition of fillets, roe, and milt. Samples labeled 1 are from Feb 3, 2 are from Feb 17, 3 are from Mar 3, 4 are from Mar 16, 5 are from Mar 31, 6 are from Jul 15, and 7 are from Aug 15.

4. General Conclusion

After assessing proximate composition, fat-soluble vitamins, fatty acids, amino acids, and minerals in Alaska pollock fillets, milt and roe over two seasons, it was determined that the nutritional composition of Alaska pollock fillets and roe changes significantly from season A to season B, with only small changes within seasons. Milt, only present in season A, did not change significantly during the season. In general, fillets, roe and milt were found to be significantly different from one another.

Because fillets contained significantly higher protein, fat, vitamin D, omega-3 fatty acids, and EAAI scores in season B than in season A, they were determined to be higher quality in season B. These changes likely occurred due to the higher availability of food and increased feeding activity common in summer months. The changes found in proximate composition could lead to higher production yields during processing for season B fish. Concerning vitamins, fatty acids, amino acids, and minerals, having this information could be used to compare Alaska pollock to competing whitefish and also serve as a marketing tool.

Roe, typically only collected during season A, was also found early in season

B. From season A to season B, roe increased in fat content. This increase was primarily associated with increases and omega-3 and omega-6 fatty acids, both noted for their health benefits. Of the three sample types tested, roe was the only one found to completely meet or exceed requirements of essential amino acids.

Although vitamins A and E did not change significantly between seasons, vitamin D

decreased throughout season A and was even lower in season B. With these composition changes, roe could be marketed differently based on catch season.

Although milt changed sporadically, there were no trends associated with the changes and no singular stood out as completely different from all others. Of the three sample types, milt was the lowest in nutritional value on a wet basis, with low vitamin and mineral content. However, it still almost completely met essential amino acid requirements, with only a 2 mg/g protein deficiency in histidine.

Alaska pollock fillet, roe and milt composition was also analyzed on a dry basis. This information could be used in the development of new products from

Alaska pollock, particularly roe and milt. On a dry basis, roe and milt contain high protein, vitamin, mineral and omega-3 content. This gives them excellent potential for creation of various supplements or functional ingredients, which would allow them to be marketed as products for human consumption. Thus, future work may include studying effects of processing on nutritional content or functional properties of the proteins present.

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Appendix A

Appendix A - Fatty acid content of Alaska Pollock fillets, roe and milt caught throughout the A and B seasons in 2015. Values were calculated on a mg/ 85 g serving basis for fillets and mg/15 g serving for roe and milt. Type Date C14:0 C16:0 C16:1 C18:0 C18:1 C18:1 C18:2 C20:1 C20:4 C20:5 C22:6 n9t n9c n6c n6 n3 n3 Fillet Feb 3 0.00 104.62 0.00 0.00 0.00 20.79 0.00 6.81 0.21 201.06 262.17 (0.00) (11.34) (0.00) (0.00) (0.00) (4.23) (0.00) (6.63) (0.61) (39.97) (25.01) Feb 17 0.00 102.48 2.60 23.59 0.40 26.65 0.00 4.96 0.00 214.53 242.86 (0.00) (17.91) (4.30) (7.83) (1.21) (7.47) (0.00) (5.26) (0.00) (51.62) (45.80) Mar 3 0.58 98.43 3.29 22.96 0.00 24.88 0.74 4.53 1.58 187.94 216.46 (1.41) (16.32) (3.68) (6.66) (0.00) (7.00) (1.95) (3.54) (4.12) (39.60) (45.97) Mar 16 0.67 88.19 4.24 22.13 0.00 23.47 0.76 7.66 0.00 161.92 191.53 (2.02) (15.38) (5.01) (5.89) (0.00) (6.25) (2.28) (4.71) (0.00) (25.48) (39.00) Mar 31 0.00 85.23 5.92 17.68 0.00 21.84 1.40 3.15 0.00 134.63 172.76 (0.00) (2.86) (5.16) (1.93) (0.00) (3.13) (1.98) (4.45) (0.00) (26.24) (14.36) Season A 0.25 95.79 3.21 17.27 0.08 23.52 0.58 5.42 0.36 180.01 217.16 Avg. (1.22) (16.43) (4.32) (11.07) (0.63) (6.61) (1.60) (5.34) (1.99) (46.62) (48.57) Jul 15 2.39 123.80 1.99 21.87 0.00 19.26 3.61 4.18 0.00 312.06 267.73 (4.14) (18.83) (5.26) (6.63) (0.00) (5.27) (6.39) (7.78) (0.00) (68.08) (56.16) Aug 15 3.07 116.69 2.82 21.72 0.00 23.99 0.96 6.60 0.00 224.78 259.42 (4.01) (15.88) (4.32) (4.81) (0.00) (6.26) (2.88) (7.72) (0.00) (39.87) (50.76) Season B 2.73 120.25 2.41 21.79 0.00 21.62 2.29 5.39 0.00 268.42 263.58 Avg. (4.08) (17.51) (4.78) (5.69) (0.00) (5.94) (4.95) (7.84) (0.00) (68.48) (53.21)

Appendix A (Continued) Type Date C14:0 C16:0 C16:1 C18:0 C18:1 C20:1 C22:1n9 C20:5 C22:6 n9c n3 n3 Roe Feb 3 4.58 58.42 11.52 0.26 17.08 5.77 0.00 94.84 96.99 (2.40) (9.01) (3.40) (0.58) (2.02) (5.89) (0.00) (19.15) (15.67) Feb 17 6.42 67.21 13.89 1.27 18.26 1.70 0.00 108.99 110.19 (2.48) (6.37) (2.45) (2.54) (3.66) (2.63) (0.00) (14.63) (8.50) Mar 3 2.23 58.55 9.85 0.00 17.45 2.66 0.00 133.41 91.38 (1.52) (5.54) (2.11) (0.00) (3.94) (2.79) (0.00) (23.51) (6.97) Mar 16 5.04 54.04 9.84 0.13 13.90 1.44 0.00 102.97 104.50 (3.18) (9.32) (5.91) (0.26) (3.89( (1.87) (0.00) (26.56) (12.32) Mar 31 3.36 70.99 13.05 0.59 13.86 0.00 0.00 103.17 93.60 (2.75) (6.93) (4.87) (1.17) (3.35) (0.00) (0.00) (26.09) (26.75) Season A 4.33 61.84 11.63 0.45 16.11 2.31 0.00 108.68 99.33 Avg. (2.90) (9.81) (4.43) (1.36) (3.93) (4.23) (0.00) (21.55) (43.00) Season B 7.02 46.06 10.59 0.41 6.85 7.32 2.23 129.04 283.80 Avg. (2.01) (9.98) (2.90) (0.71) (5.20) (2.47) (2.71) (21.55) (43.00) Milt Feb 3 0.13 33.30 0.57 0.23 14.05 2.21 0.00 52.71 62.07 (0.12) (2.49) (0.09) (0.10) (5.00) (1.31) (0.00) (15.75) (12.27) Feb 17 0.00 34.22 2.36 1.17 20.62 5.63 0.00 60.89 61.69 (0.01) (3.96) (1.02) (0.81) (4.30) (3.17) (0.00) (7.36) (7.05) Mar 3 0.09 34.99 1.51 3.74 17.88 1.24 0.00 53.93 48.89 (0.15) (10.43) (0.47) (1.72) (2.37) (1.00) (0.00) (9.89) (8.35) Mar 16 0.03 33.70 2.24 1.83 21.63 1.45 0.00 53.71 59.02 (3.18) (9.32) (5.91) (0.26) (3.89) (1.87) (0.00) (26.56) (12.32) Mar 31 0.17 38.71 2.21 4.69 19.99 3.62 0.00 56.87 49.26 (0.25) (5.97) (1.42) (1.93) (2.50) (0.76) (0.00) (12.53) (7.30) Season A 0.08 34.98 1.78 2.33 18.83 2.83 0.00 55.62 56.19 Ave. (0.16) (6.71) (1.09) (2.27) (4.66) (1.71) (0.00) (10.69) (10.09)

Appendix B

Appendix B - Amino acid composition by date and season of Alaska pollock fillets, roe, and milt on a mg/g protein basis. Type Date ALA GLY VAL LEU ILE THR SER PRO ASP MET + GLU PHE + LYS HIS CYS TYR Fillet Feb 3 4.98 29.80 44.26 73.46 37.60 25.73 20.54 19.81 53.86 17.27 65.73 47.74 66.01 6.25 (1.47) (6.12) (17.01) (20.17) (14.91) (2.78) (2.36) (4.35) (7.43) (3.73) (11.48) (16.18) (20.45) (2.32) Feb 17 4.17 25.98 33.53 58.14 26.95 25.46 25.36 18.73 43.73 17.45 46.63 45.13 45.17 4.04 (0.79) (3.18) (7.16) (15.58) (2.70) (2.75) (6.26) (4.55) (3.76) (0.89) (4.11) (11.15) (16.60) (0.74) Mar 3 4.99 28.61 38.53 61.51 29.56 32.56 23.55 19.65 51.49 17.71 69.79 32.29 66.05 5.89 (0.46) (2.48) (5.35) (8.12) (3.44) (5.38) (2.42) (1.54) (2.88) (3.25) (8.04) (2.96) (2.22) (0.00) Mar 16 4.31 28.46 37.65 61.55 32.79 25.44 23.70 18.95 49.08 19.70 72.23 38.44 65.32 2.79 (0.48) (2.80) (3.39) (5.24) (6.34) (2.01) (2.10) (2.41) (4.42) (5.21) (14.54) (13.73) (15.13) (0.00) Mar 31 3.38 26.08 31.89 51.46 26.18 20.20 15.06 18.13 37.12 15.03 42.35 41.75 39.57 7.28 (0.63) (5.03) (5.74) (9.76) (6.63) (6.45) (4.94) (4.81) (5.27) (4.99) (16.19) (7.68) (16.58) (0.96) Season A 4.37 27.79 37.17 61.22 30.62 25.88 21.64 19.05 47.06 17.43 59.35 40.89 56.43 5.56 Avg. (1.04) (1.04) (10.07) (14.79) (9.09) (5.78) (5.40) (3.82) (7.81) (4.20) (17.05) (12.95) (19.38) (2.08) Jul 15 6.99 43.26 60.32 100.19 50.33 30.50 21.57 26.21 64.53 30.44 84.41 45.89 108.69 13.94 (0.82) (2.39) (5.71) (14.30) (8.27) (4.26) (1.76) (4.14) (9.33) (1.78) (17.50) (10.55) (4.54) (0.76) Aug 15 7.12 53.57 66.46 109.13 62.35 49.75 38.64 30.87 75.44 38.90 88.76 40.40 109.77 13.52 (0.84) (2.78) (10.47) (5.94) (5.93) (10.86) (4.00) (1.84) (2.68) (7.35) (1.46) (9.57) (8.93) (3.94) Season 7.06 48.42 63.39 104.66 56.34 40.12 30.10 28.54 69.99 34.67 86.59 68.00 109.23 13.69 B Avg. (0.83) (5.77) (8.98) (11.83) (9.38) (12.68) (9.08) (3.96) (8.77) (6.81) (12.61) (10.74) (7.10) (3.09)

Appendix B (Continued) Type Date ALA GLY VAL LEU ILE THR SER PRO ASP MET + GLU PHE + LYS HIS CYS TYR Roe Feb 3 13.74 42.19 88.96 131.89 74.99 49.05 61.44 67.83 79.10 30.19 115.46 48.54 89.91 18.84 (0.19) (2.58) (2.63) (7.24) (5.62) (2.09) (0.75) (2.06) (4.38) (4.95) (8.83) (12.43) (6.19) (0.00) Feb 17 15.21 44.13 99.25 134.36 83.54 51.72 65.59 67.36 76.98 32.12 111.51 47.15 103.92 29.18 (0.48) (2.80) (3.39) (5.24) (6.34) (2.01) (2.10) (2.41) (4.42) (5.21) (14.54) (13.73) (15.13) (0.00) Mar 3 15.74 48.03 101.51 145.98 91.83 54.14 62.31 70.37 80.78 34.22 110.99 44.57 115.18 33.67 (0.62) (3.37) (3.58) (4.52) (6.66) (3.58) (6.41) (2.91) (8.51) (5.16) (20.93) (15.11) (18.16) (1.76) Mar 16 12.83 36.27 71.54 120.92 59.71 43.86 48.44 59.12 60.47 24.94 73.65 35.21 90.68 19.50 (0.53) (3.28) (1.74) (3.94) (2.84) (5.07) (6.45) (2.69) (5.64) (2.03) (14.44) (1.89) (13.86) (2.26) Mar 31 14.08 42.64 76.83 144.29 66.98 42.05 51.24 65.74 61.64 28.44 67.59 30.74 96.09 27.85 (0.63) (5.03) (5.74) (9.76) (6.63) (6.45) (4.94) (4.81) (5.27) (4.99) (16.19) (7.68) (16.58) (0.96) Season A 14.00 42.91 86.87 135.81 74.76 47.36 55.29 66.04 68.95 30.22 92.62 94.77 96.17 25.69 Avg. (1.66) (5.50) (13.09) (16.92) (13.25) (6.45) (10.15) (6.21) (11.45) (3.88) (24.75) (10.56) (12.59) (6.64) Season B 12.37 44.21 83.16 137.42 71.53 43.32 42.71 65.84 54.74 31.39 76.50 38.58 81.24 25.12 Avg. (1.78) (9.25) (16.07) (19.41) (15.19) (6.62) (4.33) (6.33) (14.76) (9.93) (28.95) (15.43) (31.00) (3.23) Milt Feb 3 6.37 65.65 83.68 98.92 51.55 43.23 40.51 41.27 56.96 22.36 73.59 64.27 81.67 12.22 (0.82) (2.39) (5.71) (14.30) (8.27) (4.26) (1.76) (4.14) (9.33) (1.78) (17.50) (10.55) (4.54) (0.76) Feb 17 5.32 55.64 73.20 89.24 43.49 39.02 29.85 38.97 50.27 17.84 57.70 65.37 52.57 12.48 (0.84) (3.45) (5.50) (10.67) (8.32) (5.90) (5.71) (4.39) (10.27) (2.06) (13.31) (9.99) (6.18) (2.43) Mar 3 4.92 41.84 60.62 76.87 38.69 33.67 27.89 31.09 40.22 17.41 61.00 74.86 74.18 11.88 (0.81) (3.69) (8.75) (7.94) (6.09) (2.62) (5.84) (1.75) (2.68) (8.92) (1.23) (9.94) (9.08) (2.61) Mar 16 6.05 63.18 74.29 99.07 49.51 40.49 36.82 37.46 50.21 22.04 61.54 71.72 83.49 16.99 (0.84) (2.78) (10.47) (5.94) (5.93) (10.86) (4.00) (1.84) (2.68) (7.35) (1.46) (9.57) (8.93) (3.94) Mar 31 5.01 46.15 65.66 84.96 40.78 32.88 29.50 32.98 39.78 19.67 56.28 86.01 64.38 11.91 (3.46) (2.70) (17.05) (24.28) (9.62) (9.36) (12.55) (21.82) (9.24) (7.50) (19.82) (14.03) (9.82) (3.08) Season A 5.53 54.49 71.49 89.81 44.81 37.86 32.91 36.35 47.49 19.86 62.02 51.96 71.26 13.32 Ave. (1.11) (14.80) (15.09) (15.66) (8.66) (9.30) (9.80) (8.80) (11.26) (4.37) (11.00) (11.89( (17.35) (5.01)