Title: The Use of the pH Shift Method to Recovery Protein and Lipids from Sardines and other Pelagic Fish Species

Oregon Sea Grant Project: 2 years. Beginning March, 2004

Principle Investigators: Michael T. Morrissey and Jae Park, Oregon State University Seafood Laboratory, Astoria, OR.

Problem/Opportunity: The large stocks of sardine ( Sardinas sagax ) and other pelagic fish species off the Pacific
Northwest coast have received increased attention as potential resources for functional proteins and valuable omega-
3 oil. In Oregon, 49 million lbs. of sardines were harvested in 2002, the vast majority of which were directed
towards non-human consumption such as long-line tuna bait. Preliminary studies found that these sardines are large in
size (ave. 180 gm) and oil content will vary from 6% to 20% during the harvest season. Isolation of functional
proteins and omega-3 oil from pelagic fish for food production would be a more responsible way of using a nutritious
and abundant raw material. Studies were successfully carried out to evaluate acid and alkali solubilization (pH-shift)
for Pacific whiting. This new protein recovery method provides good functionality and efficient separation of fish oils.
The pH-shift method is a new revolutionary process that maximizes protein and fish oil recovery. Unlike the
conventional method of surimi manufacturing, no washing and dewatering are involved and sarcoplasmic proteins and
other protein materials are recovered and can be utilized as functional proteins similar to recovering and using isolated
soy proteins. Furthermore, the pH-shift method, unlike conventional surimi processing, allows for the recovery of fish
oils by non-thermal centrifugation. The increased yield for functional protein s and recovery of fish oils with important
omega-3 fatty acids are critical for the economic success of this new fishery in the Pacific Northwest.

The objectivs of this study are the following: 1) What is the recovery and quality of proteins through the pH-shift
method; 2) What happens to sarcoplasmic proteins retained through pH-shift process ; 3) Can the conformational
integrity of myofibrillar proteins be maintained during long term frozen storage; 4) What is the recovery of lipids and
the concentration and stability of omega-3 fatty acids in the pH-shift method?

Approach: Protein fraction - separation and evaluation : Rinsed sardine fillets/mince will be subjected to
conventional, acid-aided (pH 2 and 3), and alkali-aided (pH 10.5, 11, and 12) processing methods. In the pH-shift
method the homogenate will be centrifuged at 4,000 ×g for 20 min for each washing step. For acid- or alkali-aided,
2 N HCl or 2N NaOH, will be used to first solubilize the fish proteins after mixing the fillets with distilled water 1 to 9
ratio. Acidic or alkaline homogenates will be centrifuged at 8,000 × g for 25 min followed by pH adjustment to the
isoelectric point (pH 5.5). Neutral and membrane lipids, skin, and bone will be removed using cheesecloth followed
by mechanical dewatering using centrifugation (4,000 ×g, 20 min). Temperature will be maintained below 5 °C . Fish
protein samples will be vacuum-packed and stored at -30 °C until tested. At each step, samples will be subjected to
biochemical, enzymatic, and rheological analyses to measure conformation changes of myofibrillar proteins, as
affected by acid, proteolytic enzymes, and other sarcoplasmic proteins. For rheological properties, the pH will be
adjusted to approximately 7.0 using 2N NaOH.

Oil fraction - separation and evaluation : The oil fraction will be recovered after the first centrifugation of the protein
separation method. Further purification of the oils will be accomplished through crystallization techniques.
Identification of omega-3 fatty acids will be analyzed using gas chromotogrpahy. Separation of docosahexaenoic
(DHA) and eicosapentaenoic (EPA) acids will be accomplished through several methods including liquid
chromatography, molecular distillation and urea complexation. DHA and EPA concentrations will be enhanced
through a new lipase-assisted hydrolysis method. Stability of the recovered oils will be tested over a six month
period.

Target audiences: There are currently seven sardine processing operations in the Lower Columbia River basin who
will benefit directly from this research. Pelagic species represent 30% of the world's catch and the research will be
important at the international level. The annual OSU Surimi Technology School, which is founded by PI s and taught
both locally and at three international sites, will be an ideal place to demonstrate the project results.

Anticipated benefits: The local sardine fishery has limited markets for harvested sardines. The pH-shift method will
allow for extraction of food-grade functional protein and fish oils and will increase market potential and economic
benefits. The research will also show fundamental principles for protein and lipid extraction from pelagic species.

Potential Outcomes: The successful completion of the project will make a processor of sardines more profitable by upgrading bait/meal to food grade food and functional ingredients.