Michael T. Morrissey

Marine Biotechnology for Production of Food Ingredients

The marine world represents a largely untapped reservoir of bioactive ingredients that can be applied to numerous aspects of food processing, storage, and fortification. Due to the wide range of environments they survive in, marine organisms have developed unique properties and bioactive compounds that, in some cases, are unparalleled by their terrestrial counterparts. Enzymes extracted from fish and marine microorganisms can provide numerous advantages over traditional enzymes used in food processing due to their ability to function at extremes of temperature and pH. Fish proteins such as collagens and their gelatin derivatives operate at relatively low temperatures and can be used in heat-sensitive processes such as gelling and clarifying. Polysaccharides derived from algae, including algins, carrageenans, and agar, are widely used for their ability to form gels and act as thickeners and stabilizers in a variety of foods. Besides applications in food processing, a number of marine-derived compounds, such as omega-3 polyunsaturated fatty acids and photosynthetic pigments, are important to the nutraceutical industry. These bioactive ingredients provide a myriad of health benefits, including reduction of coronary heart disease, anticarcinogenic and anti-inflammatory activity. Despite the vast possibilities for the use of marine organisms in the food industry, tools of biotechnology are required for successful cultivation and isolation of these unique bioactive compounds. In this chapter, recent developments and upcoming areas of research that utilize advances in biotechnology in the production of food ingredients from marine sources are introduced and discussed.

Advances in DNA-Based Techniques for the Detection of Seafood Species Substitution on the Commercial Market

Increased worldwide trade and processing of seafood has increased the potential for species substitution on the commercial market. To detect and prevent species substitution, several deoxyribonucleic acid (DNA)-based methods have been developed that can be used to identify species in a variety of food types. For large-scale applications, such as regulatory screening, these methods must be rapid, cost-effective, reliable, and have high potential for automation. This review highlights recent technological advances in DNA-based identification methods, with a focus on seafood species identification in automated, high-throughput settings. Advances in DNA isolation methods include silica-based columns for use in high-throughput operations and magnetic bead particles for increased and targeted recovery of DNA. The three most widely used methods for seafood species identification (polymerase chain reaction [PCR] sequencing, PCR-restriction fragment length polymorphism, and species-specific PCR) will be discussed, with a focus on the incorporation of technologies such as rapid PCR cycling, microfluidic chips, and real-time PCR. Emerging methods, including DNA microarrays and next-generation sequencing will also be explored for their potential to identify seafood species on a large scale. Overall, many of the technological advances discussed here offer complementary properties that will enable species identification in a variety of settings and with a range of products.

Finite element analysis as a tool for crossflow membrane filter simulation

Finite element analysis (FEA) is a very powerful tool in analyzing many engineering problems. In this study, FEA was used to simulate the development of concentration polarization in ultrafiltration of protein solutions. A miniature crossflow membrane filter was developed to verify the FEA models. Polysulfone membrane disks (47 mm) were used in this study. Bovine serum albumin (BSA) solutions of different concentrations were pumped across the membrane flow channel. The crossflow velocity of the feed solution was carefully controlled at the laminar region. With the flow velocities within the flow channel estimated by a perturbation solution, the protein concentration on the membrane surface and the mass transfer coefficient were accurately predicted by FEA. This simulation method may provide a useful tool in engineering analysis and design of a membrane filtration process.

The use of isolated hepatocytes from rainbow trout (salmo gairdneri) in the metabolism of acetaminophen

Abstract 1. 1. Hepatocytes were isolated from rainbow trout. Cytrochrome P-450 and glutathione (GSH) were determined and found to be considerably lower than values reported for rat hepatocytes. 2. 2. Acetaminophen was incubated with rainbow trout hepatocytes; and glucuronide, glutathione and sulfate conjugates were analyzed. 3. 3. The formation of GSH conjugates were lower than that of glucuronide conjugates and both were several fold lower than those reported for rat hepatocytes. 4. 4. In contrast to rat hepatocytes, sulfate conjugates were not detected. 5. 5. These results demonstrate the ability of isolated fish hepatocytes to metabolize xenobiotics and further illustrates differences in the formation of metabolites between trout and mammals.

Parameters for the recovery of proteases from surimi wash water.

Proteases are important bioactive compounds that have many applications in food processing. In this study, laboratory scale experiments were performed to establish conditions for recovery of a heat stable, acid protease from Pacific whiting (Merluccius productus) surimi process water. Reduction of proteinaceous solids and recovery of protease activity was maximized when process water was pre-treated with acid (pH 4) followed by heat (60 degrees C). In addition, acid plus heat treatment of process water appeared to improve membrane flux and concentration of protease activity (10-fold) was achieved in half as much time as treatments using acidification but not heat. Neither purification nor concentration of protease was effective using either 300 and 1000 kDa ultrafiltration or 0.3 microm microfiltration membranes. However, concentration of protease using 50 kDa ultrafiltration membranes was successful in recovering about 80% of original protease activity. Results provide conditions for further investigations into pilot plant recovery of protease from surimi process water.

Quantification and Distribution of Lipid, Moisture, and Fatty Acids of West Coast Albacore Tuna (Thunnus alalunga)

Abstract Intrinsic quality characteristics, including lipid, moisture, and fatty acid content, of West Coast albacore tuna (Thunnus alalunga) was investigated. West Coast albacore tuna were troll caught off the Oregon coast, delivered to the Oregon State University Seafood Laboratory, held at -30°C and analyzed for lipid distribution in different areas of the muscle. Additional albacore were troll caught off the Oregon coast and analyzed fresh for fatty acid content. Core samples were taken from six designated body zones of the fish. The lipid content ranged from 3.9 ± 0.2 to 36.3 ± 1.1%, with a distribution of higher lipid towards the head and lower lipid towards the tail. Total omega-3 content averaged approximately 40% of the identified fatty acids for each body zone, with average omega-3 (g/100 g tissue) ranging from 2.1 ± 0.5 to 3.5 ± 0.4. An inverse correlation (R2 = −0.95) was found for lipid and moisture content.

A Review of Mercury in Seafood

ABSTRACT Mercury is a toxic heavy metal released into the environment from both natural and anthropogenic sources. It is of great interest to consumers as to whether it can cause neurological effects at low dose levels. The effects of organic mercury exposure at high levels have been demonstrated in several large-scale poisonings, particularly those in Japan and Iraq in the 1950s, ′60s and ′70s. These epidemics showed that organic mercury, in sufficient concentrations, is a potent neurotoxin that is especially harmful to the developing nervous system. Since the most common form of human exposure to organic mercury is through fish consumption, several epidemiological studies have examined the relationship between maternal fish intake and health effects in humans, especially the fetus. Levels of mercury in fish vary depending on factors such as: trophic level in the food chain, size, and habitat location. For this reason, it is important to gather information on mercury levels in different types of fish in various parts of the world. Results of recent studies have caused the Food and Drug Administration (FDA) and the Environmental Protection Agency (EPA) to issue new advisories on the fish consumption for pregnant women and young children. However, there is concern that other individuals will significantly reduce their fish consumption also, thereby decreasing the potential health benefits of seafood. This review is meant to promote understanding of current issues regarding mercury in seafood and provides a compilation of up to date information on the following topics: background information on mercury; large scale mercury poisonings; epidemiology studies and risk assessment; and studies on mercury in tuna in different geographical locations.

Mercury Content in Pacific Troll-Caught Albacore Tuna (Thunnus alalunga)

Abstract Ninety-one albacore tuna (Thunnus alalunga) captured during the 2003 commercial fishing season were tested for total mercury content in muscle tissue. The fish were harvested between 32.72°N (off Southern California) and 48.30°N (off the northern tip of Washington) between July and November. Fish weighed from 3.14 to 11.62 kg and were 50.8-86.4 cm long. Total mercury content in the albacore muscle tissue ranged from 0.027 ppm (μg/g) to 0.26 ppm. The average total mercury content was 0.14 ± 0.05 ppm, which is below the U.S. Food and Drug Administration action level and Canadian standards (1.0 ppm methylmercury and 0.50 ppm total mercury, respectively). Total mercury concentrations showed positive correlations with length and weight of albacore (R2= 0.40 and 0.38, respectively), but there was no correlation with date of capture or lipid content. Results indicate that Pacific troll-caught albacore have low levels of total mercury in the edible flesh and are well within international safety standards for mercury levels in fish.

Pilot plant recovery of catheptic proteases from surimi wash water

Recovery of bioactive compounds, such as proteolytic enzymes, from waste streams is a means to both recuperate value and reduce environmental pollution. Previously optimized lab-scale parameters for the recovery of a stable crude protease fraction from Pacific whiting (Merluccius productus) surimi wash water were tested using pilot plant equipment. Pretreatment of surimi wash water with 60 degrees C heat, acidification to pH 6, and centrifugation doubled ultrafiltration membrane flux and significantly improved protease purity by reducing a majority of the 35-205 kDa proteins. Concentrated crude protease obtained from wash water contained predominantly cathepsin L activity. Enzyme purity was increased about 100-fold, and yield was approximately 80%. Stability (frozen and freeze-dried protease) was maintained for 9 weeks at -80 degrees C. Freeze-dried preparations were also stable for 9 weeks at 4 and -15 degrees C. Successful application of pilot plant conditions allows for sufficient production of protease for further investigations into their applicability.

Molecular cloning, sequence analysis and expression distribution of rainbow trout (Oncorhynchus mykiss) cystatin C

Cystatin C is one of a family of proteinase inhibitors of cathepsins and other cysteine proteinases. Among warm-blooded vertebrates, small functional regions of cystatin amino acid sequences are well conserved among species, but major portions of cystatin amino acid sequences vary evolutionarily. Although considerable attention has been given to mammalian and avian cystatins, little data exist on cystatins from other vertebrates. A cDNA clone for trout cystatin C was isolated from a lambda gt11 cDNA library of rainbow trout (Oncorhynchus mykiss) liver. An apparently full-length cDNA clone of 674 bp encoding 132 amino acid residues was obtained. Sequence analysis indicated that trout cystatin C contains an N-terminal signal sequence extension of 21 amino acids and a mature sequence of 111 amino acid residues, with amino acid residues conserved in functional regions relative to mammalian and avian cystatin C. Using cloned cDNA as a probe, we investigated expression of the cystatin C gene in trout tissues, several cell lines of trout liver or liver tumor, and cell cultures of liver tumor origin. Cystatin C mRNA was in high abundance in trout embryo tissue, a tumor-derived liver cell line and some normal adult tissues. Southern hybridization analysis indicated one copy of the trout cystatin C gene per haploid genome, and sequence comparisons indicated considerable divergence in large portions of the coding region of the trout cystatin C gene relative to a variety of species.