Date of Award

12-2008

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Food Science and Human Nutrition

Advisor

Scott Collins

Second Committee Member

Brian L. Perkins

Third Committee Member

Rosemary Smith

Abstract

Since the mid-1800s the lobster industry in the U.S. and Canada has represented a source of revenue and a way of life for generations of families. Maine produces more than 80% of the entire landings of Homarus americanus lobster (American lobster) species in the U.S. lobster fishery, with a value of over $307 million. Given that the American lobster is such an important fishery in New England and Canada with a high export value to local and foreign markets, it is not surprising that the metrics which quantify lobster vitality are of interest to the lobster industry. The vitality of lobsters are measured Lobster vitality measurements are used to minimize losses and the resulting data are used to determine the ideal use of each lobster. One Maine lobster dealer placed the mortality rate of shipped lobsters at >2% for hard shell and 5 to 6 % for new/soft shell lobsters (newly molted) (Eaton 2003). The concentration of total hemolymph protein (THP) in the lobster hemolymph (lobster blood) correlates to muscle mass, which has lead to the conclusion that measurement of the serum protein concentration in hemolymph is a viable method for the determination of health and/or vitality of the lobster (Stewart et al. 1969). Refractometry, an invasive technique, is currently the industry standard for determining lobster vitality but this technique is invasive, time consuming and induces stress on the lobster. The major focus of this thesis was to study the optical properties of the American lobster (Homarus americanus) hemolymph and its major protein constituent, hemocyanin (Hc). These studies will be performed both in vitro and in vivo. Information gained from the results of this research will be critical in the design and development of a non-invasive optical sensor for lobster vitality. Hemolymph was analyzed using both ultraviolet/visible (UV/VIS) and fluorescence spectroscopy, to identify the most prominent feature(s) related amenable to lobster vitality. This information was used to assemble a benchtop spectrometer with fiberoptic reflection probe to non-invasively validate the ability of optical spectroscopy to assess the vitality of live lobsters. In addition, an analytical method using highperformance liquid chromatography (HPLC) with fluorescence and UV photo diode array detectors will be developed for a potential quality control procedure for the purification of Hc in lobster hemolymph. Based on in vitro work, optical sensing using absorbance spectroscopy appears to work the better than the fluorescence spectroscopy in the in vitro studies as it provides accurate and reproducible results. Besides providing THP and Hc concentration, absorbance spectroscopy can also determine the oxygenation status of hemolymph. Fluorescence spectroscopy yielded convoluted spectra that was further complicated by oxygen and self-quenching and required complicated analysis and interpretation. Fluorescence spectroscopy was far more problematic than absorbance spectroscopy, leaving absorbance as a far superior technique for determining lobster vitality. In vivo optical probe UV absorbance measurements using the fixed 280nm, 290nm and 295nm wavelengths yielded some potentially useful sampling methods, Absorbance at 280nm yielded results that were consistent with in vitro THP concentration measurements. The fluorescence emission peaks that resulted from the 280nm, 290nm and 295nm excitations were not consistent with in vitro analyses. In addition, 334nm analysis showed the same light intensity, at all sample concentrations. This reaffirms the in vitro results, and indicates absorbance spectroscopy might be better for non-invasive optical measures for the determination of lobster vitality. HPLC analysis of Hc and hemolymph was easily performed using a diode array detector (DAD). Samples had to be substantially diluted to fit the dynamic range of the fluorescence detector (FLD). Coupling two size exclusion columns worked very well in separating hemolymph and Hc proteins. The major oligmers of hemolymph were determined to be dodecamers followed by hexamers. 24-mers and 48-mers were detected, but in low concentrations. HPLC/DAD provided the best quality control analysis based on ease of sample preparation and the spectrum analysis. This technology provides separation sample cleanup and quality control monitoring.

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