CHARACTERIZATION OF SALMON (ONCORHYNCHUS KETA) AND STURGEON (ACIPENSER TRANSMONTANUS) CAVIAR PROTEINS MURAD A. AL-HOLY 1 and BARBARA A. RASCO 2,3 1 Department of Clinical Nutrition and Dietetics Faculty of Allied Health Sciences Hashemite University Zarqa, Jordan 2 Department of Food Science and Human Nutrition Washington State University P.O. Box 646376, Pullman, WA 99164-6373 Received for Publication September 6, 2005 Accepted for Publication October 29, 2005 ABSTRACT The solubility of protein components in salmon (Oncorhynchus keta) and sturgeon (Acipenser transmontanus) caviar in distilled water, 5% (w/v) NaCl, 70% (v/v) ethanol at 65C, and 0.2% (w/v) NaOH was determined. The salt soluble proteins were the predominant fraction and constituted 84.2% of the recovered protein in salmon and 86.1% in sturgeon samples. The two most prominent protein fractions (12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis) for sturgeon caviar are most likely vitellin (96 kda) and ovomucoid or phosvitin (28 kda). For salmon roe, small proteins, possibly lysozyme or phosvitin (10 kda), are also present. INTRODUCTION Little information is available on the biochemical characteristics of caviar, although some compositional information is available (Bledsoe et al. 2003). Crude lipid content varies from 5 to 20% by weight with a mean value for salmon of around 10%. Fish roe has a high concentration of protein (16 to 30% by weight) with a range of 26 28% in sturgeon (Sikorski 1994) and 29% in chum salmon on wet weight basis. The total protein content of 22 sturgeon species was examined by Wirth et al. (2000). The average protein content 3 Corresponding author: TEL: 509-335-1858; FAX: 509-335-4815; EMAIL: rasco@wsu.edu 422 Journal of Food Biochemistry 30 (2006) 422 428. All Rights Reserved. 2006, The Author(s) Journal compilation 2006, Blackwell Publishing
SALMON AND STURGEON CAVIAR PROTEINS 423 varied between 26.2 and 31.1% on wet matter basis. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (ca. 12% SDS-PAGE) has been used to differentiate sturgeon caviar species (Scobbie and Mackie 1990; Chen et al. 1996). Heating prior to extraction in SDS had a little effect on the separation patterns obtained for a range of commercially important raw and cooked fish species (Scobbie and Mackie 1988). No reported studies were found on the protein solubility for fish roe products or on the likely composition of these proteins, even though this information would be important for predicting functional properties and possibly biochemical or microbiological stability. Determining protein solubility has widespread use in studies of food proteins. From Lund and Sandstorm (1943), methods for fractionating cereal and legume proteins were developed to classify proteins based upon their solubility in water (albumins), in aqueous salt solutions (globulins), alcohol (prolamin in cereals) and alkaline solutions (glutelins for cereals). The objective of this work was to partially characterize protein constituents of salmon and sturgeon caviar. MATERIALS AND METHODS Fractionation of Salmon and Sturgeon Roe Proteins A 5-g sample of salmon roe (0.7% salt) (Onorhynchus keta) (Mayco Fish Co., Tacoma, WA) and sturgeon caviar (0.2% salt) (Stolt Sea Farms, Elvira, CA) were crushed and then defatted by extracting several times with petroleum ether using a Goldfish fat extraction method (Min 1994). The caviar extracts were dried overnight at 55C. Proteins were recovered based upon their solubility using the method described by Lund and Sandstorm (1943). Duplicate 2-g samples of salmon and sturgeon caviars were finely ground with a mortar and pestle and then transferred to 50-mL centrifugal tubes and suspended in 25 ml of distilled water. The tubes were vortexed for about 1 min to extract the proteins (Mini Vortexer, VWR Scientific, Model 945300, Henry Troemner, LLC, Thorofare, NJ). The suspensions were then centrifuged at 4000 rpm (Centra CL-2, Model 120, Thermo IEC, Needham Heights, MA) for 10 min at approximately 22C and the supernatant recovered and saved. The residues were re-extracted twice more with distilled water and the recovered supernatants designated as the water-soluble fraction. Thereafter, the residues were extracted successively with 5% (w/v) NaCl at approximately 22C, 70% (v/v) ethanol at 65C in a water bath, and 0.2% (w/v) NaOH at approximately 22C in a similar manner as for the water-soluble fraction. The total nitrogen content of the dried caviar samples, collected supernatant fractions and the
424 M.A. AL-HOLY and B.A. RASCO residue remaining after sequential extraction was determined using a Nitrogen Analyzer (Leco Corp., St. Joseph, MI). Gel Electrophoresis of Salmon and Sturgeon Caviar Proteins SDS-PAGE was conducted on 12.5% polyacrylamide and stacking gel with N, N, N, N-tetramethylenediamine (TEMED), and ammonium persulfate (APS) cross-linking agents. The gels contained the following: (stacking gel: 0.85-mL H 2 O, 0.36 ml 0.5-M Tris buffer (ph 6.9), 2.25 ml of 38% acrylamide, 7.5 ml of 20% SDS, 2.5 ml of TEMED and 15 ml 10% APS) and running gel (12.5% w/v) (1.37-mL H 2 O, 0.84 ml 2-M Tris buffer (ph 8.8), 2.25 ml of 30% acrylamide, 22.5 ml of 20% SDS, 5.0 ml of TEMED, 35 ml of 10% APS). Caviar (2 g) was suspended in 5 ml of 20-mM Tris-glycine buffer (ph 7.5) containing 0.1% SDS and sonicated (Branson Sonifier 450, VWR Scientific, Danbury, CT) intermittently several times to disperse the protein. The following samples were prepared: (1) 50 ml of the slurry was suspended in 50 ml of 20-mM Tris buffer (ph 7.5), boiled for 5 min; (2) soluble protein: the slurry was centrifuged for 15 min at 13,200 rpm (Eppendorf, 5415 D, Brinkman Instrument, Westbury, NY) to separate soluble and insoluble fractions of the protein, 50 ml of the supernatant was suspended in 50 ml of 20-mM Tris buffer (ph 7.5), the mixture was boiled for 5 min and centrifuged for 10 min at 13,200 rpm; (3) precipitate: a few micrograms of the precipitate (insoluble protein) was suspended in 50 ml of 20 mm of Tris buffer (ph 7.5), boiled for 5 min and then centrifuged at 13,200 rpm for 10 min. Samples (100 mg) were loaded into wells of the polyacrylamide gel and separated using electrophoresis system FB 400 at 20 45 ma (Ne Xagen, Inc, Fisher Scientific, Pittsburgh, PA). Electrophoresis was completed when the dye front reached the bottom of the gel. The proteins were then stained for 45 min with 0.125% (w/v) Coomassie brilliant blue R-250 (Bio-Rad Life Sciences, Hercules, CA) in 40% ethanol and 7% acetic acid, then destained in 5% methanol and 7% acetic acid. Molecular weights were estimated using a low molecular weight standard protein kit (96, 60, 38 and 28 kda) (School of Molecular Biosciences, Washington State University, Pullman, WA). RESULTS AND DISCUSSION Proteins can be classified into four main groups by their solubility as an indication of their hydrophobic and ionic properties. Hydrophobic interactions promote protein protein interactions decreasing solubility, whereas ionic interactions promote protein water interactions and result in increased solubility (Damodaran 1996). Proteins can be soluble in water at ph 6.6 (e.g.,
SALMON AND STURGEON CAVIAR PROTEINS 425 TABLE 1. PROTEIN FRACTIONS OF SALMON AND STURGEON CAVIAR ACCORDING TO THEIR SOLUBILITY* Protein fraction Fraction of total nitrogen (%) Salmon caviar Sturgeon caviar Water soluble 0.00 ± 0.00 0.00 ± 0.00 Salt soluble 84.20 ± 1.30 86.10 ± 0.15 Alcohol soluble 0.00 ± 0.00 0.00 ± 0.00 Alkaline soluble 0.00 ± 0.00 0.00 ± 0.00 Residue 12.33 ± 1.60 10.16 ± 1.00 Nonrecoverable 3.47 ± 1.40 3.84 ± 0.58 * Results are mean ± SD (n = 4), dry weight basis. serumalbumin, ovalbumin and a-lactalbumin), dilute salt solution at ph 7.0 (e.g., glycinin, phaseolin and a-lactoglobulin), in acid (ph 2) and base (ph 12) (e.g., wheat glutelins and prolamines, and in 70% ethanol (e.g., wheat gliadin and barely hordein) (Damodaran 1996). The total nitrogen content of salmon and sturgeon caviar on a defatted dry matter basis was 10.6 and 9.41%, respectively. The relative proportions of protein fractions of salmon (O. keta) and sturgeon (Acipenser transmontanus) caviar separated by solubility are presented in Table 1. Surprisingly no watersoluble protein fractions of salmon and sturgeon roe were recovered. The salt soluble fractions of salmon and sturgeon roe comprised the majority of the recovered protein, with the percentage of total nitrogen at 84.2% in salmon and 86.1% for sturgeon caviars. No alkaline soluble or 70% ethanol soluble fractions were recovered. Although these are typical constituents in cereals and legumes, proteins with these chemical properties would not be expected to be present in an aquatic food. The nitrogen content of the insoluble residue comprised 12.3 and 10.2% of the total nitrogen in salmon and sturgeon roe, respectively, and is most likely the collagen from the outer egg membrane and inner membranous structure that was not soluble in any of the extracting solutions used in this study. Sikorski (1994) reported that in fish roe, the albumins contribute to about 11% of total nitrogen; ovoglobulin constitutes about 75%, and collagen about 13% of the crude protein. The proportion and type of proteins in fish muscle and reproductive tissues are variable, changing in the course of the year by several percent in anadromous fishes such as salmon and sturgeon (Hart 1973). For example in the fish muscle, during the maturation of the gonads and in the periods of depletion, the muscle tissue becomes richer in proteins insoluble in salt solution (Sikorski 1994). During spawning, salmon mobilizes lipid from the muscle tissue and transfers it to the
426 M.A. AL-HOLY and B.A. RASCO 1 2 3 4 5 6 7 8 96 60 38 28 FIG. 1. TYPICAL PROTEIN PROFILES OF SALMON AND STURGEON CAVIAR ON 12.5% SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS (1) Salmon caviar, extracted in 20-mM Tris/glycine; (2) supernatant from 1; (3) supernatant from heated salmon caviar suspended in 20-mM Tris; (4) sturgeon caviar, extracted in 20-mM Tris/glycine; (5) supernatant from 4; (6) supernatant from heated sturgeon caviar suspended in 20-mM Tris, 7 and 8; protein molecular weight markers. eggs. Sturgeon deposits lipid within proteinaceous layers inside the eggs and are unique in that they will reabsorb their eggs (atresia) under conditions of environmental stress. Figure 1 shows electrophoretic patterns for extracts of salmon and sturgeon caviar with distinctive low molecular weight bands ( 25 28 kda), confirming that protein electrophoretic patterns can be used to differentiate fish species (Scobbie and Mackie 1990; Chen et al. 1996). The prominent bands in sturgeon caviar were at approximately 96 and 28 kda. The electrophoretic patterns observed here for sturgeon (A. transmontanus) roe were similar to those observed by Chen et al. (1996) for American sturgeon (species unidentified). There were four distinctive protein bands of salmon (O. keta) roe electrophoretic profile (10 100 kda) with prominent protein bands in the insoluble fraction at 90 96, 20 and 10 kda with minor bands between 30 60 kda. The protein at approximately 96 kda may be a vitellin-like protein similar to that in chicken egg yolk (deman 1999). A protein with similar properties was also isolated from the freshwater giant prawn (Macrobrachium rosenbergii). Similar bands were also obtained for salmon roe (Salmo salar) (Scobbie and Mackie 1990) and for different types of sturgeon roe (Chen et al. 1996). Vitellin is composed of two major polypeptide components with estimated molecular weight (MW) from 83.1 to 95.9 kda (Chen and Kuo 1998).
SALMON AND STURGEON CAVIAR PROTEINS 427 Lysozyme is a globulin with a MW of 14 17 kda, and may represent some of the bands between 0 20 kda. Notwithstanding, bands that appeared between 10 and 30 kda could also be phosvitin (Losso et al. 1993). The soluble fraction of sturgeon caviar exhibited an electrophoretic band at approximately 27 kda. This band may possibly represent ovomucoid, a glycoprotein, which normally has a MW of 27 29 kda. Other likely components of fish roe including collagen (360 kda) and ovomucin (7600 kda) were not recoverable on these gels. CONCLUSIONS Proteins in caviar are primarily salt soluble with a substantial amount of insoluble collagen, which composes the shell. SDS-PAGE confirms the presence of two prominent fractions in sturgeon caviar at approximately 96 and 28 kda, most likely vitellin and ovomucoid glycoprotein or phosvitin. For salmon caviar, three prominent bands were seen at 96, 20 and 10 kda and may be vitellin and possibly lysozyme or phosvitin. Having a better understanding of the protein composition of caviar products may help to improve thermal, superchilling and freezing processes, and to better understand factors affecting product stability during refrigerated storage. ACKNOWLEDGMENT This research was sponsored by the USDA International Marketing Program for Agricultural Commodities and Trade, Western Regional Aquaculture Consortium, Washington Idaho Aquaculture Consortium, Washington State University, Hashemite University (Zarqa, Jordan), Stolt Sea Farms, LLC, National Fisheries Institute and Puget Sound Institute of Food Technologists. We would also like to thank Dr. Chulhee Kang from the School of Molecular Biosciences, Washington State University for kindly allowing us to use his lab. REFERENCES BLEDSOE, G.E., BLEDSOE, C.D. and RASCO, B.A. 2003. Caviar and fish roe products. Crit. Rev. Food Sci. 43(2), 233 271. CHEN, Y-N. and KUO, C.M. 1998. Purification and characterization of vitellin from the freshwater giant prawn, Macrobrachium rosenbergii. Zool. Studies 37, 126 136.
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