Sep 3, 1989 - Selection of a Purification Procedure for. Production of the Rock Crab Cancer irroratus Protein Ingredient. JOHN D. CASTELL'. AND J. C. KEAN.
Vol. 20, No. 3 September, 1989
JOURNAL OF THE WORLD AQUACULTURE SOCIETY
A Standard Reference Diet for Crustacean Nutrition Research. 11. Selection of a Purification Procedure for Production of the Rock Crab Cancer irroratus Protein Ingredient JOHN D. CASTELL’ AND J. C. KEAN Department of Fisheries and Oceans, Fisheries and Environmental Sciences, Halifax Fisheries Research Laboratory, P.O. Box 550, Halifax,Nova Scotia B3J 25‘7, Canada
D. G. C. MCCANN Biology Department, Dalhousie University, Halifax Nova Scotia B3H 4H6. Canada
ANDREW D. BOGHEN Department de Biologie, Universite de Moncton, Moncton, New Brunswick E l A 3E9, Canada
DOUGLAS E. CONKLIN University of California, Davis Campus, Bodega Bay Marine Laboratory, P.O. Box 247, Bodega Bay, California 94923 USA LOUIS
R. D ’ h R A M O
Department of Wildlge and Fisheries, Mississippi State University, P.O. Drawer L W,Mississippi State, Mississippi 39762 USA
Abstract Obtaining reliable results from comparative crustacean nutrition studies requires a dietary reference protein which is available world-wide and is nutritionally optimal for growth and survival. Traditional protein sources include “vitamin free” casein, egg albumin, whole egg protein, soybean protein, and others. These have proven to be inadequate for juvenile lobster (Homrus americanus). Purified rock crab (Cancer irroratus) protein is proposed to be nutritionally superior to casein for inclusion in semi-purified lobster diets. Eighteen week feeding trials with juvenile H.americanus were conducted to compare diets containing protein obtained from rock crabs (Cancer irroratus), and produced by several different purification processeswith a control diet having casein as its primary protein source. Diets containing purified rock crab protein were found to be superior to the control diet. Among the differently derived purified rock crab protein products, the precooked, isopropanol-purified was the most effective dietary ingredient.
During the last two decades, a number of semi-purified diet formulations for research on crustacean nutrition have been published. While “vitamin free” casein was often used as the major source of protein in these diets, there were indications that this ingredient would not be reliably satisfactory. A study with lobsters (Hornarus americanus) by Mason and Castell (1980) suggested that casein was deficient in thre-
Corresponding author.
onine. Furthermore, the quality of the casein available from commercial sources proved to be inconsistent. For example, methionine, an essential amino acid for lobsters (Gallagher and Brown 1975), varied from 0.03% to 3.0% (Castell and Boghen 1979). At the low end of this range, the methionine level was insufficient, and significant mortality occurred. Boghen, Castell, and Conklin (1 982) tested purified protein concentrates from a number of marine organisms and reported that diets containing rock crab (Cancer irrora-
0 Copyright by the World Aquacultw Society 1989
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CSRD 11: PRODUCTION OF ROCK CRAB PROTEIN
tus) protein gave superior growth and survival among juvenile lobsters. The nutritive value of the crab protein concentrate varies depending upon how the crab meat is processed. For example, lysine can become nutritionally unavailable during processing in the presence of reducing sugars because of the Maillard reaction. Consequently, different processing methods have been employed on deboned, whole rock crab to determine which process yields the best protein concentrate for incorporation in lobster diet formulations.
ROCK CRAB
COOkad in Boiling Sea Watw
+
uoimlmd
FIGUREI. Purification methods for the production of the crab protein Concentrate ingredient.
Materials and Methods mixture was vacuum filtered through WhatProtein Purification man #1 filter paper. Subsequently, 500 ml In addition to the protein moiety, fish or and 250 ml of reagent grade acetone were crab meals contain a variety of lipids, min- poured over the filtrate with refiltration ocerals, carbohydrates, and other nutrients, curring each time. The residue was then and purification is necessary to remove these scraped off the filter paper and blended with other compounds. The quality of the resul- 250 ml of reagent grade methanol for 30 tant protein concentrate depends upon the seconds followed by vacuum filtration as purification procedure. before. The filtrate was washed twice with One hundred eighty kilograms of live rock 250 ml aliquots of reagent grade methanol crabs (Cancer irrorutus) were purchased followed by a third wash with 500 ml of from fisherman in Pictou, Nova Scotia and reagent-grade acetone. The crab protein transported to the Halifax laboratory of concentrate was then air-dried until all solFisheries and Oceans, Canada. The batch vent vapors were completely evaporated. was divided into three lots (Fig. 1). One lot This product was designated spray-dried was maintained alive in ambient sea water, fresh. one lot was frozen at -40 C, and a third lot The other half of the fresh deboned crab was cooked in boiling sea water. Each of the was purified by a modified method of Power three lots was then “deboned” in a meat (1962). Five hundred grams of crab was separator (Bibun Co., Hiroshima, Japan: blended in an explosionproof blender for Type SDX16-#228) at the Technical Uni- one minute with 1.5 1 of boiling isopropanol versity of Nova Scotia (TUNS). The frozen (IPA) and water in a 70:30 (v:v) ratio assuming the crab to be 80%water. This mixand cooked deboned lots were held at -40 C for further processing. The fresh, deboned ture was filtered as above and the residue crab was sub-divided equally, each half-lot blended with 1.5 1 boiling 1PA:water (70: 30) for one minute, followed by filtration. being processed differently. One-half of the fresh, deboned crab meat The procedure was repeated a third time was milled to a uniform consistency and using boiling 99Oh reagent grade isopropanol spray-dried (Noro Atomizer, Copenhagen, instead of redistilled IPA. After filtering, the Denmark; Serial #7408) at 80 C. Two protein residue was thoroughly wetted with hundred grams of this spray-dried crab distilled water, frozen at -40 C, and freezepowder was introduced into an explosion- dried [virtis FreezemobileI1(1 0- 145-VOOD) proof blender and blended with 500 ml of and drying unit (10-MR-TR)] to remove all reagent-grade acetone for 30 seconds. This water and residual solvent. The resulting
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CASTELL ET AL.
TABLE1. Ingredient composition of diets. % of dry
Ingredient Casein or purified rock crab protein product Gelatin Corn starch Cellulose DL-Methionine Mineral mix (modified Bemhardt-Tomarelli) Lecithin (soy) Glucosamine Cholesterol Cod liver oil Vitamin mix3 Vitamin E Cupric citrate
weight' 50 10 5 8.754 1
Source2
A B A A B
5 6 1
B B
1
C D
10 2 0.2 0.046
A
tic mixing bowl with a spatula. Cod liver oil and vitamins were added followed by boiling water (1.5 x dry ingredient weight) and all ingredientswere mixed with an electric hand mixer. The diets were allowed to gel at 4 C for several hours, then extruded through a 2 mm diameter die hand press. The spaghetti-like feed was quick frozen at -40 C and freeze-dried [Virtis Freezemobile I1 (10-145-VOOD) and drying unit (10-MR-TR)]. The diets were stored in sealed plastic at -40 C until fed.
Experimental Animals Wild, gravid female lobsters Homarus A americanus, captured from the Atlantic A Dry ingredient constituted 70% of the final weight Ocean near Halifax, were held in the laboratory in ambient temperature seawater and of the diet. * A = ICN Nutritional Biochemicals, Costa Mesa, then slowly acclimated to 20 C to accomCalifornia, USA, B = US. Biochemical Corporation, modate hatching. The hatched larvae were Cleveland, Ohio, USA, C = Baker Company, Philheld in seawater at approximately 20 C and lipsburg, New Jersey, USA, D = Life Brand. reared to 4th stage on newly hatched nauplii See Castell et al. (1989). of Artemia salina according to the methods of Castell (1977). When the lobsters reached crab protein concentrate product was des- 4th stage, they were moved to individual ignated IPA-fresh. compartments in trays, each capable of The frozen, deboned crab was partially housing 20 lobsters (Boghen and Castell thawed and subdivided into two equal lots. 1979). The trays were supplied with filtered One lot was purified with acetone/methanol 20 C seawater from Halifax Harbour. Lob(spray-dried frozen), and the other was pur- sters were fed frozen adult brine shrimp (R. ified with IPA as outlined previously (IPA C. Hagen, Montreal, Canada) for 10 days frozen). until feeding trials were initiated. The cooked, deboned crab was divided At the start of the experiment, each treatinto three lots. Following previously out- ment consisted of two trays, one of 18 and lined procedures, one was spray-dried, pur- the other of 19 animals. Lobsters were fed ified with acetone/methanol (spray dried three times daily on weekdays and once daicooked), and another was purified with IPA ly on holidays and weekends for 18 weeks. (IPA-cooked). The third lot of cooked de- The lobsters were exposed to approximately boned crab was also purified with IPA but 9: 15 light-dark photoperiod. Light was supall three purification steps used 99% IPA plied by cool fluorescent lighting from the (pure IPA-cooked). ceiling of the laboratory. Molts and mortalities were recorded at feedingtimes, when Diet Preparation any uneaten food from a previous feeding Eight experimental diets that contained and any mortalities were removed. At the either casein or crab protein derived from initiation of each experiment and every two one of the purification procedures as the weeks thereafter, each lobster was weighed primary protein source were prepared in the after removal of excess surface water with Halifax Laboratory (Table 1). Dry ingredi- paper toweling. The normalized biomass increase (NBI) ents were thoroughly mixed in a small plas-
CSRD 11: PRODUCT'ION OF ROCK CRAB PROTEIN
103
was calculated according to the method of Conklin et al. (1977) as follows: NBI =
final number of animals per tray; final mean wet weight of animals per tray; initial number of animals per tray; initial mean wet weight of animals per tray. The NBI for each treatment was calculated as the mean of the NBI values of each of the two trays.
Chemical Analysis The moisture content of each of the different protein sources and the experimental diets was estimated by difference in weight after 18-24 hours in a drying oven at 1 10 C. Ash content was determined by firing in a muffling furnace (Model #N30A- 1C Blue M Electric Co.) for 18-24 hours at 550 C. Protein content was estimated using the automated Kjeldahl procedure of Ferrari (1 969) with the Technicon Instruments Co., Ltd., Autoanalyzer I1 after partial predigestion in 50% concentrated sulfuric acid. Analysis of amino acid composition of samples of crab protein concentrates were conducted at the University of New Hampshire through the courtesy of Mr. Paul Chapman, Sanders Associates, Inc., Nashua, New Hampshire. Analyses were done after 24, 48 and 72 hours of mild acid hydrolysis. Separation was performed on an ion exchange resin column using Ninhydrin for color development after elution. Quantitation was based upon known amounts of each amino acid passed through the column under the same conditions as the protein hydrolysates. Statistical Analyses Statistical analyses followed methods outlined in Ott ( 1 977) and Steel and Tome
FIGURE 2. Normalized Biomass Increasesfor juvenile lobstersfed diets containing casein (Diet 8) or purified rock crab protein prepared according to diferent procedures, IPA cooked (Diet I), IPA-Fresh (Diet 2), IPA frozen (Diet 3), Pure IPA-Cooked (Diet 4). spraydried cooked (Diet 5). spray-driedfresh (Diet 6). and spray-dried frozen (Diet 7).
(1 960). Duncan's New Multiple Range Test was used to analyze differencesbetween the final mean weights and the NBIs of the dietary treatments. Prior to analysis the wet weight data were logarithimically transformed because heterogeneity of variance combined with a proportional increase in the standard deviation with an increase in the mean were observed.
Results and Discussion The method of purification significantly influenced the nutritional value of the crab protein concentrate (Table 1; Fig. 2). The mean final weights of lobsters fed Diets 6 and 7 were significantly less than those fed Diets 1, 2, 4 and 5. Notwithstanding the high average final weight, lobsters fed Diet 4 (pure IPA-cooked) suffered the highest mortality, 32.4% (Table 2). Mean survival
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CASTELL ET AL.
TABLE 2. Survival and mean final weight of juvenile lobsters reared after 18 weeks on diets containing 50% of either direrent purification products of crab protein or casein. Values followed by the same letter are not significantlydifferent (P = 0.05).
Diet 1 2 3 4 5 6 7 8
Major protein IPA-cooked IPA-fresh IPA-frozen Pure IPA-cooked Spray-dried cooked Spray-dried fresh Spray-dried frozen Casein
Mean initial weight f SD mg 196 f 184 f 194 f 194 f 193 f 189 2 192 k 192 f
61 72 63 60 61 65 66 61
exceeded 8 1.1Yo for both trays of each of the remaining treatments containing the crab protein ingredient. Differences in survival between the two trays within each treatment did not exceed 20 percent. At the termination of the experiment (1 8 weeks), Diets 5 (spray-dried cooked) and 2 (IPA-fresh) yielded significantly greater normalized weight gains. Diets based on casein, spray-dried fresh or frozen crab (Diets 8, 6, and 7) gave the poorest results. Diets containing frozen or cooked IPA extracted proteins (Diets 1, 3, 4) are all intermediate with the cooked crab extracted with IPA and no water yielding the poorest result among this group. In the purification process, deboned crab lots that were IPA purified were blended with the first two volumes of boiling IPA immediately after passing through the deboner, whereas lots that were spray-dried were stored at 4 C in a refrigerated room for up to six hours after deboning before being spray-dried. During that storage, the fresh and previously frozen crab appeared to undergo rapid deterioration becoming much darker in color. The fresh and previously frozen crab that had been stored the longest were discarded because they developed a black color and a bad odor. The precooked, deboned crab remained orange and smelled good until blending and spraydrying. This rapid deterioration of the de-
Mean final weight SD g
*
1.8 k 0.6b.C 1.9 f 0.Y 1.6 0 . 4 " ~ ~ 1.9 f 0.6b,C 2.1 f 0.7c 1.4 k 0.4" 1.2 f 0.4a 1.5 f 0.48.b
*
Percent survival
Final normalized biomass increase
83.8 91.9 91.9 67.6 88.9 81.1 91.7 75.7
1,3 1 2"sb 1,521" 1,l 60a9b9c 1 ,034b.C 1,658a 9 1 2b,c 987b,C 872c
boned fresh (Diet 6) and previously frozen crab (Diet 7) may explain the poor nutritional value of the spray-dried protein concentrate from these sources. During the deboning process it was apparent that crab meat from live and frozen animals suffered appreciable losses of fluid, presumably hemolymph (Diets 2, 3, 6, and 7). This occurred to a lesser extent in the pre-cooked crab (Diets 1,4, and 5 ) because the hemolymph had perhaps congealed. Higher yields of purified product therefore resulted from the pre-cooked lots. When the diet made from fresh, deboned IPA purified protein was left in the refrigerator to gel, it became very soupy and in fact did not gel. Apparently, the fresh crab protein contained active protease enzymes which digested the gelatin. It was difficult to extrude this diet to make pellets for freezing and freeze-drying. Freeze-dried pellets from this diet disintegrated very rapidly when fed to lobsters. This conferred some benefit, however, because the lobsters consumed the disintegratingparticles of this diet more rapidly than any of the others. In Diets 1,2, and 3 the water included in the IPA resulted in reduced mineral content of these concentrates compared with the acetone and methanol purified spray-dried concentrates (Diets 5 , 6 , and 7)as indicated by the lower ash content (Table 3). Even though no water was added to the IPA used
CSRD 11: PRODUCTION OF ROCK CRAB PROTEIN
105
TABLE 3. Ash, dry weight, and crude protein (by Kjeldahl nitrogen x 6.25) of various purified crab protein and casein. Wet weieht Diet
Dry matter
Ash
96
%
Protein IPA-cooked IPA-fresh IPA-frozen Pure IPA-cooked SD-cooked SD-fresh SD-frozen Casein
1 2 3 4 5 6 7 8
95.0 93.7 95.9 86.9 93.2 90.0 88.5 91.4
f 0.1 f 0.2
10.5 10.3 9.8 11.3 16.8 13.2 13.2 0.83
f 0.2 f 0.4 f 0.3 f 0.3 f 0.7 f 0.7
to purify the cooked crab protein in Diet 4, the natural water present during the first filtration resulted in lower ash for this protein than for any of the spray-dried concentrates. The spray-dried cooked crab (Diet 3) which yielded the highest mean final weight and normalized biomass, also had the highest ash content, 16.8%. The processing method also appeared to have an effect on amino acid composition
Protein %
f 0.1 & 0.1 f 0.1 f 0.04 f 0.05 k 0.2 k 0.04 f 0.12
81.6 k 0.1 81.4 f 0.4 84.6 0.2 73.3 f 0.5 70.0 f 0.1 75.7 f 0.9 76.8 91.1 f 1.6
*
of the crab protein (Table 4). Spray-drying slightly reduced the amount of cystine. In earlier experiments the same process had reduced cystine to undetectable levels (Boghen et al. 1982). The precooking process conserved phenylalanine. The precooked IPA purified concentrates retained the highest level of this amino acid. Differences in glycine, leucine, histidine, and arginine were also noted. Although the results
TABLE 4. Effect of extraction and purificationprocedures on amino acid compositon of crab proteins (percent of total amino acids analyzed). Tryptophan and hydroxyproline were not analyzed. ~~~
AA
Gelatin
ASP Thrb Ser Clu Pro GlY Ala CYS
6.0 1.9 3.2 10.9 15.0 20.8 9.7
6.9 4.1 4.8 21.2 10.6 1.7 2.8
9.8 4.1 4.0 15.6 3.3 4.5 5.5
VaP
2.0 1.3 1.3 3.2 0.0 2.2 0.9 4.2 1.1 8.5
5.8 2.7 4.8 9.1 5.6 4.9 2.6 7.6 1.4 3.5
4.9 3.2 4.7 7.8 3.8 4.3 2.5 8.4
Metb Isob
Led TYr Pheb Hisb Lysb NH3
Argb a
-
~
Pure IPA-water extracted Spray-dried IPA exCookeda tracted Casein (1980) Cooked Fresh Frozen cooked Cooked Fresh Frozen
-
-
-
8.6
10.8 5.0 4.6 14.7 4.2 4.9 5.2 2.3 4.8 2.7 4.4 7.7 4.2 5.1 3.0 7.6 1.2 8.1
10.7 4.9 4.4 15.2 4.1 4.9 5.4 0.7 4.6 3.0 4.4 8.1 4.2 4.5 2.8 7.9 1.3 8.9
10.8 4.9 4.6 15.0 4.0 4.7 5.3 0.8 4.7 2.9 4.5 8.1 4.3 4.6 2.9 8.3 1.2 8.4
10.3 4.9 4.5 14.8 4.4 5.1 5.3 1.o 4.8 2.9 4.6 8.0 4.2 4.6 2.8 8.0 1.3 8.4
From Boghen et al. 1982. Tentatively identified as essential for lobsters (Gallagherand Brown 1975).
10.4 4.6 4.6 14.3 4.3 6.2 5.4 0.6 4.8 2.9 4.4 7.5 5.5 5.1 3.1 6.9 1.5 9.3
10.3 4.7 4.3 15.8 5.0 5.7 5.7 0.7 4.6 2.8 4.2 7.5 4.1 4.3 2.6 7.1 1.3 9.5
10.4 4.8 4.4 15.8 6.9 5.9 5.9 0.9 4.5 2.7 4.2 7.3 3.9 4.2 2.5 6.5 1.1 8.2
106
CASTELL ET AL.
in Table 4 represent a single analysis for each protein after 24 hours hydrolysis, the analysis of proteins after 48 and 72 hours hydrolysis were very similar. The longer hydrolysis did result in increasing losses of threonine, serine, cystine, methionine and tyrosine. Although the spray-dried precooked purified crab protein concentrate (Diet 5 ) appeared to have the highest nutritional value, this product was not selected because of the high cost of spray-drying, the limited availability of spray-drying equipment, and lower protein purity, associated with the purification method. The rapid degradation of the deboned fresh (Diet 2) and prefrozen crab (Diet 3) and the apparent active proteases in the IPA purified fresh deboned crab protein led to the selection of the precooked, deboned, IPA procedure (Diet 1). This was the most suitable choice because this lot had been pre-cooked before processing, and higher product yields were therefore obtained. Cooking also destroys autolytic enzymes which can cause significant decomposition of crab material before protein purification. Additionally, the IPA purification procedure was found to be much easier, faster, and less expensive than the spraydrying techniques. For example, production costs are reduced because the IPA is reusable. The purified crab protein derived according to this procedure was therefore used as the major protein source in a proposed reference diet for crustaceans. Acknowledgments This research was conducted at the Halifax Laboratory, Department of Fisheries and Oceans, Canada and was part of an honors thesis submitted by Mr. D. G. McCann to Dalhousie University’s Biology Department. Financial support was from a grant awarded to Dr. A. D. Boghen. The authors are grateful to Dr. D. J. Scar-
ratt for his critical review of this manuscript. The technical assistance of Ms. Theresa Jones, Mr. Danny Cassavechia and Mr. Fred Rahey was greatly appreciated. Literature Cited Boghen, A. D. and J. D. Castell. 1979. A recirculating system for small scale experimental work on juvenile lobsters, Hornarus arnericanus. Aquaculture 18:383-387. Boghen, A. D., J. D. Castell and D. E. Conklin. 1982. In search of a reference protein to replace “vitamin he’’ casein in lobster nutrition studies. Canadian Jo~rnalof Zoology 60~2333-2338. Castell, J. D. 1977. Production of juvenile lobsters (Hornamarnericanus)for nutrition research. Pub. Cent. Nat. Exploit. Oceans, Ser. Actes. Colloq. (Fr.). 4:277-28 1. Castell, J. D. and A. D. Boghen. 1979. Fatty acid metabolism in juvenile lobsters (Hornam arnericanus) fed a diet low in methionine and histidine. Proceedings of the World Mariculture Society 10: 720-727. Castell, J. D., J. C. Kern, L. R. D’Abramo and D. E. Conklin. 1989. A standard reference diet for crustacean nutrition research. I. Evaluation of two formulations. Journal of the World Aquaculture Society 20(3):93-99. Conklin, D. E., A. Devers and C. E. Bordner. 1977. Development of artificial diets for the lobster Homarus arnericanus. Proceedings of the World Mariculture Society 8:844-852. Ferrari, A. 1969. Nitrogen determination by a continuous digestion and analysis system. Annuals of the N.Y. Academy of Sciences 87:792-799. Gallagher, M. L. pnd W. D. Brown. 1975. Amino acid requirements of the lobster (Homarus arnericanus).Fed.Roc. An. Soc. Exp. Biol. 34800 (Abs). Mason,E. G. and J. D. Castell. 1980. The effects of supplementing purified proteins with limiting essential amino acids in growth and survival of juvenile lobsters (Hornam arnericanus). Proceedings ofthe World Mariculture Society 11:346-354. Ott, L. 1977. An introduction to statistical methods and data analysis. Wadsworth Publishing Co., Inc. Belmont, California, USA. Power, M. E. 1962. An improved method for the preparation of fish protein concentrate from cod. Journalofthe Fisheries Research Board of Canada 19~1039-1045. Steel, R. G . D. and J. H. Torrie. 1960. Principles and procedures of statistics. McGraw-Hill, New York, USA.
