FNS  Vol.2 No.6 , August 2011
A Study of the Enzymatic Hydrolysis of Fish Frames Using Model Systems
Abstract: A model system was employed to study the operating conditions and primary parameters of enzymic hydrolysis of cod proteins. Pancreatin, papain, and bromelain were used to hydrolyse minced cod fillets under controlled conditions and with the rate of hydrolysis being continually monitored via both the pH-stat and TNBS method. The two methods were compared and evaluated. The rate of protein solubilisation was plotted against the degree of hydrolysis (DH). Dry fish protein hydrolysate (FPH) powders having short, medium and high degrees of hydrolysis (DH of approximately 8%, 11% and 16% respectively) were produced and analysed for their molecular weight distribution, using size exclusion chromatography. Almost complete protein solubilisation (75 g soluble protein per kg hydrolysis solution) could be achieved within an hour, at 40oC, at 1% enzyme/substrate ratio (w/w) with papain and bromelain. The pH-stat was found capable of continuously following the rate of hydrolysis but only at low DH. The TNBS could be accurately used even at high DH to estimate the percentage of the peptide bonds cleaved, but required chemical analysis of withdrawn samples.
Cite this paper: nullA. Himonides, A. Taylor and A. Morris, "A Study of the Enzymatic Hydrolysis of Fish Frames Using Model Systems," Food and Nutrition Sciences, Vol. 2 No. 6, 2011, pp. 575-585. doi: 10.4236/fns.2011.26081.

[1]   H. G. Kristinsson and B. A. Rasco, “Fish Protein Hydrolysates: Production, Biochemical, and Functional Properties,” In: F. M. Clydesdale, Ed., Critical Reviews in Food Science and Nutrition, Vol. 40, No. 1, 2000, pp. 43-81. doi:10.1080/10408690091189266

[2]   V. Mohr, “Fish Protein Concentrate Production by Enzymic Hydrolysis,” In: J. Adler-Nissen, B. O. Eggum, L. Munlic and H. S. Olsen, Eds., Biochemical Aspects of New Protein Food, Proceedings of the 11th FEBS meeting, Federation of European Biochemical Societies, Pergamon Press, Oxford, 1978.

[3]   S. Y. Yu and L. K. Tan, “Acceptability of Crackers (‘Keropok’) with Fish Protein Hydrolysate,” International Journal of Food Science and Technology, Vol. 25, No. 2, 1990, pp. 204-208. doi:10.1111/j.1365-2621.1990.tb01075.x

[4]   Y. J. Jeon, H. G. Byun and S. K. Kim, “Improvement of Functional Properties of Cod Frame Protein Hydrolysates Using Ultrafiltration Membranes,” Process Biochemistry, Vol. 35, No. 5, 1999, pp. 471-478. doi:10.1016/S0032-9592(99)00098-9

[5]   Y. Thiansilakul, S. Benjakul and F. Shahidi, “Compositions, Functional Properties and Antioxidative Activity of Protein Hydrolysates Prepared from Round Scad (Decapterus maruadsi),” Food Chemistry, Vol. 103, No. 4, 2007, pp. 1385-1394. doi:10.1016/j.foodchem.2006.10.055

[6]   V. Klompong, S. Benjakul, D. Kantachote and F. Shahidi, “Antioxidative Activity and Functional Properties of Protein Hydrolysate of Yellow Stripe Trevally (Selaroides leptolepis) as Influenced by the Degree of Hydrolysis and Enzyme Type,” Food Chemistry, Vol. 102, No. 4, 2007, pp. 1317-1327. doi:10.1016/j.foodchem.2006.07.016

[7]   R. P. Aguilar, M. A. Mazorra-Manzano and J. C. Ramírez-Suárez, “Functional Properties of Fish Protein Hydrolysates from Pacific Whiting (Merluccius productus) Muscle Produced by a Commercial Protease,” Food Chemistry, Vol. 109, No. 4, 2008, pp. 782-789. doi:10.1016/j.foodchem.2008.01.047

[8]   S. ?li?yt?, R. Mozuraityt?, O. Martínez-Alvarez, E. Falch, M. Fouchereau-Peron and T. Rustad, “Functional, Bioactive and Antioxidative Aroperties of Hydrolysates Obtained from Cod (Gadus morhua) Backbones,” Process Biochemistry, Vol. 44, No. 6, 2009, pp. 668-677.

[9]   J. Salampessy, M. Phillips, S. Seneweera and K. Kailasapathy, “Release of Antimicrobial Peptides through Bromelain Hydrolysis of Leatherjacket (Meuchenia Sp.) Insoluble Proteins,” Food Chemistry, Vol. 120, No. 2, 2010, pp. 556-560. doi:10.1016/j.foodchem.2009.10.054

[10]   S. Zhong, C. Ma, Y. C. Lin and Y. Luo, “Antioxidant Properties of Peptide Fractions from Silver Carp (Hypophthalmichthys molitrix) Processing by-Product Protein Hydrolysates Evaluated by Electron Spin Resonance Spectrometry,” Food Chemistry, Vol. 126, No. 4, 2011, pp. 1636-1642. doi:10.1016/j.foodchem.2010.12.046

[11]   S. Nalinanon, S. Benjakul, H. Kishimura and F. Shahidi, “Functionalities and Antioxidant Properties of Protein Hydrolysates from the Muscle of Ornate Threadfin Bream Treated with Pepsin from Skipjack Tuna,” Food Chemistry, Vol. 124, No. 4, 2011, pp. 1354-1362. doi:10.1016/j.foodchem.2010.07.089

[12]   I. M. Mackie, “Proteolytic Enzymes in Recovery of Proteins from Fish Waste,” Process Biochemistry, Vol. 9, No. 10, 1974, pp. 12-14.

[13]   W. Tarky, O. P. Agarwala and G. M. Pigott, “Protein Hydrolysate from Fish Waste,” Journal of Food Science, Vol. 38, No. 6, 1973, pp. 917-918. doi:10.1111/j.1365-2621.1973.tb02113.x

[14]   M. Hale and P. E. Bauersfeld, “Preparation of Menhaden Hydrolysate for Possible Use in a Milk Replacer,” Marine Fisheries Review, Vol. 40, No. 8, 1978, pp. 14-18.

[15]   I. M. Mackie, “Fish Protein Hydrolysates,” Process Biochemistry, Vol. 17, No. 1, 1982, pp. 26-27.

[16]   M. Diaz-Casta?eda and G. J. Brisson, “Replacement of Skimmed Milk with Hydrolyzed Fish Protein and Nixtamal in Milk Substitutes for Dairy Calves,” Journal of Dairy Science, Vol. 70, No. 1, 1987, pp. 130-140. doi:10.3168/jds.S0022-0302(87)79988-3

[17]   L. R. Beuchat, “Preparation and Evaluation of a Microbiological Growth Medium Formulated from Catfish Waste Peptone,” Journal of Milk and Food Technology, Vol. 37, No. 5, 1974, pp. 277-281.

[18]   F. Guérard, L. Dufossé, D. De La Broise and A. Binet, “Enzymatic Hydrolysis of Proteins from Yellowfin Tuna (Thunnus albacares) Wastes Using Alcalase,” Journal of Molecular Catalysis B: Enzymatic, Vol. 11, No. 4-6, 2001, pp. 1051-1059. doi:10.1016/S1381-1177(00)00031-X

[19]   C. B. Martone, O. P. Borla and J. J. Sánchez, “Fishery by-Product as a Nutrient Source for Bacteria and Archaea Growth Media,” Bioresource Technology, Vol. 96, No. 3, 2005, pp. 383-387. doi:10.1016/j.biortech.2004.04.008

[20]   S. I. Aspmo, S. J. Horn and V. G. H. Eijsink, “Hydro- lysates from Atlantic Cod (Gadus morhua L.) Viscera as Components of Microbial Growth Media,” Process Biochemistry, Vol. 40, No. 12, 2005, pp. 3714-3722. doi:10.1016/j.procbio.2005.05.004

[21]   M. B. Hale, “Relative Activities in Commercially-Available Enzymes in the Hydrolysis of Fish Protein,” Food Technology, Vol. 23, No. 1, 1969, pp. 107-110.

[22]   D. P. Sen, N. V. Sripathy, N. L. Lahiry, A. Sreenivasan and V. Subrahmanyan, “Fish Hydrolysates. No. 1: Rate of Hydrolysis of Fish Flesh with Papain,” Food Technology, Vol. 16, No. 5, 1962, pp. 138-142.

[23]   C. G. Beddows and A. G. Ardeshir, “The Production of Soluble Fish Protein Solution for Use in Fish Sauce Manufacture. No. 1: The Use of Added Enzymes,” Journal of Food Technology, Vol. 14, No. 6, 1979, pp. 603-612. doi:10.1111/j.1365-2621.1979.tb00907.x

[24]   S. I. Aspmo, S. J. Horn and V. G. H. Eijsink, “Enzymatic Hydrolysis of Atlantic Cod (Gadus morhua L.) Viscera,” Process Biochemistry, Vol. 40, No. 5, 2005, pp. 1957-1966. doi:10.1016/j.procbio.2004.07.011

[25]   C. G. Beddows, M. Ismail and K. H. Steinkraus, “The Use of Bromelain in the Hydrolysis of Mackerel and the Investigation of Fermented Fish Aroma,” Food Technology, Vol. 11, No. 4, 1976, pp. 379-388. doi:10.1111/j.1365-2621.1976.tb00736.x

[26]   P. Hevia and S. Olcott, “Flavour of Enzyme-Solubilized Fish Protein Concentrate Fractions,” Journal of Agricultural and Food Chemistry, Vol. 25, No. 4, 1977, pp. 772-775. doi:10.1021/jf60212a044

[27]   J. Montecalvo, S. M. Constandinides and S. T. Yang, “Enzymatic Modification of Fish Frame Protein Isolate,” Journal of Food Science, Vol. 49, No. 5, 1984, pp. 1305-1309. doi:10.1111/j.1365-2621.1984.tb14975.x

[28]   S. Bhumiratana, C. G. Hill jr. and C. H. Amundson, “Enzymatic Solubilization of Fish Protein Concentrate in Membrane Reactors,” Journal of Food Science, Vol. 42, No. 4, 1977, pp. 1016-1021. doi:10.1111/j.1365-2621.1977.tb12657.x

[29]   A. Gildberg, J. A. Arnesen and M. Carleh?g, “Utilisation of Cod Backbone by Biochemical Fractionation,” Process Biochemistry, Vol. 38, No. 4, 2002, pp. 475-480. doi:10.1016/S0032-9592(02)00103-6

[30]   M. B. Hale, “Using Enzymes to Make Fish Protein Concentrates,” Marine Fisheries Review, Vol. 36, No. 2, 1974, pp. 15-18.

[31]   G. Lalasides, S. Bostrom and L. B. Sjoberg, “Low Molecular Weight Enzymatic Fish Protein Hydrolysates: Chemical Composition and Nutritive Value,” Journal of Agricultural and Food Chemistry, Vol. 26, No. 2, 1978, pp. 751-756. doi:10.1021/jf60217a045

[32]   G. Lalasides and L. B. Sjorberg, “Two New Methods of Debittering Protein Hydrolyzates and a Fraction of Hydrolyzates with Exceptionally High Contents of Essential Amino-Acids,” Journal of Agricultural and Food Chemistry, Vol. 26, No. 3, 1978, pp. 742-749. doi:10.1021/jf60217a056

[33]   F. Jacobsen and O. lykke-Rasmussen, “Energy-Savings through Enzymatic Treatment of Stickwater in the Fishmeal Industry,” Process Biochemistry, Vol. 19, No. 5, 1984, pp. 165-169.

[34]   G. B. Quaglia and E. Orban, “Influence on Enzymatic Hydrolysis on Structure and Emulsifying Properties of Sardine (Sardina pilchardus) Protein Hydrolysates,” Journal of Food Science, Vol. 55, No. 6, pp. 1990-1571.

[35]   D. R. Baca, M. T. Pena-Vera, M. Diaz-Castaneda, “Production of Fish Protein Hydrolysates with Bacterial Proteases; Yield and Nutritional Value,” Journal of Food Science, Vol. 56, No. 2, 1991, pp. 309-314. doi:10.1111/j.1365-2621.1991.tb05268.x

[36]   G. A. Gbogouri, M. Linder, J. Fanni and M. Parmetier, “Influence of Hydrolysis Degree on the Functional Properties of Salmon by Products Hydrolysates,” Journal of Food Science, Vol. 69, No. 8, 2004, pp. 615-622. doi:10.1111/j.1365-2621.2004.tb09909.x

[37]   J. Wasswa, J. Tang, X.-H. Gu and X.-Q. Yuan, “Influence of the Extent of Enzymatic Hydrolysis on the Functional Properties of Protein Hydrolysates from Grass Carp (Ctenopharyyngodon idella) Skin,” Food Chemistry, Vol. 104, No. 4, 2007, pp. 1698-1704. doi:10.1016/j.foodchem.2007.03.044

[38]   S. ?li?yte, E. Dauk?as, E. Falch, I. Storr? and T. Rustad, “Yield and Composition of Different Fractions Obtained after Enzymatic Hydrolysis of Cod (Gadus morhua) by-Products,” Process Biochemistry, Vol. 40, No. 3-4, 2005, pp. 1415-1424. doi:10.1016/j.procbio.2004.06.033

[39]   B. Liaset, K. Julshamn and M. Espe, “Chemical Composition and Theoretical Nutritional Evaluation of the Produced Fractions from Enzymic Hydrolysis of Salmon Frames with ProtamexTM,” Process Biochemistry, Vol. 38, No. 12, 2003, pp. 1747-1759. doi:10.1016/S0032-9592(02)00251-0

[40]   S. Nilsang, S. Lertsiri, M. Suphantharika and A. Assavanig, “Optimization of Enzymatic Hydrolysis of Fish Soluble Concentrate by Commercial Proteases,” Journal of Food Engineering, Vol. 70, No. 4, 2005, pp. 571-578.

[41]   S. ?li?yte, E. Dauk?as, E. Falch, I. Storr? and T. Rustad, “Characteristics of Protein Fractions Generated from Hydrolysed Cod (Gadus morhua) by-Products,” Process Biochemistry, Vol. 40, No. 6, 2005, pp. 2021-2033. doi:10.1016/j.procbio.2004.07.016

[42]   J. Adler-Nissen, “Enzymic Hydrolysis of Food Proteins,” Elsevier Applied Science Publishers Ltd., London, 1986.

[43]   M. Tamura, N. Mori, T. Miyoshi, S. Koyama and H. Kohri, “Practical Debittering Using Model Peptides and Related Compounds,” Agricultural and Biological Chemistry, Vol. 54, No. 1, 1990, pp. 41-51. doi:10.1271/bbb1961.54.41

[44]   J. Adler-Nissen, “Control of the Proteolytic Reaction and of the Level of Bitterness in Protein Hydrolysis Processes,” Journal of Chemical Technology and Biotechnology, Vol. 34, No. 2, 1984, pp. 215-222.

[45]   B. C. Saha and K. Hayashi, “Debittering of Protein Hydrolyzates,” Biotechnology Advances, Vol. 19, No. 5, 2001, pp. 355-370. doi:10.1016/S0734-9750(01)00070-2

[46]   H. Hou, B. Li, X. Zhao, Z. Zhang and P. Li, “Optimization of Enzymatic Hydrolysis of Alaska Pollock Frame for Preparing Protein Hydrolysates with Low-Bitterness,” LWT—Food Science and Technology, Vol. 44, No. 2, 2011, pp. 421-428.

[47]   S. Sathivel, S. M. Smiley, W. Prinyawiwatkul and P. C. Bechel, “Functional and Nutritional Properties of Red Salmon (Oncorhynchus nerka) Enzymatic Hydrolysates,” Journal of Food Science, Vol. 70, No. 6, 2005, pp. 401-406. doi:10.1111/j.1365-2621.2005.tb11437.x

[48]   P. Bourseau, L. Vandanjon, P. Jaouen, M. Chaplain-Derouiniot, A. Massé, F. Guérard, A. Chabeaud, M. Fouchereau-Péron, Y. Le Gal, R. Ravallec-Plé, J. P. Bergé, L. Picot, J. M. Piot, I. Batista, G. Thorkelsson, C. Delannoy, G. Jakobsen and I. Johansson, “Fractionation of Fish Protein Hydrolysates by Ultrafiltration and Nanofiltration: Impact on Peptidic Populations,” Desalination, Vol. 244, No. 1-3, 2009, pp. 303-320. doi:10.1016/j.desal.2008.05.026

[49]   L. Vandanjon, M. Grignon, E. Courois, P. Bourseau and P. Jaouen, “Fractionating White Fish Fillet Hydrolysates by Ultrafiltration and Nanofiltration,” Desalination, Vol. 244, No. 1-3, 2009, pp. 303-320. doi:10.1016/j.desal.2008.05.026

[50]   A. Chabeaud, L. Vandanjon, P. Bourseau, P. Jaouen, M. Chaplain-Derouiniot and F. Guerard, “Performances of Ultrafiltration Membranes for Fractionating a Fish Protein Hydrolysate: Application to the Refining of Bioactive Peptidic Fractions,” Separtation and Purification Technology, Vol. 66, No. 3, 2009, pp. 463-471. doi:10.1016/j.seppur.2009.02.012

[51]   A. Chabeaud, L. Vandanjon, P. Bourseau, P. Jaouen and F. Guérard, “Fractionation by Ultrafiltration of a Saithe Protein Hydrolysate (Pollachius virens): Effect of Material and Molecular Weight Cut-off on the Membrane Performances,” Journal of Food Engineering, Vol. 91, No. 3, 2009, pp. 408-414. doi:10.1016/j.jfoodeng.2008.09.018

[52]   J. Adler-Nissen, “Determination of the Degree of Hydrolysis of Food Protein Hydrolysates by Trinitrobenzenesulphonyc Acid,” Journal of Agricultural and Food Chemistry, Vol. 27, No. 6, 1979, pp. 1256-1262. doi:10.1021/jf60226a042

[53]   P. L. Pellett and V. R. Young, “Nutritional Evaluation of Protein Foods,” The United Nations University World Hunger Programme Food and Nutrition Bulletin, Supplement 4, 1980.

[54]   A. Himonides, “The Improved Utilisation of Fish Waste, with Particular Reference to the Enzymatic Hydrolysis of Fish Frames for the Production of Fish Protein Hydrolysates,” PhD Thesis, Lincoln University, Lincoln-Shire, 2001.

[55]   E. Mihalyi, “Application of Proteolytic Enzymes to Protein Structure Studies,” Vol. 2, 2nd Edition, CRC Press, Boca Raton, 1978.

[56]   T. Suzuki, “Fish and Krill Protein: Processing Technology,” Applied Science Publishers Ltd., Essex, 1981.