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STUDIES ON HYDROLYZED VEGETABLE PROTEINS (HVP) AS A SOURCE OF PEPTIDES WITH UMAMI TASTE G. Speranza a,e , D. Ubiali b,e , M. E. Cosulich c , L. Bagnasco d , C. F. Morelli a , P. Francescato a , V. M. Pappalardo a a Dip. di Chimica Organica ed Industriale, Università degli Studi di Milano, Via Venezian 21, 20133 Milano, Italy; b Dip. di Scienze del Farmaco, Università degli Studi di Pavia, Via Taramelli 12, 27100 Pavia, Italy; c Dip. di Biochimica "A. Castellani", Università degli Studi di Pavia, Via Taramelli 3/B, 27100 Pavia, Italy; d Dip. di Chimica e Chimica Industriale, Università di Genova, Via Dodecaneso 31, 16146 Genova, Italy; e Italian Biocatalysis Center, Via Taramelli 12, 27100 Pavia, Italy contacts: [email protected] GRINDING flaxseed by home-style grinder 220 kDa 100 kDa 60 kDa 45 kDa 30 kDa 20 kDa 8 kDa 12 kDa Flax Proteins 10ml 20ml 30ml 40ml SDS-PAGE 50 mL of protein solution (method A) was dialysed against distilled water (MWCO 3500) at 4 °C for 48 h and then lyophilised. This product was separated by SDS-PAGE in non-reducing conditions (stacking gel 5%, running gel 18%, running buffer: 25 mM Tris base, 192 mM glycin, 0.1% SDS, pH 8.3). Flaxseed proteins were dissolved in water (20 mg/ml). To 10-20-30-40 ml of protein aqueous solution were added appropriate volumes of non-reducing sample buffer. Each sample was heated at 97 °C for 3 min and subjected to electrophoresis. The gels were visualized using Coomassie Stain protocol. Acknowledgements This work was supported by through the project “VeLiCa – From ancient crops, materials and products for the future” (protocol n° 14840/RCC) Proteolytic reactions play an important role in the development of flavor in protein-rich foods such as cheese, meat, sausage, and fermented soy products. During proteolysis free amino acids and peptides are formed. Hydrolyzed vegetable proteins (HVP), for example, are widely used as savory ingredients in culinary products for their glutamate-like “umami” taste. The Japanese word umami means delicious and is used as a synonym for the characteristic sensory properties of monosodium glutamate (MSG) and certain purine-5’-nucleotides such as inosine-5’- monophosphate (5’-IMP) or guanosine-5’-monophosphate (5’-GMP). 1 Peculiar properties of the umami substances are their ability to enhance the flavor (aroma and taste) of savory dishes and the mutual taste synergism between MSG and purine-5’-nucleotides. 2-4 Umami compounds, identified in hydrolyzed protein from vegetable and animal sources, are acidic and low molecular weight oligopeptides containing MSG. Considering the taste role played by peptides in food, AIM OF THE WORK is the development of novel hydrolytic procedures of both flax and hemp proteins based on the use of different proteases, and the SAR assessment between the taste notes of the resulting HVP and the chemical structure of their constituting peptides. Among vegetable sources, flaxseed and hempseed constitute an interesting raw material for the preparation of protein hydrolyzates. Flaxseed (Linum usitatissimum L.) contains about 35-40% protein (mainly linin and colinin) which are comparable in amino acid compositions to that of soy with high amounts of aspartic acid, glutamic acid, arginine, lysine and branch-chain amino acids. 5 Hempseed (Cannabis sativa L.) usually contains about 25% protein (mainly edestin and albumin) which are very nutritional due to the high content in essential amino acid, and easily digested. 6 Due to their amino acid profiles, proteins from both these plants are of interest in value-added products and their HVP have good potential to be applied as a valuable source of proteins for human nutrition. Besides, HVP have other peculiarities (depending on protease specificity, degree of hydrolysis, nature of released peptides), such antioxidant and antiinflammatory properties, and antifungal activity. METHOD B 5 Flaxseed (Linum usitatissimum L., Var, Valoal) GRINDING flaxseed by home-style grinder SIEVING resulting meal by a 500 μm screen DE-OILING by stirring with a) n-hexane (1:3 w/v, rt, 24 h b) CHCl 3 (1:3 w/v), rt, 24 h Defatted flaxseed meal cake BIOTRANSFORMATIONS to enriched w-3 and w-6 fatty acids MUCILAGE REDUCTION 1.Suspending meal in deionized water (5% w/v,) by stirring 2.Adding cellulase (1.35% w/w; activity of powder, 1,07 U/mg), pH 5 with 0,5 M HCl, 37 °C, O.N. METHOD A 7 MUCILAGE ELIMINATION 0.5 M NaHCO 3 (1/8 w/v), 50 °C, 1 h, stirring WASHING abundantly with water, AIR- DRYING (48 h) and in oven (45 °C,48 h) B A Flax and hemp fields). May 20, 2011 CNR – Istituto di Biologia e Biotecnologia Agraria (IBBA), Milano DE-OILING by stirring with n-hexane (1:3 w/v), rt, 24 h PROTEIN EXTRACTION 8 1.Cooling to 4 °C 2.Alkali solubilization (pH 10.00) with 0.5 M NaOH, rt, 30’ 3.Acid precipitation (pH 4,2) with 0,5 M HCl 4.Risuspending protein precipitate in deionized water, pH 7 with 0.5 M NaOH 5.Freeze-drying of suspension 1) Schlichtherle-Cerny H. and Amado R., J. Agric. Food Chem., 2002, 50, 1515; 2) Kurihara, K., Am. J. Clin. Nutr., 2009, 90, 719S; 3) Yamaguchi, S. J. Food Sci. 1967, 32, 473; 4) Morelli, C.F., Manitto, P., Speranza, G., Flav. Fragr. J., to be published; 5) Udenigwe, C.C., Lu, Y-L., Han, C-H., Hou, W-C., Aluko, R.E., Food Chem., 2009, 116, 277; 6) Wang, X.S., Tang, C.H., Yang, X.Q., Gao, W.R., Food Chem., 2008, 107, 11; 7) Marambe P. W. M. L. H. K., Shand P. J. and Wanasundara J. P. D., J. Am. Oil Chem. Soc., 2008, 85, 1155; 8) Dev D.K. and Quensel E., J Food Sci, 1988, 52, 1834. SIEVING resulting meal by a 500 μm screen Oil Oil
