Efeito de taninos da lentilha sobre a hidrólise da albumina pela tripsina

Effect of lentil tannins on albumin hydrolysis by trypsin

EFFECT OF LENTIL TANNINS ON ALBUMIN HYDROLYSIS BY TRYPSIN¹

NEVES², Valdir A.; LOURENÇO², Euclides J. & SILVA², Maraiza A.

SUMMARY

The tannins of lentil seed coat were extracted and purified, allowed to interact with isolated lentil albumin and casein and studied turbidimetrically. The interactions of lentil albumin and casein with tannin at various tannin-to- protein ratios proved to be pH-independent and pH-dependent, respectively. In vitro trypsin hydrolysis of the proteins without tannins showed that the heating at 99°C/15 min. reduced the susceptibility of albumin and increased the susceptibility of casein to the enzyme. The influence of tannin-to-protein ratios (1:40; 1:20; 1:5. 1:2,5) on the in vitro tryptic hydrolysis was more inhibitory for casein than for lentil albumin when unheated or heated at 99°C/15 min. After heating both proteins were more hydrolyzed at all tannin-to-protein ratios. SDS-PAGE of the products of hydrolysis of native albumin-tannin interactions show that the extent of proteolysis is dependent of the protein-tannin ratio.

Key words: lentil, tannin-protein interaction, in vitro hydrolysis, Lens culinaris.

RESUMO

EFEITO DE TANINOS DA LENTILHA SOBRE A HIDRÓLISE DA ALBUMINA PELA TRIPSINA. Os taninos da casca da semente de lentilha foram extraídos e purificados, levados à interação com albumina isolada de lentilha e com caseína; e estudados por turbidimetria. As interações da albumina e caseína com taninos purificados, a várias relações tanino-proteína, mostraram ser independente e dependente do pH, respectivamente. Hidrólise in vitro com tripsina das proteínas sem taninos indicou que o aquecimento a 99°C/15 min reduzia a susceptibilidade da albumina e aumentava a da caseína à tripsina. A influência de diferentes relações tanino:proteína (1:40; 1:20; 1:5; 1:2,5) na hidrólise mostrou maior inibição para caseína que para albumina de lentilha, independente de aquecimento. Após aquecimento ambas proteínas foram mais hidrolizadas para qualquer das relações tanino proteínas estudadas. A eletroforese em gel de poliacrilamida-dodecilsulfato de sódio do transcurso da hidrólise da interação tanino-albumina nativa mostra a dependência da relação tanino:proteína.

Palavras-chave: lentilha, interação tanino:proteína, hidrólise in vitro, Lens culinaris.

1 — INTRODUCTION

Condensed tannins are polymeric phenolic compounds present in a great variety of edible plants such as legume seeds, which form insoluble complexes with proteins (24, 28). These complexes are reported to be responsible for growth depression, low protein digestibility in vitro and in vivo, decreased amino acid availability and increased fecal nitrogen (9, 10, 24). The type of tannin-protein interaction has not been completely elucidated, but there are evidences suggesting the involvement of non-covalent bonding depending on the molecular characteristics of both substrates (14, 17). The relative affinity of various proteins for condensed tannins was shown to depend on the composition, structure and size of the protein, suggesting that the tannin-protein interaction is a selective process (2, 13, 28). Bothin vitro and in vivo studies indicate that the condensed tannins present in edible plants interact with proteins affecting their digestibility (3, 6, 9, 16, 24, 25).

Lentils (Lens culinaris) are a valuable source of protein in many areas of the world. However like other legumes, they also contain condensed tannins on the hulls that have been related to poor digestibility. The major proteins in lentils are globulins and albumins; although albumins are present in smaller amounts they are richer in sulfur amino acids and lysine (4, 5, 21)

The objectives of the present study were: (1) to fractionate lentil proteins, (2) to extract and purify the tannins from the hulls, (3) to study the albumin-complexing characteristics of purified lentil tannins and in vitro trypsin hydrolysis of the albumin-tannin complex.

2 — MATERIAL AND METHODS

2.1 – Material

Lentils (Lens culinaris Medik), cv. Precoz, were purchased from Centro Nacional de Pesquisas em Hortaliças-CNPH-EMBRAPA, Brasília , Brazil. Lentil seeds were soaked in cold water at 4°C before being manually dehulled. The air-dried cotyledons and seed coats were separately ground to pass through a 100-mesh sieve and the cotyledons defatted in n-hexane at room temperature. Trypsin, casein, Sephadex LH-20, (-) catechin, 2,4,6-trinitrobenzenesulphonic acid (TNBS), acrylamide, bys-acrylamide, bovine serum albumin (BSA), egg white albumin, carbonic anhydrase, soybean trypsin inhibitor and cytochrome C were purchased from Sigma Chemical Co.; St. Louis, M.O. All other chemicals were reagent grade or the best grade available.

2.2 – Methods

2.2.1 – Protein fractionation

Albumins and globulins were successively extracted from defatted lentil flour (1:10 flour to solvent ratio) with deionized water and 0.5 M NaCl solution as described by SATHE & SALUNKHE (26). The residue remaining after salt extraction was successively extracted with 70% ethanol and 0.1 M NaOH to separate the prolamin and glutelin fractions, respectively. The albumins and globulins were ressuspended in distilled water and lyophilized.

2.2.2 – Tannin extraction

One g of seed coat flour was dissolved in 10 ml of methanol, methanol-1% HCl or an aqueous acetone (7:3) mixture; homogenized (30 min. at 4°C, protected from light), centrifuged at 15000 g/20 min and filtered through Whatman n.2 filter paper. The residue was reextracted (2 X), the filtrates were combined and the tannins determined.

2.2.3 – Tannin purification

The lentil tannins were isolated from seed coat according to the procedure described above with acetone:water (7:3) containing 0.1% ascorbic acid. The final extracts were combined, rotoevaporated to remove the solvent and then freeze-dried. The lyophilized powder was dissoved in a mixture of methanol:water (1:1) containing 0.1% ascorbic acid and loaded onto a Sephadex LH-20 column (2.0 x 9.0 cm) previously equilibrated with methanol:water. The column was then washed with methanol:water (1:1) and the tannic substances were eluted with acetone-water (7:3) containing 0.1% ascorbic acid. The collected fractions were combined, rotoevaporated and lyophilized yielding a "white-brown" powder.

2.2.4 – Chemical analysis

The tannins were determined by the modified vanillin-HCl method as recomended by DESHPANDE & CHERYAN (7) using (-)catechin as a standard. Proteins were determined by the method of LOWRY et al. (19) with bovine serum albumin as a standard. Nitrogen content was determined by the microKjeldhal method (1) and nitrogen was converted to protein using 6.25 as the conversion factor.

2.2.5 – Tannin-protein interaction

The characteristics of tannin-protein interaction were studied in two stages: (1) effect of pH and tannin to protein ratios on the interaction; (2) effect of tannin on the in vitro protein hydrolysis with trypsin. The pH effect on the interaction was determined turbidimetrically as described by HOON-IL et al. (15). To perform the assays appropriated buffered solutions of albumin and casein were made, as well as a fresh aqueous solution of purified tannins containing 0,5 % Na bisulfite. The following buffers were used at the indicated pH ranges: pH 3.0-6.0 (0.1M citrate-phosphate ), pH 7.0-8.0 (0.1M Na-phosphate ). Assay: aliquots in triplicate of lentil tannin solution (10 mg/ml distilled water) were added to the sample cuvette (3.0 ml) containing the proteins (0.5 to 2.0 mg/ml), mixed and the resulting turbidity was determined as the absorbance at 450 nm for 5 minutes when it was fully developed. Tannin to protein ratios ranging from 0.5:1.0 to 2.0:1.0 and in the pH range of 3.0 to 8.0 were run. Casein (Hammarsten) was used under the same conditions for comparative purpose. Control tannin solutions were run in the same manner in the absence of protein.

2.2.6 – In vitro protein hydrolysis

Trypsin was used for studies of the in vitro hydrolysis of lentil albumin and casein. The unheated proteins, lentil albumin and casein, were dissolved in 50 mM phosphate buffer, pH 7,8. The proteins were also heated at 99°C/15 min. Aliquots in triplicate (0.5-1.0 mg/ml) of unheated and heated proteins were separetely incubated with trypsin at 1:10 enzyme to protein ratio, at 37°C in wrapped test tubes. The digested samples (from 0 to 60 min) were diluted to 8.0 ml with deionized water and allowed to stand in an ice bath for 10 min. The extent of hydrolysis was determined by the increase of free amino groups using TNBS according to the method of FIELDS (11) as modified by SPADARO et al. (27). The percent of peptide bond hydrolysis was calculated from the changes in the ratio of new amino groups in the digestion to the total number of peptide bonds in the mixture. The molar extinction coefficient for TNP-alpha-amino groups of 16500 M^-1 cm^-1 and an average weight of 113 g/mol for amino acid residues in protein were used for the calculation. All hydrolysis assays were performed in triplicate.

2.2.7 – In vitro hydrolysis of the tannin-protein interaction

Assays were performed in triplicate with unheated and heated proteins at lentil tannin to protein ratios of 1:40; 1:20; 1:5 and 1:2.5 and incubated with trypsin as described above for the proteins without tannins.

2.2.8 – Gel electrophoresis

The digests of unheated albumins without tannin and at tannin-to-protein ratios of 1:20 and 1:5 were examined during the course of hydrolysis by SDS-PAGE (12.5% gel, 124x258x1 mm). The incubated tubes were removed from the water bath at 15 and 30 sec. and at 1, 5, 15, 30, 60 and 120 min, diluted with Tris-SDS-mercaptoethanol buffer and heated at 99°C/3 min. Electrophoresis experiments were performed according to the method of LAEMMLI (18). Bovine serum albumin (MW 67 kD), egg white albumin ( MW 43 kD), carbonic anhydrase (MW 29 kD), soybean trypsin inhibitor (MW 21.5 kD) and cytochrome C (MW 12.4 kD) were used as molecular weight markers. All hydrolysis assays were performed in triplicate.

3 — RESULTS AND DISCUSSION

The content of crude protein of dehulled lentil flour was found to be 26.4 g/100 g , determined by the microKjeldhal method (1). Lentil proteins are mainly globulins and albumins. Both groups of proteins are soluble in salt solutions and fractionated by extensive dialysis of a salt extract against distilled water. The albumins, globulins, prolamins and glutelins represented 14.09, 42.67, 5.33 and 14.01% of the lentil meal protein, respectively (22). The major differences between legume protein fractions seem to occur in the water soluble albumins. BHATTY (5) reported a range of 8.1 to 14.1% for the albuminic N of 8 different legume species and suggested that the discrepancies are most likely due to cross-contamination of the water- and salt-soluble proteins. Variations in the content of albumins of legume seeds have been reported (5, 8, 21), and the extraction procedure adopted may explain the different results (8).

The tannins of lentil seed coat were extracted and purified. The amounts of tannins recovered from 1.0g seed coat meal on a dry basis as catechin equivalents, were 8.66 mg for absolute methanol, 72.19 mg for 1% HCl in methanol and 146.25 mg for 70% aqueous acetone as the solvents. Tannins purified on Sephadex LH-20 were lyophilized and produced a white-brown powder.

The interactions of albumin and soluble casein with purified lentil tannins were measured by turbidity (see methods).The albumin and casein-lentil tannin interaction shown to be dependent on the relative amounts of the two substrates (Figures 1, 2). An increase in the tannin-to-protein ratio did not produce a stoichiometric increase in turbidity with either protein, a fact that appears to be related to the formation of soluble complexes as cited by HOON-IL & HOFF (17). The lentil tannin-albumin precipitations at the pH range of 3.0 to 8.0 were approximately equivalent at the same tannin-protein ratio, with the exception of pH 3.0 (Figure 1). However, maximum casein precipitation was observed at pH 5.0 for all tannin-casein ratios (Figure 2). The behaviour of the complex formation was different for each protein as a function of pH and it was not altered with the tannin-protein ratios studied.

The mechanisms proposed for the condensed tannin-protein interaction were based on non-covalent binding and although the type of interaction has not been completely elucidated, there is evidence of the occurrence of hydrogen and hydrophobic bonding (13, 15, 28) depending on the types of sites available on the surface of the protein and therefore as a function of size, conformation and charge of the molecule (2, 12, 14, 28).

The percent hydrolysis of total peptide bonds of unheated albumin corresponded to 94.7% of that obtained with the unheated casein after 60 min. of hydrolysis. Heating reduced the hydrolysis of albumins to 48.7% of heated casein hydrolysis and lower than unheated albumin hydrolysis (Table 1 and 2, controls). The reduced hydrolysis of heated albumins was due to factors other than trypsin inhibitors, which were present in very low concentrations in the lentil albumin fraction (21); on the other hand aggregates and undigestible complexed protein formed during heating were detected on PAGE (not shown) and they were the most probable factors responsible for decreased hydrolysis.

Unheated albumin of lentil and from other leguminous seeds, such as bean, are well digested by trypsin as reported previously (23, 20); however, a reduction of tryptic hydrolysis after heating was always observed for bean albumins (20) although no reports about lentil albumin are available in the literature.

The effect of different purified lentil tannin-protein ratios on the tryptic hydrolysis of the casein and lentil albumin are shown in Tables 1 and 2. The inhibitory effect was stronger for casein than for albumin relatively to the unheated controls.

Heating of the proteins at 99°C/15 min followed by tannin interaction had a minor inhibitory effect on both proteins hydrolysis. The percentage of the heated protein hydrolysis were increased comparatively to unheated ones at all of the tannin to protein ratios, although lentil albumin was shown to be less hydrolyzed after heating and in the absence of tannins (Table 1, 2). The tannin-protein association indicates that it is largely a surface phenomenon depending on the interaction between residues in the polypeptide chain and tannin molecules (28). In this case, the conformation of the protein molecule seems to be a considerable factor in the association. The aggregates, detected after albumin heating, may be responsible for a lower tannin-albumin interaction and consequently may not affect hydrolysis, comparatively to unheated protein.

The lentil albumins subjected to tannin interaction, heating at 99°C/15 min. and hydrolysis with trypsin are shown in Table 3. The tannin-protein interaction followed by heating increased protein digestion at 1:40 and 1:20 tannin-protein ratios and did not affect it at higher ratios; comparatively to heating of the protein followed by tannin interaction and hydrolysis. These results seem to indicate that the tannin interaction at lower ratios provoked possible protein structure alterations that facilitated enzyme attack ; or the interaction followed by heating reduced the aggregation; contrary to observed at higher ratios (1:5 and 1:2.5) that presented % of inhibition of 22 and 45.4, respectively. However, a reduction of the tannin content has been observed due to heating (6, 21, 24).

The SDS-PAGE of unheated lentil albumins without tannin and at 1:20 and 1:5 tannin-albumin ratios after hydrolysis for up to 120 min. is presented in Figures 3, 4 and 5. The increase in the tannin-protein ratio similarly reduced the tryptic hydrolysis of all albumin bands. These data suggest that the interaction and extent of proteolysis may be dependent on the tannin-to-protein ratio. However, the bulk of lentil seed proteins are globulins and studies are under way in our laboratory to establish the nature of their interaction with purified lentil tannin and the effect on proteolysis.

4 — REFERENCES

ACKNOWLEDGEMENTS

The authors thank to: Mrs. Ana L.P. Falco for technical assistance. This research was supported by the FCF-UNESP research Comission and the CNPq-PIBIC program.

² Departamento de Alimentos e Nutrição. Faculdade de Ciências Farmacêuticas de Araraquara - UNESP. Rod. Araraquara-Jaú, km 01. Campus Universitário. CEP 14801-902. C.P. 502. Fax [phone]. Araraquara SP.

• (1) AOAC. Official Methods of Analysis (13^th ed.). Association of Official Analytical Chemists Washington, D.C., 1980.
• (2) ARTZ, W.E.; BISHOP, P.Q.; DUNKER, A.K.; SCHANUS, E.G., SWANSON, B.G. Interaction of synthetic proantocyanidin dimer and trimer with bovine serum albumin and purified globulin fraction G-1. J. Agric. Food Chem., v. 35, p.417-21, 1987.
• (3) AW, T.L. & SWANSON, B.G.  Influence of tannin on Phaseolus vulgaris protein digestibility and quality. J. Food Sci, v.50, p.67-71, 1985.
• (4) BHATTY, R.S.  Composition and quality of lentil (Lens culinaris Medik): A review. Can. Inst Food Sci. Technol. J, v.21, p.144-60, 1988.
• (5) BHATTY, R.S.  Albumin proteins of eight edible grain legume species: electrophoretic patterns and amino acid composition. J.Agric. Food Chem., v.30, p.620-22, 1982.
• (6) BRESSANI, R.; ELIAS, L.G.; WOLZAK, A., HAGERMAN, A.E. Tannin in common beans. Methods of analysis and effects on protein quality. J. Food Sci, v.48, p.1000-3, 1983.
• (7) DESHPANDE, S.S & CHERYAN, M.  Evaluation of vanillin assay for tannin analysis of dry beans. J. Food Sci. , v.50, p.905-10, 1985.
• (8) DESHPANDE, S.S. & NIELSEN, S.S. Nitrogenous constituents of selected grain legumes. J.Food Sci. , v.52, p.1321-6, 1987.
• (9) DURIGAN, J.F.; SGARBIERI, V.C. , BULISANI, E.A. Protein Value of Dry Bean cultivars: factors interfering with biological utilization. J.Agric. Food Chem. v.35, p.694-8, 1987.
• (10) ELIAS, L.G.; DE FERNANDEZ, D.G. , BRESSANI, R. Possible effects of seed coat polyphenolics on the nutritional quality of bean protein. J.Food Sci, v.44, p.524-7, 1979.
• (11) FIELDS, R. The rapid determination of amino groups with TNBS. Meth Enzymol. , v.25, p.464-8, 1972.
• (12) HAGERMAN, A.E. & BUTLER, L.G.  Condensed tannin purification and characterization of tannin-associated proteins. J. Agric. Food Chem, v.28, p.947, 1980.
• (13) HAGERMAN, A.E. & BUTLER, L.G. The specificity of proanthocyanidin-protein interactions. J. Biol. Chem, v.256, p.4494, 1981.
• (14) HAGERMAN, A.E. & KUCKLER, K.V. Tannin-protein interactions. Plant Flavon. in Biol. and Medic. Biochem. Pharmacol. and Structure- activity relationship, (Ed.) A.R. Lissed p. 67-76, 1986.
• (15) HOON-IL, O.H.; HOFF, J.E.; ARMSTRONG, G.S., HAFF, L.A. Hydrophobic interaction in tannin-protein complexes. J. Agric. Food Chem, v. 28, p.394-8, 1980.
• (16) HOON-IL, O.H. & HOFF, J.E. Effect of condensed grape tannins on the in vitro activity of digestive proteases and activation of their zymogens. J. Food Sci, v.51, p.80, 1986.
• (17) HOON-IL, O.H. & HOFF, J.E. pH dependence of complex formation between condensed tannins and proteins. J. Food Sci,v.52, p.1267-9, 1987.
• (18) LAEMMLI, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T-4. Nature , v.227, p.680-85, 1970.
• (19) LOWRY, O.H.; ROSEBROUGH, N.J.; FARR, A.L.; RANDALL, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem., v.193, p.265-75, 1951.
• (20) MARQUES, U.M.L. & LAJOLO, F.M. Composition and quality of albumins, globulins and glutelins from Phaseolus vulgaris J. Agric. Food Chem, v.29, p.1068-74, 1981.
• (21) NEVES, V.A. 1991. Isolamento e digestibilidade in vitro da proteína de lentilha (Lens esculenta). Doctoral thesis. FCF-USP, 140p.
• (22) NEVES, V.A. & LOURENÇO, E.J. Isolation and in vitro hydrolysis of globulin G.1 from lentils (Lens culinaris, Medik). J. Food Biochem, v.19, p. 109-120, 1995.
• (23) NEVES, V.A.; LOURENÇO, E.J., SILVA, M.A. Isolation and in vitro hydrolysis of lentil protein fractions by trypsin. Arch. Latinoamer. Nutric, v.46 (3), p 238-42, 1996.
• (24) REDDY, N.R.; PIERSON, M.D.; SATHE, S.K., SALUNKE, D.K. Dry beans tannins: a review of nutritional implications. J. Am. Oil. Chem. Soc, v.62, p.541-9, 1985.
• (25) ROMERO, J. & RYAN, D.S. Susceptibility of the major storage proteins of the bean, Phaseolus vulgaris L. to in vitro enzymatic hydrolysis. J. Agric. Food Chem. v.26, p.784, 1978.
• (26) SATHE, S.K. & SALUNKHE, D.K. Solubilization and electrophoretic characterization of the great northern bean (Phaseolus vulgaris L.) proteins. J. Food Sci,v.46, p.82-87, 1981.
• (27) SPADARO, A.C.C; DRAGHETTA, W.; LAMA, S.N.D.; CAMARGO, A.C.M, GREENE, L. A convenient manual trinitrobenzenesulfonic acid method for monitoring amino acids and peptides in chromatographic column effluents. Anal. Biochem, v.96, p.317-321, 1979.
• (28) SPENCER, M.C.; CAI, V.A.; MARTIN, R.; GAFFNEY, S.M.; GOULDIN, P.N.; MAGNOLLATO, D.; LILLEY, T.H., HASLAM, E. Polyphenol complexation- some thoughts and observations. Phytochemistry, v.27, p.2397-2409, 1988.

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Recebido para publicação em 01/04/97. Aceito para publicação 30/09/97.

Datas de Publicação

• Publicação nesta coleção
  14 Dez 2004
• Data do Fascículo
  Dez 1997

Histórico

• Aceito
  30 Set 1997
• Recebido
  01 Abr 1997