LENTIL TANNIN-GLOBULIN INTERACTIONAND AND IN VITRO HYDROLYSIS

INTERAÇÃO TANINO-GLOBULINA DE LENTILHA E HIDRÓLISE IN VITRO

LENTIL TANNIN-GLOBULIN INTERACTION AND IN VITRO HYDROLYSIS¹

Valdir A. NEVES²,^*, Euclides J. LOURENÇO²

SUMMARY

Protein fractions were isolated from lentil cotyledons and tannins were isolated and purified from lentil seed coats. The globulin fraction corresponded to 42.7% of the total lentil flour nitrogen, representing the major protein fraction. Acetone:water (7:3) was the best extractant for seed coat tannins compared to methanol or methanol-HCl 1%. Native and heated (99^oC/15 min.) isolated lentil globulin and casein were hydrolyzed with trypsin and pepsin in the absence of tannins and at 1:40, 1:20, 1:10, 1:5 and 1:2.5 tannin-to-protein ratios. The tryptic and peptic hydrolysis of the unheated proteins were reduced with increasing tannin-to-protein ratios. Unheated casein showed to be more susceptible to trypsin than globulin and the opposite effect was observed with pepsin. Heating followed by tannin interaction and hydrolysis had a more pronounced effect on tryptic than peptic digestion for both proteins.

Keywords: Lens culinaris, lentil, tannin, globulin, in vitro hydrolysis

RESUMO

INTERAÇÃO TANINO-GLOBULINA DE LENTILHA E HIDRÓLISE IN VITRO. As frações protéicas foram isoladas dos cotiledones e os taninos isolados e purificados da casca da lentilha. A fração globulina correspondeu a 42,7 % do nitrogenio total da farinha de lentilha representando a fração protéica majoritária. Comparativamente ao metanol e metanol-HCl 1% a mistura acetona:água (7:3) representou o melhor meio extrator para os taninos da casca. A fração globulina isolada, nativa e aquecida (99^oC/15 min), e caseína foram hidrolisadas com tripsina e pepsina na ausência de taninos e na presença de relações tanino:proteína de 1:40, 1:20, 1:10, 1:5 e 1:2,5. A hidrólise tríptica e péptica das proteínas não-aquecidas foram reduzidas com o aumento da relação tanino-proteína. A caseína não aquecida mostrou ser mais susceptível à tripsina que à globulina, o oposto sendo observado com a pepsina. O aquecimento seguido de interação com os taninos e hidrólise teve um efeito mais pronunciado sobre a digestão com tripsina que com pepsina para ambas proteínas.

Palavras chave: Lens culinaris, lentilha, tanino, globulina, hidrólise in vitro

1 — INTRODUCTION

In the seeds of cereals and legumes the presence and consequent interaction of tannins and proteins have been believed to be one of the factors involved in reduced protein digestibility [3, 5, 11, 22]. There is evidence that tannin-protein complexes can be formed by differents mechanisms depending on the molecular characteristics of both substrates and as a function of protein concentration, pH and salt [9, 14, 16, 17,25]. The interactions that lead to the precipitation of proteins through a complex formation were believed, in the recent past, to result from multiple hydrogen bond formation between carbonyl functions of the peptide linkages of proteins and the phenolic groups of the tannins [16, 18, 25]. The hydrophobic character of tannin molecules and the presence of aliphatic and aromatic amino acids in the internal structure of proteins indicates that hydrophobic interactions are a predominant mechanism [13, 14, 15]. Experimental evidence that might be interpreted as involvement of both interactions in the complex formation is available in the literature. Therefore, the diversity of the structures and solvent interactions seems to indicate the occurrence of the two types of interaction in a mixed form [9, 25]. Although studies have been conducted on legume proteins and the effect of their interaction with tannins, most of the studies are related to beans [2, 3, 5, 10] and few have been devoted to other legume species.

Studies about lentil seed tannins have been reported [4, 20]. However, the characteristics and effects of tannin lentil-protein interaction have not been explored. The major globulin of lentil proved to be resistant to in vitro trypsin and chymotrypsin hydrolysis in the native form and was more susceptible when heated [21]. In the present study we examined the isolation and purification of lentil tannins, and the effect of the purified lentil tannin-globulin complex formation on the in vitro susceptibility to hydrolysis at various tannin-to-protein ratios using trypsin and pepsin.

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^oC before being manually dehulled. The air-dried cotyledons and seed coats were ground, separately, to pass a 100-mesh sieve and the cotyledons deffated in n-hexane at room temperature. Trypsin , pepsin, casein, Sephadex LH-20, (-) catechin, trinitrobenzenesulphonic acid, were purchase ffrom 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

The lentil globulin was extracted as described by NEVES & LOURENÇO [21]; resuspended in distilled water and lyophilized.

2.2.2 - Tannin Extraction

One g of seed coat flour was dissolved in 10 ml methanol, methanol- HCl 1% or an aqueous acetone (7:3) mixture, homogenized for 30 min. at 4^oC protected from light, centrifuged at 10,000 rpm/20 min. and filtered through Whatman n^o2 filter paper. The residue was re-extracted (twice), the filtrates were combined and the tannins determined.

2.2.3 - Tannin Purification

The lentil tannins were isolated from seed coat meal according to the method of TERRIL et al. [26], with acetone:water (7:3) containing 0.1% ascorbic acid. The final extracts were combined, evaporated to remove the solvent, and freeze-dried. The lyophilized powder was dissolved in a mixture of methanol:water (1:1) containing 0.1% ascorbic acid and loaded onto a column of Sephadex LH-20 (2 x 9 cm) previously equilibrated with methanol:water (1:1). After the column was washed with the same solvent, the tannic substances were eluted with acetone:water (7:3) containing 0.1% ascorbic acid. The collected fractions were combined, evaporated and lyophilized, producing a white-brown powder.

2.2.4 - Tannin Determination

The tannins were determined by the modified vanillin-HCl method of DESHPANDE & CHERYAN [6] using (-) catechin as a standard.

2.2.5 - Protein Determination

Proteins were determined by the method of LOWRY et al. [19] using bovine serum albumin as a standard.

2.2.6 - Nitrogen determination

Nitrogen content was determined by the microKjeldhal method (AOAC)[1] and the nitrogen converted to protein using 6.25 as the conversion factor.

2.2.7 - In vitro Protein Hydrolysis

Trypsin (Sigma T 8003) and pepsin (Sigma P-7000) were used for studies of in vitro hydrolysis of lentil globulin and casein. The unheated proteins were dissolved in 50 mM phosphate buffer, pH 7.8. The proteins were also heated at 99^oC/15 min. Aliquots in triplicate (0.5-1.0 mg/ml) of unheated and heated proteins were incubated separately with trypsin at the 1:10 enzyme to protein ratio, at 37^oC in tubes sealed with plastic wrap. At time intervals between 0 to 120 min., tubes of the digest were diluted to 8.0 ml with deionized water and allowed to stand in an ice bath for 10 min. For pepsin hydrolysis the protein solutions were adjusted to pH 2.0 with 0.05 M HCl and incubated at 37^oC at the same enzyme to protein ratio, and tubes of the digest were treated as described for trypsin from 0 to 180 min. The extent of hydrolysis was determined by the increase of free amino groups using 2,4,6-trinitrobenzenesulphonic acid (TNBS) according to the method of FIELDS [8] as modified by SPADARO et al. [24]. The percentage 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-a-amino groups of 16500 M^-1 cm^-1 and an average weight of 113 g/mol for amino acids residues in protein were used for the calculation. All hydrolysis assays were performed in triplicate.

2.2.8 - In vitro Hydrolysis of the Tannin-Protein Interaction

The assays were performed with unheated and heated proteins at lentil tannin to protein ratios of 1:40; 1:20; 1:10, 1:5 and 1:2.5 for trypsin and 1:20, 1:10, 1:5 and 1:2.5 for pepsin, followed by incubation with the enzymes as described above for the proteins without tannins. All hydrolysis assays were performed in triplicate.

3 — RESULTS AND DISCUSSION

The salt-soluble proteins of lentil meal represented 70.18% of the flour nitrogen, whereas the globulin fraction corresponded to 42.7% of the total nitrogen. Acetone:water (7:3) proved to be the best extractant for the tannins of lentil seed coat compared to methanol or methanol-HCl 1% (Table 1). The tannnins used in this experiment were purified to remove all nontannin phenolics. The material eluted from the Sephadex LH-20 column represented 90% of the tannins applied and produced a white-brown powder after lyophilization.

TABLE 1.
Effect of different solvents on the extraction of tannin from lentil seed coat meal.

SOLVENT	TANNIN*(mg/g of meal)
Methanol	8.66 ± 0.26
Methanol/HCl 1%	72.19 ± 1.10
Acetone:water (7:3)	146.25 ± 3.60

*Tannin content is expressed as catechin equivalents.

Mean values ± standard deviations of five replicates.

The percent of total peptide bond hydrolysis of lentil globulin in the native form corresponded to 44.4% of that obtained with casein after 60 min hydrolysis with trypsin. The heating at 99^oC/15 min increased its susceptibility, although only up to 70% of that of casein (Table 2). The results of the tryptic hydrolysis of the lentil tannin-interaction at various tannin-to-protein ratios for unheated and heated proteins are shown in Tables 3 and 4.

TABLE 2.
Proteolysis of lentil globulin and casein by trypsin and pepsin.

ENZYME	% TOTAL PEPTIDE BOND HYDROLYSIS*
protein	unheated	heated**
TRYPSIN
casein lentil globulin	10,17 ± 0,13 4,52 ± 0.08	10,90 ± 0,21 7.53 ± 0.09
PEPSIN
casein lentil globulin	7,80 ± 0,16 7,24 ± 0.09	8,51 ± 0,09 8,27 ± 0.12

*Proteins were digested for 60 min with trypsin and for 180 min for pepsin.

**Heated at 99^oC/15 min.

Mean values ± standard deviations of three replications.

TABLE 3.
Effect of purified lentil tannin on casein hydrolysis by trypsin at various tannin-casein ratios.

Tannin to protein ratio	% TOTAL PEPTIDE BOND HYDROLYSIS*
	unheated	heated**
Control	10.17 ± 0.13	10.90 ± 0.21
1:40	7.88 ± 0.08	10.53 ± 0.10
1:20	7.76 ± 0.22	8.22 ± 0.08
1:5	6.74 ± 0.12	7.93 ± 0.11
1:2.5	6.50 ± 0.07	7.80 ± 0.06

*Proteins were digested for 60 min.

**Heated at 99^oC/15 min.

Mean values ± standard deviations of three replicates

TABLE 4.
Effect of purified lentil tannin on lentil globulin hydrolysis by trypsin at various tannin-globulin ratios

Tannin to protein ratio	% TOTAL PEPTIDE BOND HYDROLYSIS*
	unheated	heated**
Control	4.52 ± 0.08	7.53 ± 0.09
1:40	4.24 ± 0.12	6.14 ± 0.10
1:20	5.50 ± 0.24	5.63 ± 0.12
1:10	2.70 ± 0.08	5.24 ± 0.09
1:5	1.80 ± 0.09	5.07 ± 0.05
1:2.5	1.60 ± 0.05	4.90 ± 0.12

*Proteins were digested for 60 min.

**Heated at 99^oC/15 min.

Mean values ± standard deviations of three replicates.

Unheated casein proved to be more susceptible to the enzyme than lentil globulin for all tannin-protein ratios studied when compared to the proteins hydrolyzed whitout tannins (Tables 3 and 4). It is interesting to note that unheated globulin showed a higher percent of hydrolysis at 1:20 tannin-protein ratios than in the absence of tannin. This fact may indicates that the tannin interaction provoked possible protein structure alterations that facilitated enzyme attack, as observed by HOON-IL & HOFF [16]. Compared to the controls whithot tannins, heated casein followed by interaction and hydrolysis was more susceptible to trypsin than globulin.

After 3 h of incubation with pepsin, the percent of hydrolysis of total peptide bonds of native lentil globulin corresponded to 92.8% of that obtained with casein. Heating at 99^oC/15 min increased the hydrolysis of both proteins, with the heated globulin corresponding to 97 % of the casein hydrolysis (Table 2 ).

The peptic hydrolysis of lentil tannin-complexed proteins at different tannin-to-protein ratios is presented in Tables 5 and 6. Contrary to trypsin, the increase in tannin-to-protein ratios (1:20 to 1:2.5) had a much more pronounced inhibitory effect on casein than on lentil globulin hydrolysis in the native forms. At tannin-to-protein ratios of 1:20 and 1:10, native globulin hydrolysis was pratically the same in the absence of tannin, whereas native casein was reduced by 25% and 23% in relation to the protein in the absence of tannin, respectively. At higher ratios of 1:5 and 1:2.5, casein was much more resistant to the enzyme.

TABLE 5.
Effect of purified lentil tannin on casein hydrolysis by pepsin at various tannin-casein ratios

Tannin to protein ratio	% TOTAL PEPTIDE BOND HYDROLYSIS*
	unheated	heated**
Control	7.80 ± 0.16	8.51 ± 0.09
1:20	5.90 ± 0.08	5.72 ± 0.12
1:10	6.02 ± 0.11	5.81 ± 0.09
1:5	3.68 ± 0.09	3.93 ± 0.11
1:2.5	3.19 ± 0.06	3.31 ± 0.13

*Proteins were digested for 180 min.

**Heated at 99^oC/15 min.

Mean values ± standard deviations of three replicates.

TABLE 6.
Effect of purified lentil tannin on lentil globulin hydrolysis by pepsin at various tannin-globulin ratios.

Tannin to protein ratio	% TOTAL PEPTIDE BOND HYDROLYSIS*
	unheated	heated**
Control	7.24 ± 0.09	8.27 ± 0.12
1:20	7.39 ± 0.11	7.56 ± 0.08
1:10	7.56 ± 0.14	8.11 ± 0.06
1:5	4.43 ± 0.07	4.74 ± 0.13
1:2.5	4.11 ± 0.10	3.72 ± 0.10

*Proteins were digested for 180 min.

** Heated at 99^oC/15 min.

Mean values ± standard deviations of three replicates.

Lentil protein was less resistant to peptic digestion than casein, following both heating treatment and tannin interaction at various tannin-to-protein ratios, in contrast to the results observed with trypsin. Although hydrolysis behavior was similar for native proteins, the heating followed by tannin interaction reduced the digestion for both proteins compared to unheated ones. This fact may be accounted for by the denaturation of the proteins that increases accessibility of lentil tannin to peptide amino acids and may cause greater affinity for tannin and consequently protection of the protein from digestion.

The specificity of the tannin to protein interaction is a function of size, conformation and charge of the protein molecule [13, 25]. The lentil tannin effect on casein hydrolysis seems to result from these structural and conformational characteristics of the molecule comparatively to lentil globulin. In contrast, pepsin digestion of lentil globulin at 1:20 and 1:10 tannin to protein ratios may be attributable to a possible deformation of the native protein structure in complex formation, with a consequent exposure of the protein groups to enzyme attack. HOON-IL & HOFF [16] observed that even at a tannin concentration of 1% the tryptic hydrolysis of bovine serum albumin was more extensive than without tannins in the mixture. Contrary to the results reported here, AW & SWANSON [3] observed that bean tannin-globulin G-1 complexes were less digested by pepsin than bean tannin-casein complexes. The authors suggested that the hydrophobic bonding and the compact structure of the bean tannin-globulin complex could be responsible for the resistance to pepsin digestion. ROMERO & RYAN [23] verified that the pepsin and chymotrypsin digestibility of bean globulin G-1 was not affected by the tannin content of high and low tannin seed lines, but the addition of tannin to G-1 protein significantly reduced tryptic hydrolysis.

Despite conclusive evidence for a negative effect of tannin on protein digestibility, if one assumes that the concentrations of tannins in lentil seed coats is lower than 1 %, all of the inhibitory effects encountered would be less significant than they really appear.

4 — REFERENCES

[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. and 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] 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.

[6] DESHPANDE, S.S & CHERYAN, M.. Evaluation of vanillin assay for tannins analysis of dry beans. J. Food Sci., v.50, p.905-10, 1985.

[7] 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.

[8] FIELDS, R. The rapid determination of aminogroups with TNBS. Meth. Enzymol., v.25, p.464-8, 1972.

[9] FOO, L.Y. & PORTER, L.J.. The phytochemistry of proanthocyanidin polymers. Phytochemistry, v.19, p.1747- 1754, 1980.

[10] GUEGUEN, J.. Relation between conformation and hydrophobicity of pea (Pisum sativum L.). J. Agric. Food Chem., v.37, p.201-28, 1987.

[11] GUPTA, Y.P. Anti-nutritional and toxic factors in food legumes: A review. Plant Foods Hum. Nutr., v.37, p.201-28, 1987.

[12] HAGERMAN, A.E. & BUTLER, L.G. Protein precipitation methods for the quantitative determination of tannins. J. Agric. Food Chem., v.26, p.809, 1978.

[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, A.R. Liss ed., 1986, p. 67-76.

[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.577-80, 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] KOSHIYAMA, I. Comparison of acid-induced conformation changes between 7 S and 11 S globulins in soybean seeds. J. Sci. Food Agric., v.23, p.853-9, 1972.

[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] NEVES, V.A.. Isolamento e digestibilidade in vitro da proteína de lentilha (Lens esculenta). São Paulo, 1991. 140 p. Doctoral thesis, Faculdade de Ciências Farmaceuticas-Universidade de São Paulo,

[21] 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.

[22] 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.

[23] 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.784-8, 1978.

[24] SPADARO, A.C.C., DRAGHETTA, W., LAMA, S.N.D., CAMARGO, A.C.M., GREENE, L. J. A convenient manual trinitrobenzenesulfonic acid method for monitoring amino acids and peptides in chromatographic column effluents. Anal. Biochem., v.96, p.317-321, 1979.

[25] 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.

[26] TERRIL,T.H., ROWAN, A.M., DOUGLAS, G.B., BARRY, T.N. Determination of extractable and bound condensed tannin concentrations in forage plants, protein concentrate and cereal grains. J. Sci. Food Agric., v.58, p.321-29, 1992.

5 — ACKNOWLEDGEMENTS

The authors thank to: Mrs. Ana P. Tarcinali for technical assistance. This research was supported by the PADC-FCF-UNESP and CNPq-PIBIC programs.

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

^* A quem a correspondência deve ser endereçada

¹

Recebido para publicação em 10/05/98. Aceito para publicação em 06/08/98.

Publication Dates

• Publication in this collection
  20 May 1999
• Date of issue
  Aug 1998

History

• Accepted
  06 Aug 1998
• Received
  10 May 1998