The influence of extrusion of faba bean seeds and supplementation of sulphur amino acids on performance, pancreatic trypsin activity, and morphological parameters of the jejunum in rats

The effect of extrusion of faba bean seeds and supplementation of sulphur amino acids (AA) on trypsin inhibitor (TI) and lectin activity, and the histology and microbiology of the jejunum were studied. Male Wistar rats were fed semi-purified diets containing 10% faba bean seeds, either raw (R) or extruded at 160°C (E); the diets were either unsupplemented or supplemented with 0.3% sulphur A A (met + cys, 1:1). Extrusion reduced TI activity of seeds from 1.8 (FR) to 0.5 with TlU/mg (FE) and completely eliminated agglutination activity against human red blood cells (RBC). Extrusion of faba bean seeds did not affect rat performance or pancreatic trypsin activity (5255 vs 4065 U/ 100 g protein), and decreased (PO.05) dry matter digestibility (89.7 vs 92.9%). Supplementing sulphur amino acids improved performance of rats fed both raw and extruded seeds. Extrusion of faba bean and supplementing sulphur amino acids significantly improved intestinal crypt depth, villi length and width.


INTRODUCTION
Although faba bean seeds have fewer protease inhibitors and lower lectin activity than soyabean seeds (Valdebouze et al., 1980;Leontowicz et al., 2001b), they contain tannins, which reduce digestibility by forming complexes with proteins and/or digestive enzymes (Griffiths and Moseley, 1980) and also depress animal performance.Different technologies (boiling, autoclaving, extrusion) are used for inactivation of thermolabile ANFs present in legumes (Liener, 1994).The low level of sulphur amino acids limits growth of animals fed on diets with legumes but supplementation with synthetic amino acids usually improves performance.
The aim of our study on rats was to estimate the effect of extruding faba bean seeds and supplementing sulphur amino acids on performance, protein digestibility, pancreatic trypsin activity, morphometry of the jejunal mucosa, and total counts of bacteria colonizing mucosa.
Lectins were extracted from raw and extruded seeds according to Paredes-Lopez et al. (1989) and the agglutination test with human RBC (groups O, A, B, AB) was performed according to Liener (1989).
In the jejunum, the depth of crypts, length and width of villi, and thickness of the tunica mucosa were measured and total bacteria and E. coli counts were determined (except group FE) as described in a previous paper (Leontowicz et al., 2001 a).

Statistical analysis
The results were evaluated by ANOVA and differences between arithmetic means (x±SD) were evaluated by the Tukey multiple range test.

RESULTS
Trypsin inhibitor activity in faba bean seeds was reduced by extrusion from 1.8 to 0.5 TlU/mg (Table 1).The lectin extract obtained from raw seeds agglutinated human red blood cells without a clear-cut affinity for a specific blood group; the smallest activity was found against group B (Table 2).Extrusion eliminated hemagglutination activity.Digestibility, % dry matter 92.9 ± 1.5 b 89.7 ± 2.0 a 92.7 ± 0.9 b 92.8 ± 0.9 b crude protein 85.0 ±2.9 86.0± 1.1 85.2 ±2.1 85.5 ±2.2 ab,c means in the rows tagged with different letters differ significantly at PO.05 1 faba bean raw 2 faba bean extruded 3 faba bean row +amino acids 4 faba been extruded + amino acids The protein content in the diets amounted to: 12.12; 12.15; 12.77; 12.59% on a DM basis in FR, FE, FRAA and FEAA, respectively.Extrusion of faba bean seeds did not affect feed intake, body gain, or FER (Table 2).Sulphur AA supplementation of diets with raw and extruded seeds significantly improved performance.Dry matter (DM) digestibility was lower (P<0.05) in rats fed FE than FR (89.7 vs 92.9), AA supplementation significantly increased DM digestibility in FEAA.Crude protein digestibility was not affected by either extrusion or AA supplementation.
The proteolytic activity in the pancreas tended to be higher in rats fed extruded beans (Table 3).The protein content in the pancreas increased significantly in group FEAA in comparison with group FE.  ,3,as in Table 2 Extrusion of seeds and addition of AA to the diet with raw faba bean significantly increased crypt depth, suggesting an increase of proliferative processes in the mucosa epithelium.Villi length, width, as well as thickness of the tunica mucosa significantly increased due to extrusion (Table 4).576 ± 15 b a ' b,c means in the raws tagged with different letter differ significantly at PO.05 and SD ' • 2 ' 3 -4 as in Table 2 Total culturable bacterial counts decreased (P<0.05) in group FEAA in comparison with group FRAA, but there was no difference (P>0.05) in E. coli counts between groups (Table 5).4.16 ± l.ll a 1.23 ±0.60 0.296 * CFU -colony forming unit ab means in the rows tagged with different letter differ significantly at P<0.05 13,4 as in Table 2 DISCUSSION Extrusion of faba bean seeds at 160°C eliminated about 72% of trypsin inhibitor activity and total hemagglutination activity towards human RBC.Similar effects of extrusion (Kostyra et al., 1999) or boiling (Leontowicz et al., 1998) of faba bean and pea seeds have been observed.Soyabean seeds are less sensitive to extrusion at the same temperature than faba bean (Leontowicz et al., 2001a).Sulphur AA (0.3%o) significantly improved performance of rats fed both raw or extruded faba bean seeds, which is in agreement with other studies.The lack of a positive effect of extrusion of faba bean on digestibility parameters and body gain results from its lower ANF activity or possible negative effects of temperature on lysine and protein availability.Also AA supplementation did not increase pancreatic trypsin activity (P>0.05), which was reported by Friedman (1994).
Lower lectin activity in raw faba bean and lower E. coli number in the rat jejunum caused less significant morphological changes in the small intestine than in rats fed on raw soyabean (Leontowicz et al., 2001a) or pea (Leontowicz et al., 2000).Elimination of lectin activity during extrusion of faba bean seeds and AA supplementation significantly improved intestinal crypt depth, villi length and width.

CONCLUSIONS
Extrusion of faba bean seeds (160°C) completely eliminates lectin activity, but TI activity only partially.Rat performance is not affected by extrusion but is significantly improved by amino acid supplementation of both raw and extruded seeds.A synergistic effect of extrusion and amino acid supplementation is observed in respect to feed intake.Both extrusion and amino acid supplementation improve morphological parameters of the small intestine (jejunum).

TABLE 1
Trypsin inhibitors and haemagglutination titre of protein extract from faba bean seeds Haemagglutination titre of human red blood cells

TABLE 3
The influence of raw or extruded seeds and amino acids on pancreatic parameters in rats (n=T0)

TABLE 5
Microbiological parameters of the jejunum of rats (n=7)