The chemical composition and nutritive value of low-and high-tannin faba bean varieties

A comparison was carried out o f the chemical composition and nutri t ive value o f three genotypes o f faba beans: two wi th coloured flowers, Nadwiś l ańsk i and the self-ending Tinos varieties, and one wi th white flowers, Caspar. Caspar seeds were heavier and had a lower hul l p ropor t ion and lower crude protein content (27.2% D M ) . The largest differences were in the content o f proanthocyanidins (condensed tannins) which ranged f rom 0.02% (Caspar) to 1.11% D M (Tinos). The proanthocyanidin content in dehulled seeds o f coloured flower varieties was low (0.02%) and similar to that in the low-tannin variety, Caspar. The antitrypsic activity o f the seeds varied, ranging f rom 3.4 T I U / m g (Caspar) to 7.4 T I U / m g (Tinos). The inositol phosphate (1.05-1.15% D M ) and agalactoside (2.1-2.4% D M ) contents d id not differ significantly. The new varieties, Caspar and Tinos, contained almost twice as much vicine and convicine (0.75% and 0.92%, respectively) as the tradi t ional variety, Nadwiś l ańsk i (0.36% D M ) . True digestibility coefficients ( T D ) for seeds determined on rats differed significantly and were: for Caspar, 83.9; Tinos, 77.3 and Nadwiś l ańsk i , 80.0. Replacing whole seeds wi th dehulled seeds significantly increased the digestibility o f dietary protein, to 88.1, 88.3 and 84.9, respectively. The net protein uti l ization ( N P U ) o f the Nadwiś l ańsk i and Tinos seeds were similar (55.4 and 55.0, respectively) and significantly lower than the N P U o f Caspar seeds (61.0). The protein efficiency rat io (PER) o f whole seeds supplemented w i th methionine and tryptophan were similar (Caspar, 2.21; Nadwiś l ańsk i , 2.18; Tinos, 2.03) and insignificantly lower than the PER o f dehulled seeds (2.37, 2.32 and 2.25, respectively). K E Y W O R D S : faba bean, antinutrients, proanthocyanidins, nutri t ive value, rats, protein 402 FREJNAGEL S. ET AL.


INTRODUCTION
The high content of antinutritional factors in the seeds of coloured-flower faba bean varieties lowers nutrient digestibility and utilization in the digestive tract of monogastric animals (Longstaff and Mac Nab, 1991;Jansman et al., 1994).For this reason, interest in cultivating varieties with lower contents of these compounds, so-called "zero ANFs" is growing in many European countries.The first low-tannin variety of a white-flowering faba bean imported from Holland has been introduced in Poland.The cultivation of the low-tannin variety, as well as of new "self-ending" varieties, points to the need for fuller characterization of their feed value.
The objective of this study was to compare the chemical composition of seeds from a high yielding traditional variety and of white-flowering and "self--ending" varieties, and to determine to what degree the differences in the content of phenolic compounds affect the nutritive value of the protein in the seeds of the new varieties, as compared with the traditional variety, Nadwiślański.Moreo ver, the aim of the study was to examine the effect of dehulling the seeds on the differences in nutritive value as related to the differences in the level of phenolic substances in the seed hull.

Material
Seeds of the Nadwiślański, Tinos and Caspar varieties from the 1992-1994 harvests, purchased from Polish Plant Cultivation Stations and imported from Holland (Caspar 1993) were used in this study.The weight of the seeds, seed hull proportion and basic chemical composition were analysed during three years, while the remaining components were determined in samples from one year (1993).The feeding experiments were carried out using seeds from the 1993 harvest.

Physical characteristics of seeds
Average seed weight was measured by weighing 1000 randomly chosen seeds ten times.Specific density was determined by a conventional method by measuring the volume of water displaced by a weighed sample of 20 seeds.The percentage proportion of seed hull was determined by manual separation of cotyledons and hulls in a sample of seeds weighing about 100 g.To standardize the conditions and facilitate the separation of cotyledons and hulls, the samples were dried at 40°C

Analytical methods
The chemical composition, i.e. dry matter, ash, crude protein, crude fat, crude fibre and dietary fibre contents were determined by standard methods (AOAC, 1990).Neutral detergent fibre (NDF), acid detergent fibre (ADF) and lignin were determined according to Van Soest and Wine (1967).The cellulose content was calculated from the difference between the ADF and lignin contents, while hemicellulose was computed as the difference between the NDF and ADF contents.
Amino acid composition was assayed using an automatic Beckman Model 6300 analyzer after hydrolyzing samples in 6 N HC1.Sulphur-containing amino acids were determined after oxidizing the samples in formic acid, tryptophan was assayed after hydrolysis in barium hydroxide.Protein quality was characterized by the essential amino acid index according to Oser (EAAI), and the limiting amino acid score (CS) according to Block and Mitchell, using the composition of egg white as the standard.
Total phenolic compounds were determined according to Naczk and Shahidi (1989), based on the reaction of these compounds with the Folin-Ciocalteau reagent.The flavanol content was determined using the vanillin method (Price et al., 1978), and proanthocyanidins (condensed tannins) were assayed according to Oszmiahski et al. (1988) using the coloured reaction of cyanidins in butanol-HCl.Trypsin inhibitor activity (TIA) was determined by the method of Kakade et al. (1974).HPLC was used to assay the following components after the appropriate preparation of the samples: according to Sandberg and Ahderinne (1986) to determine inositol phosphates, according to Bjerg et al. (1985) to determine vicine and convicine, and according to Muzquiz et al. (1992) to determine sugars from the raffinose family (a-galactosides).

Nutritive value of protein of whole and dehulled faba beans
A control diet containing casein and diets containing whole or dehulled seeds of the faba bean varieties were used in the feeding experiments.The diet compositions are given in Table 1.The methionine and tryptophan deficiency in the faba bean diets were offset by the addition of synthetic amino acids (0.25% and 0.05%, respectively), according to Pastuszewska et al. (1985).The protein content of the diets was about 10%.
The experiments were carried out on 56 Wistar rats aged 28-30 days weighing about 65 g.Each experimental group comprised 4 males and 4 females.The rats were housed individually in organic glass cages in rooms with controlled lighting (12 h), temperature 22-23°C and relative humidity 70%.The experiment lasted 4 weeks and the results, i.e. feed consumption, body weight gains, were used to Composition of diets, % FREJNAGEL S. ET AL.    calculate the protein efficiency ratio (PER).True digestibility of protein (TD) was determined on the basis of a 5-day faeces collection in the last week of the experiment.The amount of metabolic nitrogen excreted in the faeces, i.e. 46.3 mg/rat/5 days, was taken from Rakowska et al. (1978).In evaluating net protein utilization (NPU), the animals were kept in groups in cages with two males and two females per cage.The nitrogen content in the bodies of rats was determined on the basis of their water content after ten days of the experiment.The significance of differences among groups was assessed using the Duncan test.

Weight and basic chemical composition of seeds
Tinos seeds were lighter while Caspar seeds were distinctly heavier than the seeds of Nadwiślański variety.The average weight of 1000 seeds (from 448 to 522 g) was lower than that of 22 varieties of faba beans (560+ 170 g) in the study by Wang and Uberschar (1990).These authors also found that the low-tannin varieties had higher average weights of 1000 seeds, even over 700 grams.The share of seed hull was inversely proportional to the weight of the seeds (from 12.8% in Caspar to 14.3% in Tinos) and was within the 13-15% limits given by other authors as characteristic of faba beans (Bjerg et al., 1988;Wang and Uberschar, 1990).According to Helsper et al. (1993) the proportion of faba bean hulls may range from 11.6 to 15.7%.
The crude protein content of the seeds was between 27.2 and 28.9% DM, which is slightly lower than that found in most European varieties (Bjerg et al., 1988).Caspar seeds had slightly less crude protein (27.2% DM).According to the data of Grosjean et al. (1995) the crude protein content in Caspar seeds was 26.5%, while in the seeds of another white-flowering variety, 34.7%.This may indicate that the Caspar variety stands out with its low protein content among the Dutch low-tannin varieties.The dietary fibre content of the beans varied proportionately to the proportion of the hull, and ranged from 19.7% (Caspar) to 21.05% DM (Tinos).The dietary fibre content corresponded to the maximum levels found in faba beans by Bjergegaard and Sorensen (1992).Dehulling increased the protein content from 29-30% to 32-33% in Nadwiślański and Tinos, and from 24 to 26% DM in Caspar seeds, and decreased the dietary fibre content from 21-22% to 13-14% DM, so that the ratio of protein to fibre increased from 1.4 g/g in whole seeds to 2.3 g/g in dehulled seeds (Table 3).

Amino acid composition of protein
The protein of the varieties under study had a similar amino acid composition and similar essential amino acid index (EAAI, 66.0-66.9)and limiting amino acid score(CS) (Table 4).The content of lysine, phenylalanine and tyrosine in protein was similar to the values given by other authors (Pastuszewska et al., 1993).All seeds had the low methionine and cystine contents that are characteristic of faba beans and are responsible for the relatively low CS that varied from 34.7 (Nadwiślański) to 38 (Caspar).The tryptophan content ranged from 0.91-1.01g/16g N and was intermediate between the values given by Jansman et al. (1993), and Smulikowska and Chibowska (1993).Dehulling reduced the content of some amino acids to a certain extent (including lysine, phenylalanine, tyrosine, and tryptophan), and as a result caused a slight decrease in the essential amino acid index (65.2-66.2).The results of Smulikowska and Chibowska (1993) also show that dehulling faba beans does not affect negatively the amino acid composition of protein.

Antinutritional factors content
The content of phenolic compounds (Table 5) in Caspar seeds (0.27% DM) was five to six times lower than in varieties with coloured flowers (Nadwiślański, 1.35%, Tinos, 1.69% DM).The proanthocyanin and flavanol contents were similar to the tannin content found by Wang and Uberschar (1990) and Pastuszewska et al. (1993).
The total phenolic compound content, as well as of flavanols, in Nadwiślański and Tinos seeds was similar to the values determined by the same methods by Ortiz et al. (1993).The proanthocyanidin content differed clearly among varieties and ranged from 0.85% (Nadwiślański) and 1.11% (Tinos) to 0.02% (Caspar).In comparison with data from the literature (Wang and Uberschar, 1990;Longstaff et al., 1993) the studied Polish coloured flowered varieties had a lower proanthocyanidin content than found in many European varieties.
Dehulling reduced the phenolic compound content to the levels characteristic for low-tannin varieties (0.02% DM).The total polyphenol and flavanol contents in dehulled seeds of all varieties were lower than found by Ortiz et al. (1993).
Whole Nadwiślański and Tinos seeds had two-fold higher antitrypsic activity, 6.3 and 7.4 TIU/mg, respectively, than Caspar seeds (3.4 TIU/mg).No major differences were found in antitrypsic activity among dehulled seeds (3.9-4.3TIU/mg).This shows that the antitrypsic activity of faba beans determined by the method of Kakade et al. (1974) resulted to a large extent from their content of phenolic compounds.A similar conclusion can also be drawn from the results of Helsper et al. (1993).
The vicine and convicine contents in faba beans (Table 5) were within the range given by Griffths and Ramsay (1992), and the two new varieties, Caspar and Tinos, had almost twice as much glycosides as the traditional Nadwiślański (0.75%, 0.92% and 0.36% DM, respectively).No major differences were found in the content or composition of phytates in the seeds of the compared varieties (Table 5).Total inositol phosphates in the seeds were close to 1% DM.The content of sugars from the raffinose family was similar in all varieties (2.05-2.36%DM) and was within the range given by Lattanzio et al. (1986), i.e. 1.4 to 2.62%.

Nutritive value of protein from whole and dehulled faba beans
Table 6 presents the results of the feeding studies in which whole and dehulled faba beans were used.A distinct, albeit not statistically proven, differences in feed consumption were found.The intake of diets containing Caspar seeds was the highest (355 ±31.2 g), that of the Tinos seed containing diets was the lowest (298 + 20.3 g).These results partially support the observations of other authors that feed consumption significantly decreases as the polyphenol content in the feed increases (Jansman, 1993).
A significant effect of faba bean variety was found on the protein digestibility of the diets.Similarly as in the studies of other authors (Pastuszewska, 1985;Jansman, 1993), true digestibility (TD) of protein in seeds with higher polyphenol contents was lower.Similar TD values for faba bean protein in rats were obtained by other authors in experiments on the effect of polyphenols on protein digestibility (Bjerg,1988;Pastuszewska, 1993).Dehulling caused a highly significant rise in TD by about 8 percentage units in the groups receiving diets containing Tinos and Nadwiślański seeds, and about 5 percentage units in the diet with Caspar seeds.
The NPU did not differ between the Nadwiślański and Tinos varieties (55.4 and 55.0, respectively), but was significantly higher in Caspar (61), which could have been the result of the higher sulphur amino acid content in the protein of this variety.Dehulling Caspar seeds significantly lowered the NPU from 61 to 56.1, i.e., proportionately to the differences in the CS of whole and dehulled seeds (38 and 35.5, respectively).No correlation was found between the polyphenol content in seeds and NPU, although such relationship was found in the study by Pastuszewska (1993).Alzueta et al. (1992) found a negative effect of phenols on NPU and TD but no effect on BV of diets containing tannin extracts from faba beans.No significant differences were found in PER of the compared varieties, both if the diet contained whole or dehulled seeds.PER was the highest in diets with whole or dehulled Caspar seeds (2.21 and 2.37, respectively), and the lowest with whole or dehulled Tinos seeds (2.03 and 2.25, respectively), which contained larger amounts of proanthocyanidins.A similar tendency, i.e. a higher nutritive value of diets containing faba bean varieties with lower phenolic contents, was also found in studies on pigs (Grala, 1993) and on poultry (Smulikowska and Chibowska, 1993;Ortiz et al., 1994).
Dehulling seeds caused a distinct, although statistically nonsignificant, increase in the PER (Table 6).The highest rise, about 10%, the diet containing whole seeds, was noted for the Tinos variety that has the highest polyphenol content.It can be presumed that the rise in PER was the result of both the increased energy content of the diet with dehulled seeds (with a lower dietary fibre content) as well as of the lower content oT polyphenols.

CONCLUSIONS
As compared with the Nadwiślański and Tinos varieties, the seeds of the white-flower variety, Caspar, were heavier and had a lower hull proportion, and a lower crude protein content.They contained small amount of polyphenols, had the lowest antitrypsic activity, and relatively high vicine and convicine contents.

TABLE 2
Weight, specific weigh, proportion of hull and chemical composition of seeds 1

TABLE 3
Chemical composition of whole and dehulled seeds, % DM

TABLE 5
Content of antinutritional factors in whole and dehulled seeds, % DM

TABLE 6
Results of nutritional experiments on rats