J. Sci. Food Agric.

1980, 31, 255-259

The Effect of Diets Containing Field Beans of High
or Low Polyphenolic Content on the Activity of
Digestive Enzymes in the Intestines of Rats
D. Wynne Griffiths and Gwyn Moseley
Welsh Plant Breeding Station, Aberystwyth, Wales
(Manuscript received 9 August 1979)
Saline extracts of the intestinal contents of 36 rats, which had been randomly allocated
into six dietary groups, were assayed for trypsin, a-amylase and lipase. The activities
of both trypsin and a-amylase were significantly reduced in rats consuming diets
containing testa from Dylan, a coloured flowered variety of field bean, whilst those
receiving diets containing testa from the tannin-free, white flowered variety Triple
White had comparable activities with those on a control testa-free diet. Polyvinyl-
pyrrolidone-saline extracts of the gut contents, however, had similar trypsin activity for
all diets and it was concluded that the original observed reduction in digestive enzyme
activity was due to the presence of field bean polyphenolics, which are known to be
present in the testa of all coloured flowered varieties. I n contrast, lipase activity was
increased in rats which received Dylan testa diets. It is suggested that the presence of
field bean tannins stimulates an increased pancreatic secretion of all digestive
enzymes; but in the gut, the affinity of tannin for lipase is less than for either dietary
protein or the other digestive enzymes.
1. Introduction
Feeding trials with both poultry1 and rats2 have shown that a considerable increase in nutritive
value resulted from the removal of the testa from field beans prior to their inclusion in animal diets.
Although it has been suggested that some of the increase is a consequence of increased metabolis-
able energy2 it has been clearly demonstrated that the presence of condensed tannins in the testa
contributes considerably to a decrease in the nutritive value of beans for poultry.3 These polyphen-
olic compounds have also been shown to inhibit in vitro the enzymic activity of fungal and rumen
microbial cellulase4 and also the digestive enzymes trypsin, a-amylase and lipase.5
A comparison of the effects of feeding the seed coats from a coloured flowered variety of bean
with those from a tannin-free white flowered variety revealed that the former produced not only a
significant reduction in dietary nitrogen digestibility but also a small but statistically significant
reduction in the apparent digestibility of available carbohydrates and lipids6 Since the role of field
bean polyphenolics in vitro may not be totally restricted to their effect on the availability of dietary
protein, the present work was designed to examine the effects of varietal variation in testa poly-
phenolic content on the activity of gut enzymes.
2. Experimental
2.1. Animals and diets
Thirty-six Sprague-Dawley weanling rats selected for uniformity of body weight and individually
housed in stainless steel metabolism cages, were randomly allocated to six treatment diets, the con-
stituents of which have been previously reported and are fully summarised in Table 1.
0022-5142/80/0300-0255 $02.00 0 1980 Society of Chemical Industry
255
256 D. W. Griffiths and G. Moseley
Table 1. Composition of the experimental diets (g kg-1)
Component Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6
. _ _ ~
- - Dylan cotyledons 300 300 300 -
300 300 300
Triple White cotyledons - - -
Dylan testa - 100 - - 100 -
100
Triple White testa - - 100 - -
Wheat straw 100 - - 100 -
The daily allocation of food (approximately 12 g day-l) was offered twice daily at 09.00 and
16.00 h over a 15 day experimental period. Following this, the rats were weighed, fed and killed
approximately 2 h later. The pancreas and intestines were removed and weighed, and the intestinal
contents collected and immediately stored at - 20C until required for analysis.
Separate extracts for the enzyme assays were prepared by macerating 500 mg of gut content
in 10 ml physiological saline (0.85 % NaCl solution) or a 5 % solution of polyvinylpyrrolidone
(PVP) in saline and centrifuging until clear. Aliquots of the supernatant were diluted with saline
to achieve an appropriate concentration for the enzyme assays.
2.2. Enzyme analysis
Trypsin (E.C. 3.4.4.4) activity was determined using the synthetic substrate N-benzoyl-DL-arginine-
p-nitro-aniline by the method of Erlanger, Kokowsky and Cohen7 in 0.1 M Tris buffer. cr-Amylase
(E.C. 3.2.1 . l ) activity was estimated by the colorimetric method of Bernfelds using soluble starch
as substrate. Lipase (E.C. 3.1.1.3) activity was determined by the method of Seligman and Nachlasg
using 2-naphthyl laurate as substrate. Lipase activity taken as the difference i n absorbance between
samples in the presence and absence of taurocholic acid.
All enzyme assays were conducted at 37C and in each case one unit of activity was defined as that
producing an increase of 10-2 in absorbance under the assay conditions used.
2.3. Chemical analysis
Nitrogen content was determined by the macro-Kjeldhal method and expressed as crude protein
using a conversion factor of 6.25. Lipid content was determined by gas-liquid chromatography
according to the method of Welchlo and total soluble carbohydrates using the anthrone method as
described by Thomas.ll Total phenolic content was determined by the Folin-Dennis method and
polyphenols by the vanillin-HCl method as reported by Burns.12 Ash content was taken as the
percentage of material remaining after heating overnight at 450C.
3. Results
3.1. Diets
All six diets contained similar levels of ash, lipid, soluble carbohydrate and crude protein (see
Table 2). However, diets 2 and 5, both of which contained testa from the coloured flowered variety
Table 2. Major constituents and phenolic content of experimental diets (g kg-1)
Diet 1 Diet 2 Diet 3 Diet 4
~
Diet 5 Diet 6
Testa type Nil Dylan
_____-
Crude protein 115 117
Total soluble carbohydrate 868 842
Lipid 42 44
Ash 21 21
Total phenolicsQ 7 14
Polyphenolicsb Trace 4
Triple White Nil
~
116 119
850 853
48 38
21 21
6 6
Trace Trace
~
Dylan
127
826
41
16
13
4
_____
~
Triple White
I 22
832
39
17
6
Trace
a Figures based on commercial tannic acid standards.
b Figures based on catechin standards.
Effect of bean diets on gut enzyme activity
257
Dylan, had approximately twice the phenolic content of the other four diets. An examination of
the tannin content of the various diets showed that diets 1, 3, 4 and 6 which included either straw or
testa from Triple White, contained negligible amounts of these polyphenolic compounds; the levels
present being too low to be determined accurately by the vanillin-HC1 method. In contrast, diets
2 and 5 contained significant amounts of tannin; accounting for approximately 0.4% of the
total diet.
3.2. Pancreas weights
Despite considerable variation in pancreas weights (see Table 3) no clear relationship emerged
between mean pancreas size and testa treatment. Similarly cotyledon type (i.e. Dylan or Triple
White) had little effect on mean pancreas size.
Table 3. Effect of the experimental diets on the mean pancreas weight (g kg-1 body wt) of rats
Dylan cotyledons Triple White cotyledons
. -
Constituent Diet Number Pancreas wt Diet number Pancreas wt
Wheat straw 1
Dylan testa 2
Triple White testa 3
S.e.d.
3.6 4 2.9
3.5 5 2.9
2.9 6 3.1
k0.78 k0.78
3.3. Enzyme activity
The mean lipase, trypsin and a-amylase activity g-I of intestinal content of the rats fed in the six
experimental diets are given in Table 4. These results show that the activity of trypsin was signi-
ficantly depressed in rats fed the Dylan testa based diets as compared with those fed Triple White
testa or straw based diets. Similarly cc-amylase activity was also significantly less in rats receiving
the Dylan testa based diets as compared with the Triple White testa based diets; the values for
which were slightly higher than those for the straw based diets.
Table 4. Effect of experimental diets on the digestive enzyme activities (as defined in section 2.2) in the intestines
of rats
Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6
Testa type Nil Dylan Triple White Nil Dylan Triple White S.e.d.
Trypsin 19.7 12.3 18.6 17.0 12.5 17.2 k2. 61
a- Amylase 12.1 4.1 14.0 10.5 4.3 16.6 22.16
Lipase 4.7 18.7 4 . 8 7.5 18.6 8 . 8 52.35
- -~
I n complete contrast, lipase activity was significantly enhanced in the animals fed diets 2 and 5
(i.e. the Dylan testa based diets) compared with those receiving either the straw (diets 1 and 4) or
the Triple White testa based diets (3 and 6).
A statistical examination of the results showed that cotyledon type had no significant effect on
the activities of any one of the three enzymes examined.
The results of determining the trypsin activity of the PVP-saline extracts of the intestinal con-
tents (see Table 5) revealed that the presence of the tannin complexing agent increased the trypsin
activity of the extracts from rats receiving diets 2 and 5. Statistical analysis showed that within
each cotyledonary type no significant differences were to be seen between the three testa treat-
ments (i.e. Dylan, Triple White or straw).
18
258 D. W. Griffiths and G. Moseley
Table 5. Trypsin activity (as defined in section 2.2) of PVP-saline extracts of rat intestinal contents
Dylan cotyledons Triple White cotyledons
~ - -.
Dietary constituents Diet Number Trypsin activity Diet Number Trypsin activity
.. ~- - _____.
Wheat straw 1
Dylan testa 2
Triple White testa 3
S.e.d.
17.4
17.2
17.7
f 2. 9
4
5
6
20.1
20.3
23.9
22.9
Statistically significant relationships were found between the total phenolic intake of the individual
rats and the corresponding intestinal enzyme activity (see Table 6). Negative correlations were
found for both trypsin and a-amylase activity but as expected lipase was positively correlated with
intake values.
Table 6. Correlation coefficients for digestive enzyme
activity with total phenolic intake on an individual rat basis
~ _ _ _ _ _ _
Enzyme n r-Value Significance
Trypsin 36 -0.546 ***
a-Amylase 36 -0.639 ***
Lipase 36 0.787
***
***P<0.001.
4. Discussion
I t has been demonstrated previously4, 13, l4 that the testa of field beans from white flowered plants
have a low phenolic content and are completely devoid of tannins, whilst testa from coloured
flowered varieties may have a varying but substantial phenolic content, composed partly of con-
densed tannins. Analysis of the diets in this experiment confirms these results and the adverse effects
of the diets containing testa from the coloured flowered variety on growth rates, nitrogen digest-
ibility and apparent digestibility of other nutrients have been reported previously.6
It is generally accepted that ingested tannins may reduce the nutritive value of feed by forming
indigestible complexes with dietary protein. However, it has also been shown that field bean testa
extracts5 and carob bean tannins15 will inhibit the activity of digestive enzymes in vitra. The results
shown in Table 4 clearly demonstrate that both trypsin and a-amylase activity were significantly
reduced in the intestines of rats fed field bean diets containing the higher levels of polyphenolics,
indicating that enzyme inhibition may also play a part in reducing nutritive value in viva.
I t is possible that inhibition of proteolytic enzymes in the gut of rats fed field bean diets may be
due either to non-specific complexing with polyphenolic compounds or to specific inhibition by the
proteinic protease inhibitor as described by Warsy et a1.16 However, it has been shown that the
levels of protease inhibitor present in field beans is too low to account for the observed reduction
in nutritive value17 and comparatively little difference in protease inhibitor activity has been found
between coloured and white flowered varieties5 Furthermore, the results in Table 5 show that,
whereas the saline extracted gut contents from dietary groups 2 and 5 showed a significant decrease
in trypsin activity, there was a complete restoration of trypsin activity when the same gut samples
were extracted in the presence of PVP. This indicates that the decrease in trypsin activity was due
to the formation of a reversible tannin-enzyme complex produced by a reaction between the poly-
phenolics present in the testa of the coloured flowered variety Dylan and the proteolytic enzyme
trypsin. Although earlier in-vitro studies5 had shown that both the field bean tannin-trypsin and
Effect of bean diets on gut enzyme activity 259
tannin-cr-amylase complexes were re-activated in the presence of PVP, attempts to demonstrate a
similar effect for the in-vivo produced a-amylase-tannin complex proved impossible as amylase
activity appeared to be completely destroyed when the intestinal contents were extracted with the
PVP-saline solution.
In contrast to the reduction in trypsin and cr-amylase activity observed in the intestinal contents
of rats consuming diets containing Dylan testa, there was a significant increase in intestinal lipase
activity. This is not readily explained but appears to be analogous to that observed by Gertler and
Nitsanl* in the intestines of chicks fed raw soya bean meal. They found that whereas trypsin and
chymotrypsin activities were reduced due to the presence of active protease inhibitors, pancreato-
peptidase E activity in the intestines was enhanced. From these results they conclude that the pre-
sence of the inhibitor stimulates an increased production of all pancreatic enzymes but the effect
of the inhibitor is considerably greater on trypsin and chymotrypsin compared with pancreato-
peptidase E and, consequently, results in increased levels of activity of this enzyme. I t may be,
therefore, that the presence of field bean polyphenolics in the rat intestines stimulates a similar
increase in production of all digestive enzymes but under gut conditions the affinity of tannins for
lipase is less than that for other enzymes or dietary proteins.
In conclusion, it would appear that the lower nutritive value of diets containing field bean poly-
phenolics was due to a reduction in both dietary protein availability and digestive enzyme activity.
The development, therefore of new varieties of field beans low in tannin content would appear to be
nutritionally beneficial, although the consequences of eliminating seed coat tannins on the suscepti-
bility of the plants to pathogenic attack and other factors should also be considered.
References
1. Marquardt, R. R.; Campbell, L. D. Raw and autoclaved faha beans (Vicia faba L. var. minor) in chick diets.
J. Sci. Food Agric. 1974, 25, 741-746.
2. Nitsan, Z. Viciu faba vs soyabean meal as a source of protein. J. Sci. Food Agric. 1971, 22, 252-255.
3. Martin-Tanguy, J .; Guillaume, J .; Kossa, A. Condensed tannins in horse bean seeds. Chemical structure and
apparent effect on poultry. J. Sci. Food Agric. 1977, 28, 757-765.
4. Griffiths, D. W.; J ones, D. I. H. Cellulase inhibition by tannins in the testa of field beans (Vicia faba). J. Sci.
Food Agric. 1977, 28, 983-989.
5. Griffiths, D. W. The inhibition of digestive enzymes by extracts of field beans (Vicia fabu). J. Sci. Food Agric.
1979,30,458462.
6. Moseley, G.; Griffiths, D. W. Varietal variation in the anti-nutritive effects of field beans (Vicia faba) when
fed to rats. J. Sci. Food Agric. 1979, 30, 772-778.
7. Erlanger, B. F.; Kokowsky, N.; Cohen, W. The preparation and properties of two new chromogenic suh-
strates for trypsin. Arch. Biochem. Biophys. 1961, 95, 271-278.
8. Bernfeld, P. Amylase, OL and p. In Methods of Enzymology (Colwick S. B.; Kaplan, N. O., Eds) Academic
Press, New York, 1955, pp. 149-150.
9. Seligman, A. M.; Nachlas, M. M. Lipase. In Methods of Enzymatic Analysis (Bergmeyer, H. U., Ed.) Academic
Press, New York, 1963, pp. 776-778.
10. Welch, R. W. A micromethod for the estimation of oil content and composition in seed crops. J. Sci. Food
Agric. 1977, 29, 635-638.
11. Thomas, T. A. An automated procedure for the determination of soluble carbohydrates in herbage. J. Sci.
Food Agric. 1977, 29, 639-642.
12. Burns, R. E. Methods of tannin analysis for forage crop evaluation. Georgia Expt Sf a. Tech. Bull. 1963 NS, 32.
13. Bond, D. A. In vitro digestibility of the testa in tannin-free field beans (Vicia faba L.). J. agric. Sci. Camb.
14. Rowlands, D. G.; Corner, J . J . Genetics of pigmentation in broad beans (Vicia fuba L.). Eucurpiu 1962,
3,229-234.
15. Tamir, M.; Alumot, E. Inhibition of digestive enzymes by condensed tannins from green and ripe carobs.
J. Sci. Food. Agric. 1969, 20, 199-202.
16. Warsy, A. S. ; Norton G.; Stein, M. Protease inhibitors from broad bean, isolation and purification. Phyto-
chemistry 1974, 13,2481-2486
17. Abbey, B. W.; Neale, R. J . ; Norton, G. Nutritional effects of field bean (Vicia faba L.) proteinase inhibitors
fed to rats. Br. J. Nutr. 1979, 41, 31-38.
18. Gertler, A.; Nitsan, 2. The effect of trypsin inhibitors on pancreatopeptidase E, trypsin, chymotrypsin and
amylase in the pancreas and intestinal tract of chicks receiving raw and heated soya-hean diets. Br. J. Nufr.
1976, 86, 561-566.
1970,24,893-904.