Plant Proteins - American Chemical Society

11 Protein-Procyanidin Interaction and Nutritional Quality of Dry Beans 1

W. E. Artz , B. G. Swanson, B. J. Sendzicki, A. Rasyid, and R. E. W. Birch

Downloaded by MONASH UNIV on December 8, 2014 | http://pubs.acs.org Publication Date: June 18, 1986 | doi: 10.1021/bk-1986-0312.ch011

Food Science and Human Nutrition, Washington State University, Pullman, WA 99164-6330

Thermodynamic analysis of the temperature dependance of procyanidin binding to bovine serum albumin (BSA) and bean glycoprotein G-1 suggested predominantly hydrophobic and hydrophilic binding, respectively. A cis-parinaric acid fluorescence assay for surface hydrophobicity supported amphiphilic interactions of procyanidin. Heat denatured G-1 had a surface hydrophobicity greater than native G-1. Procyanidin dimer and trimer inhibited trypsin digestion of BSA. In vitro digestibility and Tetrahymena-Protein Efficiency Ratio (t-PER) were inversely related to procyanidin concentration. Procyanidin intubation restricts rat growth and damages intestinal villi. Procyanidins intubated with food or as dry beans were not as inhibitory as procyanidins intubated alone. Digestibility and PER of tempeh prepared with red beans and corn were less than the digestibility and PER of soybean tempeh. Tempeh, Rhizopus oligosporus, fermentation did not improve digestibility or nutritional quality of dry black beans.

The common dry bean, Phaseolus v u l g a r i s , is a grain legume consumed in large quantities around the world. Black and other colored beans provide appreciable protein, vitamins, minerals and calories f o r r u r a l and urban populations of developing countries. The n u t r i t i o n a l importance of beans is great since access to protein of animal o r i g i n is limited. Legumes and cereals, which contain complementary proteins, provide protein of greater quality than consumption of legumes or cereals alone. However, consumption of beans and cereals in a favorable n u t r i t i o n a l quality r a t i o and amount tends to be infrequent in developing countries. World production of legumes appears to be declining compared to production and greater y i e l d s of cereals. Legume production, however, is s t i l l encouraged i n t e r n a t i o n a l l y to f i x atmospheric nitrogen and contribute to increased s o i l f e r t i l i t y in developing countries. Dry 1

Current address: Food Science, University of Illinois, Urbana, IL 61801. 0097-6156/86/0312-0126506.00/0 © 1986 American Chemical Society

In Plant Proteins: Applications, Biological Effects, and Chemistry; Ory, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

11. ARTZ ET AL.

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127

beans are also an excellent source of complex carbohydrates, f i b r e and polyunsaturated fatty acids. However, dry beans have several undesirable attributes such as enzyme i n h i b i t o r s , phytates, flatus factors, l e c t i n s , allergens and condensed tannins that constrain n u t r i t i o n a l quality unless destroyed or removed. This paper presents research data that delineate the relationship of dry bean proteins to dry bean procyanidins, and discusses the constraints protein-procyanidin interaction places on n u t r i t i o n a l quality of dry beans.


Proteins in Legumes Proteins present in the seeds of legumes are primarily of two types: 1) enzymatic and s t r u c t u r a l metabolic proteins responsible for normal c e l l u l a r a c t i v i t i e s including the synthesis of s t r u c t u r a l proteins, and 2) storage proteins. The storage proteins and reserves of carbohydrates and o i l s are synthesized during seed development (1). Storage proteins occur within the c e l l in discreet protein bodies (Figure 1) that develop late during maturation of bean seeds (2). The quantity of protein in dry beans ranges from 18 to 25% (180 - 250 g/kg) dry weight. Protein fractionation studies of Phaseolus vulgaris L . have generated three major soluble protein fractions: phaseolin ( G l ) , globulin (G2) and albumin (3-4). Considerable confusion surrounds the nomenclature of seed proteins of common beans. Phaseolin is reportedly the preferred t r i v i a l designation for the globulin-1 ( G l ) , glycoprotein II or v i c i l i n , a 6.9S protein which aggregates to form the 18S tetramer at pH 4.5 05, Ο . Globulin-1 ( G l ) , globulin-2 (G2) and albumin represent 36-46%, 5-12% and 12-16% of the t o t a l seed p r o t e i n , respectively, although there appeared to be some contamination between the l a t t e r two fractions after usual i s o l a t i o n procedures (4). Environmental factors such as geographic location and growing season substantially influence protein content of dry beans (6). Polypeptide c l a s s i f i c a t i o n of Gl f r a c t i o n has been well documented and permits c l a s s i f i c a t i o n of bean c u l t i v a r s into three groups: Tendergreen, Sanilac and Contender, on the basis of electrophoresis banding patterns (7). The major storage protein in beans, globulin-1 ( G l ) , exhibits a pH dependent polymerization that was u t i l i z e d in p u r i f i c a t i o n (8). At pH 3.8 to 5.4, Gl exists as a tetramer, while at pH 6.4 to 10.5, Gl exists as a monomer of MW 163,000 (9). The i s o e l e c t r i c point of Gl is pH 4.4 - 5.6. Gl s o l u b i l i t y is independent of pH from pH 2.5 to 12.0 (10) in O.5F NaCl. A crude extract of Gl was prepared for p u r i f i c a t i o n on cyanogen bromide activated Sepharose. Tannins/Procyanidins Tannins are one of several a n t i n u t r i t i o n a l factors present in dry beans. Any polyphenolic compound that precipitates proteins from an aqueous solution can be regarded as a tannin (11). Tannins precipitate proteins due to functional groups that complex strongly with two or more protein molecules, building up a large cross-linked protein-tannin complex (12). Naturally-occurring food legume tannins are reported to interact with enzyme and non-enzyme proteins to form complexes that


128

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r e s u l t in i n a c t i v a t i o n o f d i g e s t i v e enzymes and p r o t e i n i n s o l u b i l i t y ( 1 3 ) . J j i v i t r o and in v i v o s t u d i e s i n d i c a t e t h a t bean t a n n i n s d e c r e a s e p r o t e i n d i g e s t i b i l i t y and p r o t e i n q u a l i t y ( 1 4 ) . Condensed t a n n i n s and p r o c y a n i d i n s a r e terms used t o d e s c r i b e the same g e n e r a l c l a s s of compounds, p l a n t p h e n o l i c s t h a t a r e polymers o f the f l a v a n - 3 - o l s ( F i g u r e 2 ) , (+) c a t e c h i n and/or (-) e p i c a t e c h i n (15). P r o c y a n i d i n s heated in a l c o h o l and a c i d w i l l produce c o l o r e d compounds s t r u c t u r a l l y r e l a t e d t o a n t h o c y a n i d i n s (16). P r o c y a n i d i n polymers c o n s i s t o f c h a i n s of 5, 7, 3 , 4 t e t r a h y d r o x y f l a v a n - 3 - o l c o n n e c t e d by C ( 4 ) - C ( 6 ) o r C ( 4 ) - C ( 8 ) bonds (15). P r o c y a n i d i n s o c c u r f r e e and not as g l y c o s i d e s ( 1 7 ) . An a d d i t i o n a l hydroxy group can sometimes be found on the Β r i n g o f the f l a v a n - 3 - o l a t the 5 p o s i t i o n . Some hydroxy groups on the Β r i n g may be m e t h o x y l a t e d (18) and the methoxyl groups may a f f e c t protein-procyanidin interaction. P r o c y a n i d i n c o n c e n t r a t i o n s range from 1.5 t o 18.6 mg o f p r o c y a n i d i n per gram o f whole bean f l o u r ( 4 ) . P r o c y a n i d i n s a r e found in g r e a t e r c o n c e n t r a t i o n s in c o l o r e d beans t h a n in w h i t e beans, most o f which a r e l o c a t e d in the seed c o a t , t e s t a or h u l l . f

?


f

Methods D i m e r i c and t r i m e r i c c a t e c h i n were p r e p a r e d by r e d u c t i o n o f d i h y d r o q u e r c e t i n w i t h sodium b o r o h y d r i d e in the p r e s e n c e o f c a t e c h i n (19). P o l y m e r i z a t i o n was f o l l o w e d on s i l i c a g e l TLC u s i n g an a c e t o n e : t o l u e n e : f o r m i c a c i d (60:30:10) s o l v e n t ( 2 0 ) . The v i s u a l i z a t i o n agent was v a n i l l i n (1 g/100 ml) in 70% v / v s u l f u r i c acid/water. C a t e c h i n , c a t e c h i n dimer and c a t e c h i n t r i m e r appeared as red s p o t s w i t h r e s p e c t i v e Rf v a l u e s of O.66, O.54 and O.43. The f l a v a n - 3 , 4 - d i o l appeared as a p u r p l e spot w i t h an Rf v a l u e o f O.63. S e p a r a t i o n o f d i m e r i c and t r i m e r i c c a t e c h i n was a c c o m p l i s h e d w i t h Sephadex LH-20 u s i n g an e t h a n o l : w a t e r (45:55) s o l v e n t . P u r i t y o f the p r o c y a n i d i n was e v a l u a t e d w i t h r e v e r s e d - p h a s e HPLC i s o c r a t i c a l l y w i t h 4% a c e t i c a c i d in w a t e r . T r i t i u m - l a b e l l e d p r o c y a n i d i n dimer and t r i m e r were s y n t h e s i z e d s i m i l a r l y w i t h 25 mCi t r i t i a t e d sodium b o r o h y d r i d e added to the r e a c t i o n m i x t u r e o v e r a 20 min p e r i o d under nitrogen. B i n d i n g constants of l i g a n d , t r i t i u m l a b e l l e d c a t e c h i n dimer and t r i m e r , t o d e f a t t e d b o v i n e serum a l b u m i n (BSA) and p u r i f i e d bean p r o t e i n G l were d e t e r m i n e d by the method d e v e l o p e d by S o p h i a n o p o u l o s e t a l . (23) w i t h an Amicon M i c r o p a r t i t i o n System MPS-1 (American Corp., Danvers, MA). S e p a r a t i o n o f the f r e e l i g a n d from the bound l i g a n d was a c c o m p l i s h e d by c o n v e c t i v e f i l t r a t i o n o f f r e e l i g a n d t h r o u g h an a n i s o t r o p i c , h y d r o p h i l i c YMT u l t r a f i l t r a t i o n membrane. The d r i v i n g f o r c e was p r o v i d e d by c e n t r i f u g a t i o n . P r o t e i n s were q u a n t i t a t i v e l y r e t a i n e d above the membrane w h i l e low m o l e c u l a r weight l i g a n d p a s s e d t h r o u g h the membrane. Binding c o n s t a n t s were d e t e r m i n e d by S c a t c h a r d p l o t a n a l y s i s (24-26). L i n e a r r e g r e s s i o n a n a l y s i s was used t o f i t the p o i n t s f o r the Scatchard p l o t . Procyanidin Binding

t o Bovine Serum Albumin

(BSA)

P o l y m e r i c p r o c y a n i d i n e x t r a c t i o n from b l a c k beans ( P h a s e o l u s v u l g a r i s L. cv. B l a c k T u r t l e Soup) and p u r i f i c a t i o n was a



ARTZ ET AL.


F i g u r e 1. S c a n n i n g e l e c t r o n m i c r o g r a p h o f P h a s e o l u s v u l g a r i s c o t y l e d o n showing p r o t e i n b o d i e s (P) and s t a r c h g r a n u l e s ( S ) . Bar = 10 Urn.

PROCYANIDIN B2 F i g u r e 2. dimer B2.

Structure

of epicatechin, catechin

and p r o c y a n i d i n



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modification of the procedure of Strumeyer and Malin (21) and the procedure of Cansfield et a l . (22) using 80:20 r a t i o of acetone:water as primary extracting solvent, and LH-20 for clean-up. The method of Kato and Nakai (27) for determining protein surface hydrophobicity was adapted for evaluating procyanidin binding to BSA and G l . The procedure is based on the fact that the fluorescence quantum y i e l d of c i s - p a r i n a r i c acid increases 40-fold when c i s - p a r i n a r i c acid enters a hydrophobic environment from a hydrophilic environment. The digestion of BSA by trypsin in the presence of procyanidin dimer, procyanidin trimer and black bean procyanidin polymer was evaluated by discontinuous sodium dodecyl sulfate (SDS) slab gel electrophoresis and a p i c r y l sulfonic acid (TNBS) assay (28). Scatchard plots were used to determine the binding constants of procyanidins to BSA and bean globulin Gl at temperatures of 19, 29 and 39°C (Figures 3 and 4). Nu (v) is moles of ligand bound per mole of protein. L is the concentration of the free ligand. The equilibrium binding constant is equal to the negative slope of the corrected curve as determined by linear regression analysis from the Scatchard p l o t . Nonspecific binding is the binding of ligand to protein s i t e s possessing low a f f i n i t y (24). High a f f i n i t y binding must be corrected for nonspecific binding (25). Nonspecific binding was determined as the y-axis intercept of the extension of the lower a f f i n i t y binding curve. The lower a f f i n i t y nonspecific binding was subtracted from binding possessing the high a f f i n i t y to produce the corrected binding curve. The negative slope of the curve is equal to the equilibrium association binding constant and the x-axis intercept is equal to the moles of ligand bound per mole of protein. Thermodynamic analysis of the binding constants of BSA and procyanidin dimer and trimer from the Van't Hoff equation (29) indicates a reaction with a positive entropy change, a positive

Table I.

Binding Constants Gl

Temperature

Trimer (k) 74,000 42,000 27,000

19 29 39

Table I I .

Procyanidin Trimer Dimer Trimer

Enthalpy, entropy and free energy

Protein Gl BSA BSA

BSA Procyanidin Trimer Dimer (k) (k) 110,000 4,000 120,000 8,200 122,000 20,490

Enthalpy (H) (kcal/mole) - 9.26 14.9 O.96

Entropy (S) (eu) - 9.41 67.5 26.3

Free Energy (G) (kcal/mole) - 6.51 - 4.81 - 6.73


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3000,

2500h+_ 2000


V/L

1500 SCATCHARO PLOT

1000 Κ - 4000

COR. SCATCHARO PLOT

500

Figure 3,

Scatchard plot of BSA and procyanidin dimer at 19C.

25000r20000 - ,

1500oL

+

+ 4

V/L

Κ - 110000 + SCATCHARO PLOT COR. SCATCHARO PLOT .5

Figure 4.

1.5

Scatchard plot of BSA and procyanidin trimer at 19C.


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PLANT PROTEINS


e n t h a l p y change and a n e g a t i v e f r e e e n e r g y , i . e . a spontaneous r e a c t i o n t h a t is t o t a l l y e n t r o p y d r i v e n ( T a b l e s I and I I ) . Since h y d r o p h o b i c i n t e r a c t i o n s a r e e n t r o p y d r i v e n , t h e b i n d i n g o f BSA t o p r o c y a n i d i n is h y d r o p h o b i c . In aqueous s o l u t i o n s , h y d r o p h o b i c i n t e r a c t i o n is v e r y i m p o r t a n t (30). Water m o l e c u l e s a t t h e s u r f a c e o f t h e h y d r o p h o b i c domain c r e a t e d by a n o n p o l a r s o l u t e r e a r r a n g e in o r d e r t o r e g e n e r a t e b r o k e n hydrogen bonds, b u t in d o i n g so c r e a t e a g r e a t e r degree o f l o c a l o r d e r t h a n e x i s t s in pure l i q u i d w a t e r , t h e r e b y p r o d u c i n g a d e c r e a s e in e n t r o p y (30). The d r i v i n g f o r c e f o r h y d r o p h o b i c i n t e r a c t i o n is the i n c r e a s e in e n t r o p y when t h e o r d e r e d w a t e r is r e l e a s e d t o t h e bulk water. Hydrophobic i n t e r a c t i o n s a r e entropy d r i v e n . C e r t a i n t y p e s o f n o n - c o v a l e n t i n t e r a c t i o n s such as hydrogen bonds, London i n t e r a c t i o n s and v a n d e r Waals i n t e r a c t i o n s a r e e n t h a l p y d r i v e n i n t e r a c t i o n s ( 2 6 ) ; heat is r e l e a s e d d u r i n g bond formation. The heat r e l e a s e d d u r i n g bond f o r m a t i o n s t a b i l i z e s t h e bonds. Hydrogen bonds, London i n t e r a c t i o n s and van d e r Waals i n t e r a c t i o n s a r e v a r i a n t s on t h e d i p o l e - d i p o l e i n t e r a c t i o n model, which i n c l u d e permanent and i n d u c e d d i p o l e s . T r i m e r i c p r o c y a n i d i n b i n d s more t i g h t l y t o BSA t h a n d i m e r i c p r o c y a n i d i n ( T a b l e I I ) . P a r t i t i o n c o e f f i c i e n t s o f d i m e r i c and t r i m e r i c c a t e c h i n between n - o c t a n o l and w a t e r i n d i c a t e p r o c y a n i d i n t r i m e r is more h y d r o p h o b i c t h a n p r o c y a n i d i n dimer. Increased b i n d i n g c o n s t a n t s o f t r i m e r r e l a t i v e t o dimer agree w i t h r e p o r t e d partition coefficients. Surface h y d r o p h o b i c i t y assays with c i s - p a r i n a r i c a c i d c o n f i r m t h e thermodynamic a n a l y s i s t h a t b i n d i n g of p r o c y a n i d i n t o BSA is h y d r o p h o b i c . Procyanidin Binding

t o Bean G l o b u l i n ( G l )

B i n d i n g o f p r o c y a n i d i n t r i m e r t o bean p r o t e i n G l was temperature dependent ( F i g u r e 5 ) . An i n c r e a s e in t e m p e r a t u r e r e s u l t e d in a l a r g e d e c r e a s e in t h e b i n d i n g c o n s t a n t ( T a b l e I ) . G l b i n d i n g t o p r o c y a n i d i n t r i m e r is spontaneous and h y d r o p h i l i c in n a t u r e . The b i n d i n g is d r i v e n by t h e l a r g e change in e n t h a l p y ( T a b l e I I ) . The type o f b o n d i n g i n v o l v e d between G l , a g l y c o p r o t e i n , and p r o c y a n i d i n is p r o b a b l y hydrogen b o n d i n g . E v a l u a t i o n of the G l i n t e r a c t i o n with p r o c y a n i d i n trimer with c i s - p a r i n a r i c a c i d confirmed that the b i n d i n g o f n a t i v e G l t o p r o c y a n i d i n t r i m e r is h y d r o p h i l i c ( F i g u r e 6). Heat-denatured Gl e x h i b i t e d a s u r f a c e h y d r o p h o b i c i t y g r e a t e r than t h a t o f n a t i v e G l . The i n c r e a s e was n o t unexpected s i n c e h y d r o p h o b i c groups a r e commonly o r i e n t e d towards t h e c e n t e r o f p r o t e i n s in aqueous s o l v e n t s . Heat d e n a t u r a t i o n o f p r o t e i n exposes h y d r o p h o b i c groups t o t h e s o l v e n t . B i n d i n g o f d e n a t u r e d G l t o bean p r o c y a n i d i n o l i g o m e r was p r e d o m i n a n t l y h y d r o p h o b i c . Common bean p r o c y a n i d i n s a r e c a p a b l e o f both h y d r o p h i l i c and hydrophobic i n t e r a c t i o n with p r o t e i n . H y d r o p h i l i c i n t e r a c t i o n s are f a v o r e d w i t h a h y d r o p h i l i c g l y c o p r o t e i n l i k e common bean g l o b u l i n G l , while hydrophobic i n t e r a c t i o n s are favored a f t e r p r o t e i n d e n a t u r a t i o n , when p r o t e i n h y d r o p h o b i c groups a r e exposed t o t h e solvent.



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1

2

3

4

V F i g u r e 5. S c a t c h a r d G l a t 19C.

p l o t o f p r o c y a n i d i n t r i m e r and bean

F i g u r e 6. F l u o r e s c e n c e cis-parinaric acid.

globulin

o f G l (O.04%), p r o c y a n i d i n dimer and


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Trypsin Inhibition

Table I I I .

Percent Inhibition of the Trypsin Digestion of BSA


Procyanidin Dimer Trimer Polymer

1 mg/ml 16.9 42.3 50.5

5 mg/ml 25.2 71.2 89.4

Trypsin digestion of BSA was inhibited by addition of procyanidin dimer, trimer and oligomer (Table I I I ) . Increased procyanidin concentration increased i n h i b i t i o n of the trypsin digestion of BSA. Increased procyanidin chain length also increased i n h i b i t i o n of trypsin digestion. Protease i n h i b i t i o n by procyanidin does not occur by i r r e v e r s i b l e binding of procyanidin to the active s i t e of the protease. Procyanidin is not a s p e c i f i c i n h i b i t o r for either trypsin or chymotrypsin, i . e . procyanidin does not i n h i b i t by binding i r r e v e r s i b l y to the active s i t e , rather procyanidin binds non-specifically to the enzyme and/or protein substrate. Since procyanidin does not bind s p e c i f i c a l l y to protease active s i t e s , but reacts n o n - s p e c i f i c a l l y , Scatchard plots indicate less than one mole of procyanidin is bound per mole of protein. With polymeric procyanidin, considerable c r o s s - l i n k i n g w i l l occur. Not a l l a n t i - n u t r i t i o n a l effects can be explained by high a f f i n i t y binding. Feeding Procyanidins Complete removal of procyanidin is not necessary to overcome anti-nutritional effects. Removal of most procyanidin or addition of s u f f i c i e n t protein w i l l overcome a n t i - n u t r i t i o n a l effects of procyanidins. Small concentrations of procyanidin can be e a s i l y overcome by adding protein. The most apparent n u t r i t i o n a l effect of feeding procyanidins at naturally occurring concentrations in plants, such as in sorghum grain (1-2%), are growth depression, poor feed efficiency ratios and increased f e c a l nitrogen (12). Protein Efficiency Ratio is a procedure to measure the r a t i o of weight gain to protein intake of weanling rats fed a diet with a single suboptimal 10% concentration of test protein. Tetrahymena-PER is a more rapid assay, using protozoa Tetrahymena pyriformis or Tetrahymena thermophila, as an alternative to the laboratory rat as a b i o l o g i c a l assay for protein quality. Good correlation between PER determined with the rat and Tetrahymena have been reported (14). In v i t r o d i g e s t i b i l i t y and Tetrahymena-PER are inversely related to procyanidin content (14). B i o a v a i l a b i l i t y , expressed as Tetrahymena growth, of bean globulin Gl in the presence of black bean procyanidins correlated well with in v i t r o d i g e s t i b i l i t y of the protein. Health consequences of procyanidins in the human diet are r e l a t i v e l y unknown, but the t o x i c i t y for human beings may be similar to the t o x i c i t y observed in experimental animals (12). Bender and Mohammidiha (31) proposed that increased fecal nitrogen from rats fed diets containing large quantities of cooked legumes was due to increased gastrointestinal mucosal c e l l turnover, rather than poor



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protein d i g e s t i b i l i t y . Fairweather-Tait et a l . (32) discovered that mucosal c e l l sloughing increased 35% in the small intestine of rats fed beans compared to rats fed a control d i e t . Physiological alterations such as damage to the mucosal l i n i n g of the gastrointestinal tract and increased cation excretion have also been demonstrated (33-35). Very great concentrations of dietary procyanidin, near 5%, can cause death (12). Increased fecal nitrogen or decreased nitrogen retention by animals fed procyanidin has been explained by either a reduced d i g e s t i b i l i t y of dietary protein or an increased excretion of endogenous protein (33). Explanations for the a n t i n u t r i t i o n a l aspects of procyanidins have centered around the a b i l i t y of procyanidin to bind protein. Rats intubated with 5.0% procyanidin developed coughing, sneezing, wheezing, o v e r a l l respiratory distress and severe dehydration, and were s a c r i f i c e d after 20 d. Gross pathological examination revealed moderate to large quantities of i n t e s t i n a l gas, distended i n t e s t i n a l walls and a translucent quality to the small i n t e s t i n a l mucosa. The duodenum was discolored, black-purple, for O.5 to 1.0 cm aboral to the p y l o r i c sphincter. The jejunum and ileum were thin-walled translucent and g a s - f i l l e d when compared to jejunum and ileum of control rats (36). H i s t o l o g i c a l examination of gastrointestinal tissues from rats intubated with 5% procyanidin revealed broad, short and fused v i l l i in the areas where the duodenal tissue was dissolved. Dietary procyanidin can damage v i l l i decreasing the absorptive surface area and a l t e r i n g the absorptive c a p a b i l i t y of the i n t e s t i n a l mucosa. Nutrient a v a i l a b i l i t y is reduced and dietary protein deficiency can r e s u l t . Gastrointestinal e p i t h e l i a l damage observed with p u r i f i e d procyanidin may be dose dependent. Intubations of 1.0 and O.5% procyanidin were not toxic over a four week period, yet resulted in areas of v i l l i shortening and broadening in some of the rats intubated. Growth rate reduction did occur with 1.0 and O.5% procyanidin intubation with food. Long term consumption of unpurified procyanidins contained in legumes had no detectable effect on the h i s t o l o g i c a l appearance of the gastrointestinal tract in rats consuming diets prepared with 40% black beans. Dry Bean Fermentation-Tempeh Tempeh, an Indonesian food generally produced from soybeans fermented by Rhizopus oligosporus, is more acceptable than cooked soybeans because, in p a r t , tempeh does not have the unacceptable beany flavor and flatus problem associated with soybeans. Tempeh prepared with small red beans or a small red bean/corn mixture were acceptable in c o l o r , sweeter and more fragrant in flavor and similar in texture to soybean tempeh. The PER and in v i t r o protein d i g e s t i b i l i t y of small red bean (1.69, 85.2) and small red bean/corn (2.15, 86.1) tempeh were less than the PER and in v i t r o protein d i g e s t i b i l i t y of soybean (2.63, 88.9) and soybean/corn (3.11, 90.2) tempehs (37). Tempeh fermentation does not improve the protein quality of common beans. The presence or absence of bean h u l l s did not s i g n i f i c a n t l y affect protein u t i l i z a t i o n from tempeh. The PER for white bean t r i a l s (1.47) improved when soaking water was discarded before the beans were cooked (1.70) and fermented (38).


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Conclusions Common bean p r o t e i n and p r o c y a n i d i n i n t e r a c t i o n s can be h y d r o p h i l i c or h y d r o p h o b i c , depending on the s i t e s on the p r o t e i n a v a i l a b l e f o r interaction. Thermal p r o c e s s i n g can d e n a t u r e the p r o t e i n and change the type o f i n t e r a c t i o n p o s s i b l e . Once bean p r o t e i n is d e n a t u r e d , h y d r o p h o b i c i n t e r a c t i o n s between the p r o t e i n and p r o c y a n i d i n a r e likely. S i n c e the s t r e n g t h o f h y d r o p h o b i c i n t e r a c t i o n s i n c r e a s e s w i t h i n c r e a s e d in t e m p e r a t u r e , the i n t e r a c t i o n between p r o t e i n and p r o c y a n i d i n w i l l be enhanced d u r i n g t h e r m a l p r o c e s s i n g . Removal o f p r o c y a n i d i n w i l l be e a s i e s t p r i o r t o t h e r m a l p r o c e s s i n g . A c u t e l o n g term doses o f p r o c y a n i d i n s and food have a reduced t o x i c i t y compared to p r o c y a n i d i n i n t u b a t e d a l o n e . D i e t a r y l o n g term doses o f p r o c y a n i d i n s a r e n o r m a l l y e n c o u n t e r e d in human d i e t a r y p a t t e r n s in v a r i o u s a r e a s o f the w o r l d . Recommendations t o i n c r e a s e common bean consumption w i l l not r e s u l t in any a d v e r s e e f f e c t s t o p o p u l a t i o n s consuming l a r g e q u a n t i t i e s o f beans. Tempeh can be s u c c e s s f u l l y fermented w i t h common beans and bean/corn mixtures. However, the p r o t e i n d i g e s t i b i l i t y o r n u t r i t i o n a l q u a l i t y o f beans is not improved s u b s t a n t i a l l y by tempeh fermentation. Acknowledgments The a u t h o r s e x p r e s s thanks f o r a s s i s t a n c e from Dr. K i e t h Dunker, C h e m i s t r y Department, Dr. Ann H a r g i s , V e t e r i n a r y M i c r o b i o l o g y and P a t h o l o g y and Dr. Robert B e n d e l , S t a t i s t i c a l S e r v i c e s , Washington S t a t e U n i v e r s i t y . P a r t i a l f i n a n c i a l s u p p o r t f o r t h i s r e s e a r c h was p r o v i d e d by USAID T i t l e X I I Dry bean/Cowpea CRSP. P r o j e c t No. 0560, A g r i c u l t u r a l R e s e a r c h C e n t e r , C o l l e g e o f A g r i c u l t u r e and Home Economics, Washington S t a t e U n i v e r s i t y , P u l l m a n , WA 99164-6330. L i t e r a t u r e Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

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R E C E I V E D February 3, 1986


Plant Proteins - American Chemical Society

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