Factors affecting the rate of hydrolysis of starch in legumes 1

Susan Wong, BSc(hons), Kathy Traianedes, BAppiSci, and Kerin ODea, PhD

ABSTRACT In an attempt to understand the mechanism for the extremely slow rate of digestion and absorption of carbohydrate from legumes, we have examined a number of factors which could potentially affect the process in vitro. The rate of hydrolysis of legume starch in vitro was not affected by the presence of fat (as either butter or an emulsion). However, it was significantly increased in commercially available canned bean preparations, suggesting that the high temperatures used in the canning process may alter the availability of starch in legumes. In vitro starch hydrolysis rate was also significantly increased by grinding legumes finely prior to cooking. Finally, the slow rate of digestion and absorption of legume carbohydrate does not appear to be due to viscosity since a) increasing the shaking rate of viscous mixture of either red kidney beans or lentils from 0 to 120 oscillations per minute did not affect the hydrolysis rate, and b) a thick viscous mixture of either of these legumes did not retard the diffusion of free glucose from a dialysis sac into the dialysate. Am I Clin Nuir l985;42:38-43. KEY WORDS Legumes, starch hydrolysis, fat, viscosity, processing, physical form

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Introduction The oral ingestion of carbohydrate of leguminous origin gives rise to very low postprandial glucose and insulin responses (1) and, as a consequence, has been recommended for inclusion in the diet of diabetics (2, 3). It has been generally assumed that such foods contain viscous or gelling fibers which increase the viscosity of the unstirred layer between the food and the brush border surface of the intestine, thereby retarding nutrient absorption (4). However, we recently demonstrated that viscosity was not an important factor influencing in vitro starch hydrolysis rates in lentils (5). Indeed, those factors previously shown to be important in determining starch hydrolysis rate in cereals (6) (physical form, cooking) were also shown to be operating in the case of lentils (5). We have now extended these initial observations with lentils to other legumes as part of a larger study of numerous factors (viscosity, physical form, addition of fat, and commercial processing) which could potentially affect the rate of digestion of leguminous carbohydrate in vitro.
The American 38 Journal of Clinical

Materials
Determination

and

methods carbohydrate

of available

The carbohydrate content of the legumes and other foods used in this study was measured as previously described (6) and is based on the method of Hudson et al (7). A portion of food containing approximately 0.2 g carbohydrate was gelatinized by boiling in 20 ml of 0.2 M sodium acetate buffer(pH 5.0) for 4 h with intermittent shaking. After gelatinization 2 ml of an enzyme mixture containing 20 mg er-amylase (Halcyon Chemicals, Australia) and 20 U amyloglucosidase (Sigma, St. Louis, MO) was added and the sample incubated with shaking at 50#{176}C overnight. After enzymic hydrolysis, the sample was analyzed for glucose by reacting with para-hydroxy benzoic acid hydrazide (Pahbah). The colored aromatic hydrazide formed had an absorbance at 415 nm proportional to the monosaccharide concentration. The result was later corrected to starch by multiplying by 0.9 and expressed as a percentage of the dry weight of food. Desiccated D-glucose (May and Baker) was used as a standard. Any monosaccharide present in a sample was determined by following the same procedure without the

From the Baker Medical Research Institute, Commercial Road, Prahran, Vic 3181, Australia (SW) and the University of Melbourne Department of Medicine, Repatriation General Hospital, Heidelberg, Vic 3081, Australia (KI, KO). Received September 25, 1984. Accepted for publication December 18, 1984. Nutrition 42: JULY 1985, pp 38-43. Printed in USA 1985 American Society for Clinical Nutrition

STARCH addition of amylases. by setting up separate Rate ofhydrolysis

HYDROLYSIS

IN

LEGUMES

39

Sucrose in a sample was detected incubations containing invertase. in foods

ofstarch

(Vitrium, Sweden) is a fat emulsion containing 20% fractionated soybean oil, 1.2% fractionated lecithin, 2.5% glycerin and water. 0.5 ml of this emulsion contains 0.1 g fat. Effect ofcommercial processing

The food was cooked by boiling in distilled water for one hour. Portions of cooked legumes, each containing about 0.2 g starch, were accurately weighed out and the experiment initiated by addition of the enzyme solution (a-amylase and amyloglucosidase as above) for 30 mm at 50#{176}C. Samples were not gelatinized as the aim was to determine the rate of hydrolysis of starch in the legumes after cooking in the usual way. The amount of starch hydrolyzed after a given incubation period was measured colorimetrically as described above. In some experiments the bean preparations were incubated under three conditions: a) without enzyme, b) with amylase and amyloglucosidase, and c) with invertase to determine separately the rate of release of monosaccharide, polysaccharide, and disaccharide. Effect of viscosity

Comparisons were made between commercially available (canned) and home-made preparations of legumes with respect to total available carbohydrate content, starch content, sugar content and rate of release of monosaccharide in vitro after incubation with a-amylase and amyloglucosidase in the presence and absence of invertase. The commercially available bean preparations used in this study were Heinz Vegetarian Baked Beans and Masterfoods Red Kidney Beans. The home-cooked baked beans contained haricot (navy) beans, tomatoes, mustard, and molasses and the home-cooked red kidney beans contained onions, tomatoes, chili, and spices. In both cases the beans were soaked overnight and cooked conventionally by boiling or baking. They were not pressure-cooked.

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These studies were designed to determine whether the slow rate of carbohydrate absorption following oral ingestion of leguminous foods was due to viscosity. In the first study 0.1 g glucose was incubated in a dialysis sac in the presence and absence of a portion of cooked, mashed legumes (red kidney beans) containing 1.0 g carbohydrate and the rate of release of free glucose from the dialysis sac into the dialysate was measured. This was done by subtracting the glucose released from a dialysis sac containing kidney beans alone from that containing kidney beans plus free glucose and comparing it with the rate of release of glucose from a dialysis sac containing glucose alone. If the presence of mashed kidney beans was increasing the viscosity of the glucose solution we would have expected a reduction in the rate of release of glucose into the dialysate in the presence of the legumes. In the second study portions of red kidney beans containing 0.2 g carbohydrate were incubated at different shaking rates (0, 60, 120 oscillations per minute (OPM)) for 30 mm and the rate of starch hydrolysis to glucose measured. If viscosity was an important determinant of the rate of hydrolysis of the starch in red kidney beans, then the inhibitory effect (ie increased barrier to diffusion between enzyme and starch) would be reduced by increasing the shaking rate of the reaction mixture. Effect of physical form

Results The mean (SD) available carbohydrate contents of the foods used in this study are given in Table 1 as mono-, di-, and polysaccharides. The values for starch content obtained for red kidney beans, haricot beans, and red and brown lentils were only about two-thirds that given in the literature (8). The discrepancy is probably due to methodological differences in determination of carbohydrate content. The food table values were obtained indirectly by difference after analysis for water, fat, protein fiber, and ash. In the present study, available carbohydrate content was measured directly by incubation with digestive enzymes (7). Carbohydrate contents of legumes determined by difference may include a) some starch which is unavailable to the starch digestive enzymes due to its chemical association with protein, and b) dietary fiber not accounted for by early crude fiber analyses. The in vitro system used in this study was checked to ensure that all available carbohydrate was hydrolyzed. This was done using pure starch. A 100% recovery of the starch as glucose indicated the validity of the method used. The enzymes used were also checked regularly to be certain that maximal activity was maintained. Incubation of sucrose with a-amylase and amyloglucosidase did not result in any measurable monosaccharide re-

The effect of physical form of beans on the rate of starch hydrolysis was studied by comparing hydrolysis rate in whole red kidney beans which had been mashed with a fork after cooking to simulate chewing, with that in beans which had been finely ground in an electric grinder prior to cooking. Effect of added
fat

The rate of starch hydrolysis in portions of cooked foods (potatoes, legumes) containing 0.2 g starch was measured in the presence and absence of 0.1 g fat in the form of butter or an emulsion (Intralipid). Intralipid

40 TABLE 1 Available carbohydrate dry weight)

WONG

ET

AL

content

(mono-,

di-, and polysaccharide)

of a variety

of legumes

(mean

SD percentage

Monosaccharide
-

Sucrose
-

Starch

Haricot (navy beans) Brown lentils Red lentils Red kidney beans Commercially Red kidney Bakedbeans canned beans beans

6 12 8 6

34.0 43.0 47.2 34.8

2.0 2.6 2.0 0.2

6 6

0.9 0.1 11.6 0.1

7.5 0.1 7.7 0.8

34.6 1.0 31.4 0.4

Home-cooked equivalents Red kidney beans Baked beans

6 6

5.9 0.1 5.1 0.1

2.9 5.7

0.5

0.5

30.0 25.2

0.2 0.2

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lease. The presence of invertase in the incubation mixture was necessary for the detection of any sucrose in a sample. The percent starch hydrolyzed in a variety of legumes which had been prepared as if they were to be eaten and then subjected to a 30-mm incubation with a-amylase and amyloglucosidase is reported in Table 2. The starch in all of these legume preparations was hydrolyzed significantly more slowly than for cereals (eg spaghetti) or root vegetables (eg

TABLE

and percent total carbohydrate available after incubation of home-cooked or canned beans at 50#{176}C for 30 mm (mean SD)
Total available carbohydrates
-

Percent starch hydrolysed

Legumes Brown Red Red lentils

n 11

Starch 12.1 1.4

lentils
kidney beans

10
8 8

11.61.0
11.42.2

Haricotbeans

9.9 0.7

Canned

beans

Baked beans (haricot) Red kidney beans

Home-cooked versions

8 8

46.9
44.1

2.1

67.1 55.1

0.9

Baked beans (haricot) Red kidney beans

Non-legume carbohydrates

8 6

8.0
7.4

0.9 0.8

34.9 28.3

Potato Sweet potato Wholemeal spaghetti Plainspaghetti

10 8 10 10

63.3 4.8 64.6 0.6 69.8 2.4 72.01.2

Totalavailablecarbohydrateincludrsmono.anddisacchatide in addition to starch.

potatoes, sweet potatoes) which have been included in Table 2 for the purposes of comparison. However, it is interesting to note that the starch from canned beans was hydrolyzed much faster than that from an equivalent home-cooked version. The total available carbohydrate includes any monosaccharide and disaccharide (sugar) in the sauces in addition to the starch from the beans. Any monosaccharides were immediately available and any added sucrose was rapidly and completely hydrolyzed by invertase. However, even when the contributions of added mono-and disaccharide were corrected for, the actual rate of starch hydrolysis was much faster in the canned beans than in those cooked conventionally by boiling. The release of free glucose from a dialysis sac was not retarded by the presence of a thick viscous mixture of red kidney beans in the sac (Fig 1). The rate of release of glucose from the sac containing both the free glucose and the mashed kidney beans was the same as the sum of the rates from the sacs contaming glucose alone and kidney beans alone. Similar results were obtained when mashed red lentils were substituted for mashed red kidney beans in the dialysis sac. The rates of starch hydrolysis in mashed red kidney beans were not affected by increasing the shaking rate from 0 to 120 OPM. With no shaking 9. 1 0.7% starch was hydrolyzed in 30 mm, at 60 OPM 10.4 0.3% and at 120 OPM 10.4 0.2% (n = 6). When red kidney beans and lentils were ground finely prior to cooking, the

STARCH

HYDROLYSIS

IN

LEGUMES

41

018 016
/
gLucose
+

red kidney beans

/
,61

014

/
/

012
4-

/
gLucose

0I

In
U

010

I, iT

008
C
-

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a
U) 0
LI 01 01
/

-i

red

kidney beans

-I-

30 Time
FIG 1. Time course of the glucose red kidney beans, or both. released from

60 (minutes)
dialysis

90
sacs containing

120
0.1 g glucose, or 1.0 g carbohydrate as

starch was hydrolyzed than when they were cooking (Table 3).

much simply

more rapidly mashed after

TABLE 3 Effect of physical form on the rate of starch hydrolysis in red lentils and red kidney beans after incubation at 50#{176}C for 30 mm. Mean SD (number of determinations)
Percent

The presence of fat, either as butter or as the emulsion Intralipid, did not affect the rate of hydrolysis of starch from either ground or whole lentils or potatoes, which were included for the purposes of comparison (Table 4).

Discussion The results of this study confirm and extend our previous findings with brown lentils (5) indicating that the slow rate of digestion of leguminous starch was not due to high viscosity. Had viscosity been responsible, in-

starch

hydrolyzed

Red lentils Red kidney beans Ground red lentils Ground red kidney

beans

11.6 1.00 (10) 9.9 0.74 (8) 83.5 2.09 (5) 75.6 3.70 (6)

42

WONG

ET

AL

TABLE 4 Effect of fat on the rate of starch hydrolysis in lentils and potatoes after incubation at 50#{176}C for 30 mm. (Mean SD (n) percent starch hydrolysed)
Food Brown lentils Without fat With butter
With intra
-

lipid

12.8 83.5
60.1

1.4 (3) 2.1 (4) 3.0

(4)

12.1 62.2

0.3 (3) 81.3 2.0

(4) 61.2

Ground Potato

lentils

2.1 (4)

1.5 (4)

creasing the shaking rate of the reaction mixture would have diminished the diffusion barrier between the enzymes and starch molecules, thereby raising the starch hydrolysis rate. This did not happen either with lentils (5) or with red kidney beans. Gelling fibers such as guar gum have been shown to slow the passage of free glucose out of a dialysis sac into the dialysate due to increasing the viscosity of the medium. However, we were unable to demonstrate a similar retardation of glucose diffusion in the presence of thick viscous solutions of either lentils (5) or red kidney beans. Thus, although it was an attractive hypothesis that leguminous fiber acted like guar gum to increase the viscosity of intestinal contents to such an extent that carbohydrate digestion and absorption would be reduced (4) our findings do not support it. These results provide further support for the hypothesis that, as for cereals, the physical form of legumes is a critical factor determining the rate of starch hydrolysis. Finely grinding red kidney beans or lentils prior to cooking resulted in a 7-fold increase in starch hydrolysis rates. The co-ingestion of fat with a carbohydrate load has been shown to result in a flattening of the postprandial glucose response (9, 10). Our results showed no effect of fat on the rate of digestion of starch from either potatoes or legumes, either when added directly as butter or when added as an emulsion in order to integrate it into the reaction mixture. Thus, the most likely explanation for the in vivo results is a fat-induced delay in gastric emptying. Traditional methods of bean cookery are time-consuming, usually involving soaking overnight and prolonged cooking. For this

reason, most beans consumed in western countries like Australia and the US are in the convenient, pre-prepared, canned form. In addition to these processed forms of beans having unnecessary added salt and sugar, our data show that their starch is hydrolyzed much more rapidly than that in beans prepared by traditional methods. It is possible that the high temperatures and/or pressures to which the beans are subjected during the canning process affect the structure of the polysaccharide in the beans to make it more accessible for enzymic hydrolysis. It is interesting to note in this context that the texture of canned beans is considerably softer than that of the home-cooked beans. At present we do not know whether the more rapidly digested canned beans give rise to greater
postprandial glucose and insulin responses

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following oral ingestion. We have previously shown that grinding lentils prior to cooking was associated with a 5-fold increase in the rate of starch digestion in vitro, but only a 2-fold increase in the postprandial glucose response after oral ingestion, and no increase in the magnitude of the insulin response (11). These results suggested that factors other than rate of starch digestion were important in determining the particularly low glucose and insulin responses to legumes. However, Jenkins and coworkers did show that one form of processing involving high temperature baking did result both in an increase in the rate of starch hydrolysis in vitro and a higher glycemic response in vivo. This raises the possibility that high temperatures do modify the structure of polysaccharides in beans, possibly by disrupting the protein-carbohydrate integration, and that this does, at least partially, negate the purported benefit associated with particularly slow digestion of dried beans, once they have been processed by canning. References
1. Jenkins DJA, Wolever TMS, Taylor RH, Barker HM, Fielden H. Exceptionally low blood glucose response to dried beans: comparison with the other carbohydrate foods. Br Med J l980;28l:578-80. 2. Jenkins DJA. Slow release carbohydrate and the treatment of diabetes. Proc Nutr Soc 198 l;40: 227-35.

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3. Jenkins DJA, Wolever TMS, Jenkins AL, Ct al. The glycaemic index of foods tested in diabetic patients: a new basis for carbohydrate exchange favouring the use of legumes. Diabetelol l983;24:257-64. 4. Jenkins DJA, Wolever TMS, Leeds AR, et al. Dietary fibres, fibre analogues and glucose tolerance: importance of viscosity. Br Med J l978;l: 1392-4. 5. Wong 5, ODea K. Importance of physical form rather than viscosity in determining the rate of starch hydrolysis in legumes. Am J Gin Nutr l983;37:66-70. 6. Snow P, ODea K. Factors affecting the rate of starch hydrolysis in food. Am J Gin Nutr 1981 ;34: 2721-7. 7. Hudson GJ, John PMV, Bailey BS, Southgate DAT. The automated determination of carbohydrate. De-

8. 9.

10.

11.

velopment of a method for available carbohydrate and its application to foodstuffs. J Sci Fd Agric 1976;27:68 1-7. Composition of Foods. Bethesda, MD: US Department of Agriculture, 1975:37. Collier G, ODea K. The effect of co-ingestion of fat on the glucose, insulin and gastric inhibitory polypeptide responses to carbohydrate and protein. Am J Gin Nutr 1983;37:94l-4. Collier G, McLean A, ODea K. The effect of coingestion of fat on the metabolic responses to slowly and rapidly absorbed carbohydrates Diabetologia l984;26:50-4. ODea K, Wong S. The rate of starch hydrolysis in vitro does not predict the metabolic responses to legumes in vivo. Am J Clin Nutr 1983;38:382-7.

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