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Department of Animal Sciences, University of Illinois, Urbana, IL 61801 and * Ross Products Division, Abbott Laboratories, Columbus, OH 43219
1To whom correspondence should be addressed. E-mail: gcfahey{at}uiuc.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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KEY WORDS: • starch • digestion • in vivo • in vitro
Starch is classified nutritionally into two main groups: digestible starch (DS) and resistant starch (RS) (1–5). These are further divided into the following six fractions: 1) rapidly digestible starch (RDS), 2) slowly digestible starch (SDS), 3) physically inaccessible starch (RS1), 4) RS granules (RS2), 5) retrograded starch (RS3) and 6) chemically modified RS (RS4) (6,7). Starchy foods may contain any combination of the first five of these fractions, in varying proportions, with the majority of the starch being in the RDS and SDS fractions. There have been many studies designed to measure the differences between DS and RS (1–3,8, 11) and to differentiate among the four classes of RS (1,10). However, there have been only a few attempts to differentiate between RDS and SDS (1,12,13) despite the fact that they are the major fractions affecting the glycemic responses of humans (12,14,15). Glycemic index trials, which are expensive and time consuming and entail health risks for both subjects and lab personnel, are not a practical method for evaluating large numbers of novel starch sources for glycemic responses. Thus, for the purpose of developing nutritional supplements for diabetics for whom an extended but lower glycemic response would be desirable, it is important to evaluate starch-containing ingredients based on differences in their RDS, SDS and RS proportions.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Experiment 1
Animal selection. Forty-two male Sprague Dawley rats (mean body weight, 308 ± 8 g; age, 3 mo) were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Prior to the experiment, rats were fed a nonpurified diet. They were housed individually in stainless steel wire-bottom cages, to deter coprophagy, in an environmentally controlled room (25°C) with a 12-h light/dark cycle. Rats were given free access to water. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Illinois.
Experimental design. Rats were allotted by weight to groups, with seven rats per group and two groups assigned to each treatment. Various starch sources were used to replace the cornstarch component of the AIN-93M control diet (16) that was presented to the rats in a powdered form. The treatments were as follows: 1) control diet (Dyets, Bethlehem, PA), 2) control diet with potato starch (Dyets) and 3) control diet with amylomaize (CrystaLean; Opta Food Ingredients, Bedford, MA). Chromic oxide was added to the diets at 2 g/kg as a digestion marker. The duration of the study was 14 d. Feed intake was determined daily. Rats were weighed at the beginning and end of the experiment.
Collection of samples. On d 1–13, rats were given free access to the diets. On the evening of d 13, feed was removed for 10–13 h. After being deprived of food, rats were given free access to their respective diets for 2 h, and intake was recorded. Rats were then killed at 1 h postprandial by placement in a CO2 chamber. Then a ventral midline incision was made, and the small intestine from the entry of the bile duct through the cecum was removed. Immediately after removal, the small intestine was measured, and its length was recorded. Mosquito clamps were applied to divide the small intestine into 15 equal-length segments. The contents of the small intestinal segments were expressed and precipitated in 800 g/L ethanol.
Chemical analyses. The contents of the small intestine were centrifuged at 25,900 x g for 20 min at 4°C. The supernatant was removed and the pellet was frozen at -20°C. After freezing, samples were lyophilized in a bulk tray dryer (FTS Systems, Stone Ridge, NY). To ensure adequate sample size, dried samples were composited by segment within the animal group prior to analysis.
Diet, starch sources, and small intestinal samples were analyzed for dry matter (DM), ash and organic matter (OM; 100% minus percentage of ash) using AOAC (17) methods. Chromium in diet and digesta samples was analyzed according to Williams et al. (18). Concentrations of chromium were measured using an atomic absorption spectrophotometer (model 2380; PerkinElmer, Norwalk, CT). Starch recovered in digesta was calculated by dividing chromium intake by the chromium concentration in the segment.
Total starch was determined using a modification of the starch method of Thivend et al. (19), which included an initial incubation in a boiling water bath with 5 mL of dimethylsulfoxide. Glucose concentration of the supernatant was measured by a glucose oxidase method (glucose test kit 510-A; Sigma-Aldrich, St. Louis, MO). Cornstarch (Sigma-Aldrich) and potato starch (Sigma-Aldrich) were used as standard controls for all starch methods used.
Starch fractions of both diets and starch sources were determined using a time-modified version of the Muir and O’Dea method (2,3). Timing commenced with the addition of the enzyme solution. Sufficient numbers of tubes were started so that incubation could be stopped prior to the addition of enzyme and for every 30 min after addition of the enzyme for the first 7 h. An additional subset of tubes was allowed to incubate for 10 and 15 h to determine the full extent of starch digestion. After incubation, all tubes were placed immediately in a boiling water bath for 5 min and then cooled to room temperature in an ice-water bath. The supernatant samples from the incubations were analyzed for glucose. The substrate pellet remaining after the 15-h incubation was lyophilized and subjected to the starch method of Thivend et al. (19) to quantify any remaining starch. The RS fraction was also calculated as the difference between the total starch value of Thivend et al. (19) and the DS measured at the 15-h incubation time.
The starch fractions of free glucose, RDS and DS were also determined using the method of Englyst et al. (1). The difference between the DS and the RDS is considered the SDS fraction. The difference between the total starch concentration value of the Thivend et al. method (19) and the DS value of the Englyst et al. method (1) represented the RS in the sample.
For all laboratory analyses, samples were analyzed in duplicate, and analyses were repeated if duplicates differed by >5%. Replication due to high variation between duplicates occurred <1% of the time. Starch assays were repeated if a deviation of >5% was observed for the starch standards.
Experiment 2
Animal selection. Animal type and numbers were the same as in experiment 1 (mean body weight, 345 ± 38 g; age, 3 mo).
Experimental design. As in experiment 1, rats were allotted by weight to groups, with seven rats per group and two groups assigned to each treatment. Various starch sources were used to replace the cornstarch component of the AIN-93M control diet (16). The treatments were as follows: 1) control diet with maltodextrin (Maltrin M180; Grain Processing Corporation, Muscatine, IA), 2) control diet with modified (hydrogenated) maltodextrin (Grain Processing Corporation) and 3) control diet with pullulan (pullulan PF10; Hayashibara, Okayama, Japan). Diets were formulated to meet or exceed the nutrient requirements of rats (16). Chromic oxide was added to the diets at 2 g/kg as a digestion marker. The duration of the study was 14 d. Feed intake was determined daily. Rats were weighed at the beginning and end of the experiment.
Collection of samples. Techniques used were the same as in experiment 1.
Chemical analyses. All methods used were the same as in experiment 1 with one exception. A further modification was made to the starch method of Thivend et al. (19) to ensure the complete quantification of the pullulan starch source. The enzyme solution was modified to contain 1000 U/L heat-stabilized pullulanase (Promozyme 400L; Novozymes; Sigma-Aldrich).
For all laboratory analyses, samples were analyzed in duplicate, and analyses were repeated if a deviation of >5% between duplicates was observed. Replication due to high variation between duplicates occurred <1% of the time. Starch assays were repeated if a deviation of >5% was observed for the starch standards.
Statistical analyses. Data were analyzed as a completely randomized design using the general linear models procedures of SAS (20). Differences in animal weights were analyzed as a paired t test within a completely randomized design (20).
Data for starch disappearance from the rat small intestine were fitted to a logistic model equation to determine the rates of disappearance and the segments in which the maximal rates of disappearance were attained for each diet. The starch disappearance data, as determined by Muir and O’Dea (2,3), were fitted to the same logistic model equation to determine the rates of disappearance and the times at which the maximal rates of disappearance were attained. This function is frequently used to model biological growth (21). It is a sigmoidal curve that can describe accelerating and, after passing through an inflection point or plateau, decelerating phases of growth or disappearance (22). The segment (for the rat data), or the time (for the Muir and O’Dea (2,3) data), at which maximal rates of disappearance occurred were calculated according to the following equation:
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where S is the percentage of starch disappearance, A1 is the first asymptote or first maximal percentage of starch disappearance, t is the intestinal segment (1–15) or time (0.5–15.0 h), C1 is the segment or time at which the first rate of starch disappearance is maximal (the inflection point), B1 is a measure of the duration of the first phase of starch disappearance, A2 is the second asymptote or second maximal percentage of starch disappearance, C2 is the segment or time at which the second rate of starch disappearance is maximal (the inflection point) and B2 is a measure of the duration of the second phase of starch disappearance.
If no significance was observed for the second set of maximums and, thus, no second plateau of starch disappearance was observed, that term was dropped and the following equation was used:
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where S is the percentage of starch disappearance, A is the asymptote or maximal percentage of starch disappearance, t is the intestinal segment (1–15) or time (0.5–15.0 h), C is the segment or time at which the rate of starch disappearance is maximal (the inflection point) and B is a measure of the duration of starch disappearance.
Variables (A1, A2, B1, B2, C1 and C2) were estimated for each group and diet combination using nonlinear regression (NLREG) (23). The model explained 95% or more of the variation in starch disappearance in all cases. Variables (A1, A2, B1, B2, C1 and C2) were estimated for each diet and starch source analyzed by the Muir and O’Dea method (2,3) using NLREG (23). The model explained 95% or more of the variation in starch disappearance in all cases.
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RESULTS AND DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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All starch sources selected for this experiment had been cold-extracted from the primary sources to prevent heat damage and any possible change to the starch fraction ratios from steam-extraction gelatinization and retrogradation. Cornstarch was selected because it is a commonly used starch. It is predominantly amylopectin (
75%), can be fully digested in the small intestine, and contains only small amounts of RS. Potato starch, in contrast to cornstarch, is predominantly a crystalline amylose and is slowly hydrated and digested in the small intestine. Amylomaize (CrystaLean) is a cornstarch extracted from a high amylose corn variety and can be composed of up to 50% amylose. It was expected to be digested only to a moderate extent in the small intestine. The two maltodextrins were made from cornstarch that had been partially hydrolyzed to change its functional characteristics. The modified maltodextrin was hydrogenated maltodextrin. Pullulan is a bacterial form of starch. It is a linear glucan consisting of repeating units of maltotriose joined by 
-(1
6) linkages. There are suggestions that pullulan contains other bonds, such as (13) linkages, but this has not been confirmed (24). Like potato starch, it is resistant to digestion by mammalian enzymes but is highly fermentable by bacteria.
Chemical composition data.
DM concentrations varied among starch sources and diets (Table 1), ranging from 81.9 to 97.1 g/100 g substrate and from 88.6 to 96.6 g/100 g substrate, respectively. OM concentrations were more consistent, ranging from 97.0 to 100.0 g/100 g DM and from 95.8 to 97.2 g/100 g DM. The total starch concentrations of the Thivend et al. (19) method varied for most of the starch sources and corresponding diets, ranging from 92.7 to 102.9 g/100 g DM and from 59.5 to 64.6 g/100 g DM, respectively. The total starch concentration for the modified maltodextrin source and diet was low, 69.8 and 49.8 g/100 g DM, respectively. This low value resulted from the carbohydrate in the compound being nonsusceptible to amyloglucosidase, -amylase and pullulanase degradation, because the hydrogenation converted the reducing end sugar of the maltodextrin to sorbitol.
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3) linkages, but this is not confirmed with lower total starch concentrations. Because this assay does not expose the samples to the higher temperatures used in the Thivend et al. (19) method, it is possible that the starch sources are slower to hydrolyze and would not have been exposed to the enzymes long enough to be fully degraded. This could result in the presence of soluble oligosaccharides that would not be detected as glucose, but would not separate from the supernatant during centrifugation to be quantified with the RS fraction. At this time, the presence of soluble oligosaccharides is the most likely explanation for the reduced RS values detected. Because this was not expected or suggested in the literature describing this in vitro method, the supernatant was not analyzed for the presence of oligosaccharides. The three methods used to determine RS concentrations resulted in very different values (Table 2). This was demonstrated previously by Champ (4) who compared the RS methods of Berry (25) and Björck et al. (26) and found major differences in RS concentrations. For samples containing low concentrations of RS, such as cornflakes, values ranged from 1.5 to 3.7 g/100 g substrate, with the method of Berry (25) resulting in the higher values. For samples high in RS, such as raw potato starch, the discrepancy was greatly magnified, with values ranging from 0.1 to 47.8 g/100 g substrate.
Animal data.
Food intake (22.3 g/d), weight gain (39.8 g/14 d) and relative small intestine length (291.4 cm/kg body) did not differ among treatments or groups.
There were distinct patterns of disappearance observed for the different starch sources, with the modified maltodextrin exhibiting an 80 g/100 g starch disappearance value by the third segment of the intestine, while amylomaize disappearance values were negative at the same segment (Fig. 5). Pullulan demonstrated a gradual increase in disappearance. Cornstarch resulted in what might be considered the best example of a gradual disappearance that was complete by segment 15. In theory, this type of digestion curve should lead to a more gradual rise in blood glucose concentrations. Starch disappearance observed at segment 15 (the terminal ileum) ranged from 62.6 to 98.8 g/100 g starch, which is comparable with the ranges of 53.6 to 102.2 g/100 g starch and 56.8 to 93.0 g/100 g starch for DS with the Englyst et al. (1) and the Muir and O’Dea (2,3) methods, respectively.
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Planned comparisons between the two methods used to generate DS values and the ileal disappearances collected from the rat model showed significant (P < 0.05) differences in most instances (Table 3). This would be expected considering the differences found in RS concentrations. In two instances (the cornstarch and pullulan diets), the DS of the two methods used were in agreement with each other, but not with the animal data. In all instances, the in vitro values noted for the diets were more closely related than were the values for the starch sources. This might be expected, because the in vitro methods were developed using food sources and not using purified starch sources.
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In future studies, the soluble oligosaccharide fragments that were not susceptible to ethanol precipitation need to be quantified. Further animal work needs to be done to collect intestinal disappearance data for different starch sources before in vitro modeling can progress. Current in vitro methods should be modified to result in DS disappearance data that are more in agreement with animal intestinal disappearance data. Furthermore, studies are needed to evaluate the effects of varying concentrations of protein, fat and fiber on starch digestion.
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FOOTNOTES |
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Manuscript received 21 November 2002. Initial review completed 29 December 2002. Revision accepted 24 March 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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1. Englyst, H. N., Kingman, S. M. & Cummings, J. H. (1992) Classification and measurement of nutritionally important starch fractions. Eur. J. Clin. Nutr. 46:S33-S50.
2. Muir, J. G. & O’Dea, K. (1992) Measurement of resistant starch: factors affecting the amount of starch escaping digestion in vitro. Am. J. Clin. Nutr. 56:123-127.
3. Muir, J. G. & O’Dea, K. (1993) Validation of an in vitro assay for predicting the amount of starch that escapes digestion in the small intestine of humans. Am. J. Clin. Nutr. 57:540-546.
4. Champ, M. (1992) Determination of resistant starch in foods and food products: interlaboratory study. Eur. J. Clin. Nutr. 46:S51-S62.
5. Asp, N.-G. (1994) Nutritional classification and analysis of food carbohydrates. Am. J. Clin. Nutr. 59:679S-681S.[Abstract]
6. Englyst, H. N. & Macfarlane, G. T. (1986) Breakdown of resistant and readily digestible starch by human gut bacteria. J. Sci. Food Agric. 37:699-706.
7. Brown, I. L., McNaught, K. J. & Moloney, E. (1995) Hi-maizeTM: new directions in starch technology and nutrition. Food Aust. 47:272-275.
8. Champ, M., Molis, C., Flourié, B., Bornet, F., Pellier, P., Colonna, P., Galmiche, J.-P. & Rambaud, J-C. (1998) Small-intestinal digestion of partially resistant cornstarch in healthy subjects. Am. J. Clin. Nutr. 68:705-710.[Abstract]
9. Tovar, J., Björck, I. M. & Asp, N.-G. (1992) Incomplete digestion of legume starches in rats: a study of precooked flours containing retrograded and physically inaccessible starch fractions. J. Nutr. 122:1500-1507.
10. Englyst, H. N., Kingman, S. M. & Cummings, J. H. (1993) Resistant starch: measurement in foods and physiological role in man. Meuser, F. Manners, D. J. Seibel, W. S. eds. Plant Polymeric Carbohydrates 1993:137-146 Royal Society of Chemistry Cambridge, UK. .
11. Stephen, A. M., Haddad, A. C. & Phillips, S. F. (1983) Passage of carbohydrate into the colon. Gastroenterology 85:589-595.[Medline]
12. Englyst, H. N., Veenstra, J. & Hudson, G. J. (1996) Measurement of rapidly available glucose (RAG) in plant foods: a potential in vitro predictor of glycaemic response. Br. J. Nutr. 75:327-337.[Medline]
13. Murray, S. M., Flickinger, E. A., Patil, N. R., Merchen, N. R., Brent, J. L., Jr & Fahey, G. C., Jr (2001) In vitro fermentation characteristics of native and processed cereal grains and potato starch using ileal chyme from dogs. J. Anim. Sci. 79:435-444.
14. Goñi, I., Garcia-Alonso, A. & Saura-Calixto, F. (1997) A starch hydrolysis procedure to estimate glycemic index. Nutr. Res. 17:427-437.
15. Wolfsdorf, J. I. & Crigler, J. F., Jr (1997) Cornstarch regimens for nocturnal treatment of young adults with type 1 glycogen storage disease. Am. J. Clin. Nutr. 65:1507-1511.
16. National Research Council (1995) Nutrient Requirements of Laboratory Animals 4th ed. 1995 National Academy Press Washington, DC.
17. Association of Official Analytical Chemists (1984) Official Methods of Analysis 14th ed. 1984 Association of Official Analytical Chemists Washington, DC.
18. Williams, C. H., David, D. J. & Iismaa, O. (1962) The determination of chromic oxide in faeces samples by atomic absorption spectrophotometry. J. Agric. Sci. (Camb.) 59:381-385.
19. Thivend, P., Christiane, M. & Guilbot, A. (1972) Determination of starch with glucoamylase. Meth. Carbohydr. Chem. 6:100-105.
20. SAS Institute Inc. (1996) SAS User’s Guide: Statistics, Version 6 1996 SAS Institute Cary, NC.
21. Koops, W. J. & Grossman, M. (1993) Multiphasic allometry. Growth Dev. Aging 57:183-192.[Medline]
22. Koops, W. J. & Grossman, M. (1991) Applications of a multiphasic growth function to body composition of pigs. J. Anim. Sci. 69:3265-3273.[Abstract]
23. Sherrod, P. H. (2002) NLREG User’s Guide, Version 5.3 2002 Phillip H. Sherrod Brentwood, TN.
24. Tsujisaka, Y. & Mitsuhashi, M. (1993) Pullulan. Whistler, R. L. BeMiller, J. N. eds. Industrial Gums, Polysaccharides and Their Derivatives 3rd ed. 1993:447-460 Academic Press New York, NY. .
25. Berry, C. S. (1986) Resistant starch: formation and measurement of starch that survives exhaustive digestion with amylolytic enzymes during the determination of dietary fibre. J. Cereal Sci. 4:301-314.
26. Björck, I., Nyman, M., Pedersen, B., Siljeström, M., Asp, N. G. & Eggum, B. O. (1986) On the digestibility of starch in wheat bread: studies in vitro and in vivo. J. Cereal Sci. 4:1-11.
27. Kararli, T. T. (1989) Gastrointestinal absorption of drugs. Crit. Rev. Ther. Drug Carrier Syst. 6:39-86.[Medline]
28. Mathers, J. C., Smith, H. & Carter, S. (1997) Dose-response effects of raw potato starch on small intestinal escape, large-bowel fermentation and gut transit time in the rat. Br. J. Nutr. 78:1015-1029.[Medline]
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