In Vivo and In Vitro Starch Digestion: Are Current in Vitro Techniques

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Biomacromolecules 2010, 11, 3600–3608

In Vivo and In Vitro Starch Digestion: Are Current in Vitro Techniques Adequate? Jovin Hasjim, Gautier Cesbron Lavau, Michael J. Gidley, and Robert G. Gilbert* The University of Queensland, Centre for Nutrition and Food Sciences, Brisbane, QLD 4072, Australia Received September 6, 2010

The time evolution of the size distributions of (fully branched and debranched) starch molecules during in vivo and in vitro digestion was analyzed using size exclusion chromatography (SEC) and compared. In vivo digesta were collected from the small intestine of pigs fed with raw normal maize starch; in vitro digestion was carried out on the same diet fed to the pigs using a method simulating digestion in the mouth, stomach, and small intestine. A qualitative difference was observed between the in vitro and the in vivo digestion. The former showed a degradation of starch molecules to a more uniform size, whereas the in vivo digestion preserved the size distribution of native starch before producing a multimodal distribution, the heterogeneous nature of which current in vitro methods do not reproduce. The use of in vitro digestion to infer in vivo digestion patterns and, hence, potential nutrition benefits need to take account of this phenomenon.

Introduction Starch, comprising branched polymers of glucose, is the main energy source in human food and animal feed. This paper compares and mechanistically interprets the changes in the structure (size distributions) of starch molecules (fully branched) and their individual branches (debranched) that occur through enzymatic degradation by two methods: a common in vitro technique and in vivo in pigs, which are close models for human digestion.1,2 Any qualitative differences in the evolution of the starch structure must be borne in mind when designing and interpreting in vitro experiments whose goal is scientific understanding of digestive processes involving starch-based foods in human and animal nutrition. Starch consists of two R-glucans: amylose and amylopectin. Amylose is the smaller molecule with a molecular weight of about 106 Da and a few long-chain branches.3 Amylopectin is the larger, highly branched molecule and has a molecular weight of about 108 Da. In plants, starch is synthesized into semicrystalline granules where the branches of amylopectin are arranged into many alternating amorphous and crystalline lamellae,4 and amylose is in an amorphous form interspersed among the amylopectin crystallites.5 In most mammals, including humans and pigs, starch digestion and glucose absorption occur mostly in the small intestine. In the intestinal lumen, starch is hydrolyzed by pancreatic R-amylase to maltose and larger oligosaccharides, which are further hydrolyzed to glucose by maltase-glucoamylase and sucrase-isomaltase on the brush-border surface (a heterogeneous process) before being absorbed.6,7 Starch digestibility has a big impact on human health. Rapidly digestible starch (RDS) is thought to promote metabolic syndrome, including insulin resistance, obesity, and diabetes,8 whereas slowly digestible starch (SDS) and enzyme-resistant starch (RS) within a caloriecontrolled diet are thought to have beneficial effects for protecting against metabolic syndrome and colon cancer.9-11 Despite the fact that starch is mostly digested in the small intestine, most human and animal studies (in vivo) of starch * To whom correspondence should be addressed. E-mail: b.gilbert@ uq.edu.au.

digestion have been carried out using the digesta collected from the terminal ileum, colon, or feces.12-14 These digesta only show the structure of RS that is not digested in the small intestine but do not provide enough information to understand the mechanism of the starch digestion in the small intestine, such as the differences in the digestion of RDS and SDS. Many in vitro studies have been designed to simulate and investigate digestion in the small intestine.15-23 These in vitro digestion methods provide practical and reproducible approaches to study starch digestibility and its relationship with starch molecular structure. Although these methods are validated by quantitative comparison with RS values typically obtained from the terminal ileum of ileostomy subjects, they might oversimplify the digestion mechanism in the small intestine. The enzymatic reactions in the in vitro digestion are carried out only in homogeneous solution/suspension, whereas the intestinal digestion takes place in the intestinal lumen and at the surface of the intestinal wall, both of which are likely to provide a heterogeneous environment. Furthermore, there are more enzymes involved in the intestinal digestion than in the in vitro digestion, for example, maltase-glucoamylase and sucraseisomaltase versus fungal glucoamylase (also called amyloglucosidase), respectively. The results from in vitro starch digestion might not reflect that of in vivo starch digestion in the small intestine (and indeed discrepancies in the hydrolysis rate between in vivo and in vitro studies have been reported19,24). The prime investigative tool used here is one commonly used in the study of both linear and branched synthetic polymers, that is, size exclusion chromatography, to analyze the size distributions of both whole starch molecules and of individual branches (after treatment with a debranching enzyme). These size distributions allow a means to understand, and indeed discover, the significant mechanistic processes in the system under study (e.g., refs 25 and 26). The objective of this study is to obtain a better understanding of the in vivo digestion of starch in the small intestine by studying the structure of starch in digesta collected from different parts of pig small intestine. The structures of the starch collected from the in vivo digesta can be compared with those of the same starch digested at different times using a standard

10.1021/bm101053y  2010 American Chemical Society Published on Web 10/20/2010

In Vivo and In Vitro Starch Digestion

in vitro method. The results from this study will provide more accurate information on the in vivo starch digestion than those obtained through in vitro analysis, and the technique developed through this study will be useful in designing improved in vitro methods, as well as the long-term aim of starch products with better nutrition value, such as higher levels of SDS. Raw starch granules were used in this study. Although starch is often consumed after cooking, granular starch is commonly found in low-moisture foods, such as biscuits and some breakfast cereals, and in some uncooked fruits and vegetables. Furthermore, raw starch is normally used for animal feed. Recently, native starch granules have been proposed as a healthy carbohydrate food ingredient because of their slowly digestible property.15,18 Thus, it is important to study the digestion of starch granules for both human and animal nutrition.

Experimental Section Materials. Raw normal maize starch was obtained from Penford Australia Ltd. (Lane Cove, NSW, Australia). Other ingredients of the pig diet (see below) were obtained from local sources. Porcine pancreatic R-amylase type VI-B (A-3176), pepsin from gastric porcine mucosa (lyophilized powder, P-6887), pancreatin from porcine pancreas (P-1750), MOPS sodium salt (M-9381), and lithium bromide (ReagentPlus, 213225) were from Sigma Aldrich Pty Ltd. (Castle Hill, NSW, Australia). D-Glucose (GOPOD Format) kit (K-GLUC), thermostable R-amylase from Bacillus licheniformis (E-BLAAM), amyloglucosidase from Aspergilus niger (E-AMGDF), and isoamylase from Pseudomonas sp. (E-ISAMY) were from Megazyme International Ltd. (Co. Wicklow, Ireland). Dimethyl sulfoxide (GR for analysis ACS) was from Merck and Co, Inc. (Whitehouse Station, NJ, U.S.A.). Other chemicals were reagent grade and used as received. In Vivo Digestion. All animal experiments were performed in compliance with the University of Queensland animal ethics requirements. In vivo digesta were collected from the small intestine of seven large white pigs (P-1-7) raised at the Centre for Advanced Animal Science, The University of Queensland, Gatton, QLD, Australia. They were given a diet containing raw normal maize starch (51.1%, wet basis (wb); 47.5%, dry basis (db)), cooked red meat (23.6%, wb), sunflower seed oil (8.9%, wb), sucrose (7.0%, wb), tallow (5.0%, wb), wheat bran (4.0%, wb), sodium chloride (0.3%, wb), and vitamin and mineral mix (0.2%, wb) on the night before sacrifice and at 2 and 4 h before sacrifice. The small intestine was removed under anesthetic and divided into four sections, SI-1-4, from the stomach end to the cecum end (Supporting Information). SI-1 was within the first meter from the stomach end. SI-2 was from the end of SI-1 until the midpoint of the whole small intestine. SI-3 was the other half of the small intestine excluding the last meter from the cecum end. SI-4 was the last meter from the cecum end. The digesta were recovered from each section by squeeze expulsion and then stored at -20 °C prior to analysis. The digesta from SI-4 section were not analyzed in this study because of the limited amount of the digesta and the very low amount of starch in the digesta. In Vitro Digestion. The pig diet was also digested in vitro using a modified enzymatic method of Sopade and Gidley21 in triplicate. The pig diet containing 10.00 g (db) starch was preincubated with artificial saliva solution (5 mL) containing porcine pancreatic R-amylase (1000 U/mL), potassium chloride (21.1 mM), calcium chloride (1.59 mM), and magnesium chloride (0.2 mM) in carbonate buffer (14.4 mM, pH 7) for 15-20 s at ambient temperature. The result was then preincubated with pepsin (4 mg/mL) in hydrochloric acid solution (25 mL, 0.02 M) in a shaking water bath at 37 °C for 30 min. It was then neutralized with sodium hydroxide solution (25 mL, 0.02 M) and diluted with acetate buffer solution (125 mL, 0.2 M, pH 6) containing calcium chloride (200 mM) and magnesium chloride (0.49 mM). The result was then digested with pancreatin (8 mg/mL) and amyloglucosidase

Biomacromolecules, Vol. 11, No. 12, 2010

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(112 U/mL) in the same acetate buffer solution (25 mL) in the shaking water bath at 37 °C. Aliquots (10 mL) were removed at 0, 1, 2, 5, and 25 h. Each aliquot was then mixed with absolute ethanol (30 mL) and centrifuged at 4000g for 10 min. The precipitate (in vitro digesta) was allowed to dry overnight in a fume hood at ambient temperature and stored in a dry place for further analyses. The concentration of glucose in the supernatant was determined using the Megazyme D-glucose (GOPOD Format) kit following the procedure given by the manufacturer. The molar mass conversion from glucose to anyhydroglucose (the starch monomer unit) is 0.9. The degree of hydrolysis was calculated using the following equation:

%hydrolysis ) total weight of glucose in supernatant × 0.9 × 100% dry weight of starch in diet Starch Content. Starch contents of the in vivo digesta were analyzed in duplicate using AACC Method 76-13.27 Digesta from SI-1 and -2 were diluted five times before analysis; that from SI-3 was analyzed without dilution. The digesta or diluted digesta (0.2 mL) was precisely weighed and then it was mixed with 3.0 mL of absolute ethanol and centrifuged at 4000g for 10 min to remove the soluble sugars in the digesta. The precipitate was allowed to dry overnight in a fume hood at ambient temperature. It was then wetted with 80% ethanol (0.2 mL) and, subsequently, dissolved with dimethyl sulfoxide (2 mL) in a boiling water bath for 5 min with stirring. The dissolved digesta was incubated with thermostable R-amylase (0.1 mL) in MOPS buffer (2.9 mL, 50 mM, pH 7.0) containing calcium chloride (5 mM) and sodium azide (0.02% w/v) in the boiling water bath for 12 min with stirring. The digesta was then mixed with acetate buffer (4.0 mL, 200 mM, pH 4.5) containing sodium azide (0.02% w/v) and incubated with amyloglucosidase (0.1 mL) in a water bath at 50 °C for 30 min with stirring. The volume of the result was adjusted to 10.0 mL. The total glucose produced from the starch in the digesta was determined using the Megazyme D-glucose (GOPOD Format) kit following the procedure given by the manufacturer. The glucose was converted to anyhydroglucose unit by a factor of 0.9. The moisture content of the digesta was determined from the difference in weight before and after drying overnight in an oven at 110 °C. The starch content (wb and db) was calculated using the following equations:

%starch content(wb) )

total weight of glucose × 0.9 × 100% weight of digesta

%starch content(db) ) total weight of glucose × 0.9 × 100% weight of digesta × (1 - %moisture content) Starch Morphology. Starch granules were isolated from the in vivo and in vitro digesta for morphological study using a modified wet milling process following the method of Hasjim et al.28 The isolation of starch granules was conducted at ambient temperature to restrict granule swelling. Amylose leaching is a common phenomenon accompanying excess swelling of starch granules when heated at elevated temperature,29,30 which was prevented as the starch granules were isolated at ambient temperature. Digesta (20 mL) was filtered through a screen with 53 µm openings. The remaining material on the screen was washed three times with sodium chloride solution (0.1 M, 50 mL) and filtered through the screen again. The filtrate was combined and mixed with toluene (50 mL). The mixture was stirred for 1 h and then allowed to stand until the starch granules settled at the bottom. The protein and lipid in the toluene and the sodium chloride solution layers were siphoned off and discarded, and this was repeated until all of the protein and lipid were removed, as indicated by a clear toluene layer after the starch granules settled at the bottom. The starch layer was washed with water several times and subsequently with absolute ethanol.

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The starch granules were collected after centrifugation at 4000g for 10 min and allowed to dry overnight in a fume hood at ambient temperature. The morphology of isolated starch granules was examined using a scanning electron microscope (SEM; JEOL JSM-6610, Tokyo, Japan) at the Centre for Microscopy and Microanalysis, The University of Queensland, Brisbane, Australia. Starch Molecular Size Distributions. Starch was extracted from the in vivo and in vitro digesta and dissolved following the method of Syahariza et al.31 with modification. The size distributions of whole (fully branched) starch and of debranched starch were obtained using a modification of the methods given elsewhere.22,26,32 The Supporting Information gives full details of the procedures. Size-exclusion chromatography (SEC, often termed gel-permeation chromatography, GPC) was used to analyze the time evolution of the molecular size distribution of the starch molecules during the course of in vivo and in vitro digestion. It is important to recall that SEC separates molecules by size, not by molecular weight. For a complex hyperbranched polymer, such as starch, there is no unique relationship between size and molecular weight. The elution parameter for SEC is the hydrodynamic volume, Vh (which is proportional to the product of the number-average weight distribution and the weight-average intrinsic viscosity33). For convenience, data are presented here in terms of the corresponding hydrodynamic radius (Rh) with Vh ) 4/3 Rh3. The SEC weight distribution, w(log Vh), was calculated from the detector signal and was plotted against Rh following the method of Cave et al.32 Rh is proportional to the radius of gyration (the geometric size), but the proportionality constant may vary with molecular weight (e.g., ref 34). The SEC distribution data presented in terms of Rh (or Vh), which is a molecular quantity, are reproducible, whereas presenting such data as elugrams in terms of elution time or volume are not (elution varies with the particular SEC setup and even from day to day with a given setup). For debranched starch, the linear molecules resulting from debranching do have a unique relationship between size and molecular weight (or equivalently the degree of polymerization, DP), and the debranched distributions are thus presented both in terms of Rh and DP, with the DP values obtained using the Mark-Houwink relationship (see Witt et al.22 for details). The resulting SEC chromatograms were analyzed using PSS WinGPC Unity software (Polymer Standard Services, Mainz, Germany) and normalized to yield the same peak height of the highest peak of the fully branched and debranched starch distributions. Although it is unavoidable that the amylopectin component will suffer from some shear scission in SEC,32 as long as a comparison is made on the samples run on the same day, this shear will be the same for every sample, and so the resulting distributions can be compared quantitatively; for example, if one sample shows larger molecules than another, then this is a true reflection of the actual size distributions of the polymers. Small day-to-day fluctuations in calibration can also affect the apparent distributions extrapolated to sizes outside the calibration range,32 and running all samples to be compared on the same day results in data that can be quantitatively compared. Because of this, the Rh values above the upper limit of the standards available (∼50 nm) are only semiquantitative. Statistical Analysis. Means and standard deviations from replicate measurements of the degree hydrolysis of the in vitro digestion and the starch content in the in vivo digesta were calculated using Microsoft Office Excel 2007 (Microsoft Corporation, Redmond, WA). Difference test was evaluated by t-test with Tukey’s adjustment using SAS (version 9.2, SAS Inst. Inc., Cary, NC). The significance level was set at p value 100 nm

similar to undigested starch greater amount of smaller molecules (Rh < 60 nm) and a new peak at Rh < 3 nm a new peak at Rh < 3 nm

branched starch distributions of the SI-1 digesta from P-2, -4, -6, and -7 as well as those of the SI-2 digesta from P-3 and -6 had a larger population of smaller molecules (with Rh similar to that of amylose) than that of the normal maize starch before digestion. New smaller dextrins with peak Rh at ∼1.5-2.5 nm appeared in the fully branched starch distributions of the SI-1 digesta from P-2, -4, and -6 as well as those of the SI-2 digesta from P-3, -6, and -7, which were similar to that observed in the in vitro digestion for 25 h with pancreatin and amyloglucosidase (Figure 5). This peak remained in the fully branched starch distributions of the SI-3 digesta from all pigs along with two smaller peaks observed in the region of 3 nm < Rh < 100 nm, whereas nothing was detectable for Rh > 100 nm.

Debranched Starch Distribution of Digesta. The debranched distributions of starch extracted from the in vivo and in vitro digesta are shown in Figure 6. Because, for a debranched (i.e., linear) molecule, there is a unique relationship between size and molecular weight, these data can also be presented in terms of degree of polymerization (DP), using the Mark-Houwink relationship. This is given as the upper X-axis in Figure 6, although this axis is only semiquantitative because of effects such as SEC band broadening and the possibility that the Mark-Houwink relation is not accurate for smaller DPs.35 The peaks can be divided into amylopectin branches (Rh e 3.5 nm, DP 200) and amylose branches (Rh ∼ 3.5-60 nm, DP ∼ e200-30000). The amylopectin branches themselves can be



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Figure 6. Debranched SEC weight distributions of starches extracted from in vitro and in vivo (seven pigs) digesta. The upper X-axis gives the degree of polymerization (DP); this is only semiquantitative for smaller DPs, as explained in the text. Starch ) raw normal maize starch before digestion. Table 2. Observations from the Size Distributions of Debranched Starches from In Vivo Digesta size distribution of debranched starch pigs

SI-1

SI-2

P-1

similar to undigested starch


P-2



P-3



P-4



P-5



P-6

larger peak area at amylose-branch region


P-7


similar to the starch in SI-3

further divided into two groups: branches confined to one lamella (A and B1 chains, peak Rh ∼ 1.5 nm or DP ∼ 12) and branches that span more than one lamella (B2, B3, ..., chains, peak Rh ∼ 2.5 nm or DP ∼ 50). The starch of the in vitro digesta after 1, 2, and 5 h of digestion using pancreatin and amyloglucosidase showed similar distributions of branches to those of the normal maize starch before digestion. The peak areas of the longer amylopectin branches (Rh ∼ 2-3.5 nm, DP ∼ 25-200) and the amylose branches were larger in these in vitro digesta than those of the normal maize starch before digestion, showing that branches of these lengths were associated with relatively slowly digested regions. The debranched starch distribution of the in vitro digesta

SI-3 a major population of branches at Rh 8 nm (DP 3000) with two major peaks at Rh ∼ 2 nm (DP ∼ 40) and at Rh ∼ 3.5 nm (DP ∼ 250) and a minor population of branches in the amylose-branch region at 8 nm Rh 70 nm (3000 DP 100000) with a peak at Rh ∼ 15 nm (DP ∼ 6000)

after 25 h showed only a single peak at Rh ) ∼2.5 nm (DP ) ∼ 50), but the peak tail covered the whole range of the amylopectin branches and amylose branches (Rh e 60 nm, DP e 30000). The observations from the size distributions of the debranched starch from the in vivo digesta are summarized in Table 2. The starch extracted from the digesta of SI-1 and -2 of five pigs (P-1 to -5) showed similar debranched starch distributions to that of normal maize starch before digestion. The debranched starch distributions of SI-1 and 2 digesta from P6j and that of SI-1 digesta from P-7 showed a larger peak area in the amylosebranch region. The debranched starch from the SI-3 digesta, in general, showed two major peaks at Rh ∼ 2 nm (DP ∼ 40, in

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between the two peaks of amylopectin-branch distribution) and at Rh ∼ 3.5 nm (DP ∼ 250, a new component not observed in the size distribution of debranched starch before digestion) as well as one minor peak at Rh ∼ 15 nm (DP ∼ 6,000, in the amylose-branch region). The debranched starch distribution of SI-2 digesta from P-7 was similar to those of SI-3 digesta.

Discussion The glucose content in the solution after preincubation with artificial saliva and pepsin and before the digestion with pancreatin and amyloglucosidase was minor (Figure 1), although the electron micrograph (at 0 h of in vitro digestion) suggests that the starch granules have been extensively hydrolyzed by enzymes (Figure 4). This is attributed to the absence of amyloglucosidase in the preincubation enzyme solutions, whence the dextrins (maltose and oligosaccharides) produced by R-amylase in the artificial saliva were not converted to glucose.36 The morphology of starch in the in vitro digesta did not show any apparent changes (Figure 4) when the diet was digested using pancreatin and amyloglucosidase for 0 to 2 h, although the production of glucose increased rapidly during this digestion period (Figure 1). This is attributed to the digestion pattern of the R-amylase on starch granules. The digestion starts from the surface of the granules, where the enzymes create pores, to penetrate into the structurally less-organized hilum (or center) of the granules and hydrolyze the starch granules from the inside to the outer layers.15,37 Thus, the surface morphology of residual starch granules might not show any apparent changes during digestion using pancreatin and amyloglucosidase. After 5 h of digestion, some granules were broken with the interior exposed. After extensive digestion by the enzymes, the interior of starch granules became hollow and the starch granules were easily fractured. The residual starch morphology of the in vitro digesta after 25 h of digestion (Figure 4) where the starch in the diet was almost completely digested (Figure 1) showed granule fragments and undigested starch granules, which was similar to that reported by others.15,37 The presence of undigested granules was confirmed by the fully branched starch distribution (Figure 5), where a long tail was observed extending to ∼103 nm revealing the presence of a small amount of native amylopectin in this digesta. This might be attributed to the absence or limited pores on the surface of the undigested starch granules that prevented the enzymes penetrating into the hilum of the granules.15,28,37 The fully branched starch distributions of the in vitro digesta (Figure 5) also reveal that amylopectin was rapidly degraded by amylolytic enzymes as the concentration of glucose in the solution increased (Figure 1). The debranched starch distributions of the in vitro digesta, however, were similar during 1-5 h of digestion with pancreatin and amyloglucosidase (Figure 6). This suggests that amylose and amylopectin in the maize starch granules were hydrolyzed at approximately the same rate by pancreatic R-amylase and amyloglucosidase, in agreement with the observations of Zhang et al.15 The results also suggest that amylopectin was rapidly hydrolyzed by pancreatic R-amylase (in the artificial saliva and pancreatin), producing partially hydrolyzed molecules similar in size to amylose without showing a significant changes in its branch chain lengths. The size distributions of both fully branched and debranched starch from the in vitro digesta after 25 h of digestion showed a peak at a similar hydrodynamic radius (Rh ∼ 2.5 nm, DP ∼ 50), which was not observed from those of either the normal maize starch before digestion or the starch extracted from the

Hasjim et al.

in vitro digesta digested for 5 h or less (Figures 5 and 6). This shows that a large population of linear dextrins was produced in the digesta after 25 h of digestion. A similar observation was reported for extruded starch digested using a similar in vitro method.22 Linear dextrins have more mobility and can arrange themselves into crystalline structure that is less susceptible to enzymatic hydrolysis than the native crystalline structure.22,38-40 The starch content of the in vivo digesta from SI-1 (Figure 2) was slightly lower than that in the diet. The morphology of starch from SI-1 digesta was similar to that of the undigested diet, with more tiny pores on the surface of the granules (Figure 3). The starch content, however, rapidly decreased and the starch morphology changed drastically as the digesta traveled from SI-1 to SI-2. The results suggest that starch digestion in the mouth and stomach was minor compared with that in the small intestine. Furthermore, the fully branched starch distribution of the in vivo digesta from SI-3 showed the disappearance of native amylopectin and most of the native amylose, whose presence could still be observed in the in vivo digesta from SI-2 (Figure 5). This suggested that the raw normal maize starch granules were rapidly and almost completely hydrolyzed in the upper part of the small intestine (SI-2), in agreement with that observed by Weurding et al.41 in the small intestine of broiler chickens, but different from in vitro results, suggesting that raw normal maize starch is a good source of SDS.15 There were some differences in the starch digestion rate among the seven pigs as seen in the starch contents of the in vivo digesta (Supporting Information) and the fully branched and debranched starch distributions from individual pigs (Figures 5 and 6 and Tables 1 and 2). The fully branched and debranched starch distributions of the SI-2 digesta from P-7 were similar to those of the SI-3 from the same pig (Figure 5), which was not observed in other pigs, consistent with the finding that starch contents of SI-1 and -2 digesta from P-7 were lower than those from other pigs (Supporting Information). Furthermore, the fully branched starch distributions of SI-1 and -2 digesta from some pigs (P-2, -3, -4, and -6) showed the presence of smaller dextrins (peak Rh at ∼1.5-2.5 nm) similar to those of the SI-3 digesta (Figure 5 and Table 1). In P-2 and -4, these smaller dextrins disappeared as the digesta traveled from SI-1 to SI-2. The results implied that the dextrins produced from the in vivo digestion of raw starch granules by pancreatic R-amylase in the intestinal lumen were transferred rapidly to the surface of the brush border where they were further hydrolyzed to glucose by maltaseglucoamylase and sucrase-isomaltase. Maltase-glucoamylase has the ability to hydrolyze R-1,4 linkages and remove single glucose unit from the nonreducing end of dextrins, whereas sucrase has the ability to hydrolyze R-1,4 linkages of smaller dextrins, such as maltose and maltotriose, and isomaltase prefers to hydrolyze R-1,6 linkages of dextrins from the nonreducing end.6,7 Thus, these dextrins were not always detected in the in vivo digesta collected from the intestinal lumen. Similar to what was observed in the in vitro digestion, amylose and amylopectin were hydrolyzed at approximately the same rate in the small intestine, as revealed by the debranched starch distributions of the in vivo digesta from SI-1 and -2 (Figure 6) showing no or small apparent change in the ratio of amylopectin to amylose during the in vivo digestion. Furthermore, the fully branched starch distributions of the in vivo digesta from the SI-3 of all pigs (Figure 5 and Table 1) showed the appearance of dextrins that seemed to be less susceptible to amylolytic enzymes than the native starch molecules, which was similar to that of the in vitro digesta after 25 h of digestion with pancreatin and amyloglucosidase. Some of these dextrins


seemed to be linear and some contained branches larger than native amylopectin branches or the same as amylose branches, as shown by the debranched starch distributions (Figure 6 and Table 2). The long branches and linear molecules can arrange themselves into condensed semicrystalline structures that are less susceptible to amylolytic enzymes than the native granular structure.22,38-40 The fully branched and debranched starch distributions of the in vivo digesta, however, had major qualitative differences from those of the in vitro digesta (Figures 5 and 6). The fully branched distributions of the in vivo digesta, in general, did not show the apparent degradation of amylopectin as the digesta traveled from SI-1 to SI-2 in such a way as seen in the in vitro digesta even for simulated oral and gastric digestion (Figure 5). In general, the size distribution of starch molecules of the normal maize starch before digestion seemed to be preserved during the in vivo digestion although the electron micrographs showed that the starch granules were evidently digested by the enzymes in the intestinal lumen (Figure 3). This implies aspects of an “all or none” mechanism in which a significant number of molecules/granules are hydrolyzed to completion before other substrates are digested in vivo, whereas more molecules/granules are hydrolyzed simultaneously in vitro. The greater homogeneity of granule morphology during in vitro (Figure 4) than during in vivo (Figure 3) digestion supports this suggestion. Furthermore, the amylopectin in the fully branched distributions of the in vivo digesta from SI-3 was undetectable, whereas a small amount of amylopectin (appearing as a peak tail) was still present in the in vitro digesta after 25 h of digestion. The starch molecules are hydrolyzed to glucose by the in vivo amylolytic enzymes in a conservative way that preserved the original size distribution of starch in the remaining unhydrolyzed starch molecules at the beginning of the digestion, whereas the starch molecules in the in vitro digestion were hydrolyzed to dextrins, not glucose, which still had a quite substantial hydrodynamic size. In addition, the distributions of debranched starch from the in vitro digesta after 25 h of digestion showed only a single peak, whereas those from the in vivo digesta from SI-3 of pigs were more complex (Figure 6 and Table 2). This might be attributed to the longer digestion time for the in vitro method, which allows the amylose and the dextrins produced from the digestion to crystallize to a more uniform crystalline structure.22,38-40 The single amylose branch peak (Rh ) ∼15 nm, DP ) ∼6000) and the new species of glucan branch (peak Rh ) ∼3.5 nm, peak DP ) ∼250) present in the debranched starch distributions of all in vivo digesta from SI-3 were not observed in the in vitro digesta after 25 h of digestion with pancreatin and amyloglucosidase. Overall, the results demonstrate that different mechanisms of enzyme hydrolysis occurred in the in vivo and in vitro digestion, which could be attributed to differences in, for example, the mixing efficiency, enzyme activity (type, concentration, and proximity), and incubation time of the two digestion methods.

Conclusion Information regarding starch digestibility, such as digestion rate and enzyme resistance, are of importance for diet-related disorders including obesity and diabetes. In vitro digestion methods are commonly used to obtain such information instead of in vivo experiments, because of better reproducibility and for practical purposes (in vivo animal or human studies are much more expensive and involve important ethical considerations).


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This study shows that the commonest type of in vitro method used to analyze starch digestibility exhibits qualitatively different behavior from that in a true in vivo system. This suggests that qualitatively incorrect inferences about nutritional properties of starch in food products may result from applying the most frequently used type of in vitro techniques, such as those proposed by Englyst et al.20 and Shin et al.,42 and that improved in vitro methods are required. This conclusion is drawn from a comparison of the structural differences of raw starch (contained within a diet mix with other components such as meat) during digestion using a common in vitro model system and in a true in vivo one (pigs, a good model for human digestion). Raw normal maize starch granules were rapidly and almost completely hydrolyzed in the upper part of the small intestine (in vivo). Both amylose and amylopectin in the starch granules were hydrolyzed at similar rates by amylolytic enzymes in the in vivo and in vitro digestion. The rapid degradation of amylopectin to similar size of amylose observed in the in vitro digestion was, however, not apparent in the in vivo results, which seemed to preserve the native structure of amylopectin and amylose during digestion. On the other hand, less amylopectin was observed after prolonged in vivo digestion than prolonged in vitro digestion. Furthermore, the dextrins in the digesta after prolonged in vitro digestion showed a uniform distribution with a single peak, whereas the dextrins in the in vivo counterpart showed a more complex distribution. The results showed that the heterogeneous and complex conditions applying during the in vivo digestion are not reproduced by current in vitro digestion protocols, which are relatively homogeneous. The different populations of starch molecules and their structures in the in vivo and in vitro digesta implied that the RDS and SDS contents analyzed using in vivo and in vitro digestion are likely to be different. These differences should be taken into an account when analyzing starch digestibility using in vitro methods, especially for the nutritional evaluation of starch-containing foods. Acknowledgment. The authors thank the Australian Research Council (DP0985694) for funding and the High Fibre Grains Cluster (supported by a grant from the CSIRO Flagship Collaboration Fund via the Food Futures Flagship) for providing the small intestine digesta samples. The authors also thank Mr. Simon Boman for his assistance with the figures. Supporting Information Available. Details of experimental techniques for extraction and obtaining size distributions. This material is available free of charge via the Internet at http:// pubs.acs.org.

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In Vivo and In Vitro Starch Digestion: Are Current in Vitro Techniques

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