Resistant Starch A Review

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1 Resistant Starch A Review M.G. Sajilata, Rekha S. Singhal, and Pushpa R. Kulkarni ABSTRACT CT: The concept of resistant starch (RS) has evoked new interest est in the bioavailability ailability of starch and in its use as a source of dietary fiber,, particularly in adults.. RS is now considered ed to provide functional proper operties and find applica- tions in a var ariety of foods. Types of RS, factors influencing their formation, consequence of such formation, their methods of prepar eparation, ation, their methods of estimation, and health benefits have been briefly discussed in this review eview. Keywor eywords: resistant starch (RS), functionality,, formation, prepar eparation, ation, determination, digestibility,, physiological effects, applications, commercial sources Introduction From the early years of emergence of nutritional science, it has been recognized that the ingested nutrients in the diet are not completely utilized in the body. An increasing volume of evidence suggests that with very few exceptions, only a proportion of the total ingested nutrients in a diet or food is available, and the term availability has come into use for this proportion (Southgate 1989). The nutrients measured by chemical analysis may not always be fully utilizable, mainly due to the indigestible cell walls, a bulky or dense structure, a low solubility, the presence of some compounds inhibiting the digestion, as well as components abundantly present in plant foods such as dietary fiber, phytic acid, and tannic acid, which may significantly reduce the absorption and utilization of some nutrients (Rosado and others 1987). During food processing, derivatization of nutrients and formation of cross linkages occur, thereby making the food inaccessible for digestion or/and metabolism. Such parts of nutrients are also unavailable (Erbersdobler 1989). Starch, which is the major dietary source of carbohydrates, is the most abundant storage polysaccharide in plants, and occurs as granules in the chloroplast of green leaves and the amyloplast of seeds, pulses, and tubers (Ellis and others 1998). The relatively recent recognition of incomplete digestion and absorption of starch in the small intestine as a normal phenomenon has raised interest in nondigestible starch fractions (Cummings and Englyst 1991; Englyst and others 1992). These are called resistant starches, and extensive studies have shown them to have physiological functions similar to those of dietary fiber (Asp 1994; Eerlingen and Delcour 1995). The diversity of the modern food industry and the enormous variety of food products it produces require starches that can tolerate a wide range of processing techniques and MS Submitted 2/28/05, Revised 8/2/05, Accepted 10/29/05. The authors are with Food Engineering and Technology Dept., Inst. of Chemical Technology, Matunga, Mumbai , India. rekha@udct.org. preparation conditions (Visser and others 1997). These demands are met by modifying native starches with chemical, physical, and enzymatic methods (Betancur and Chel 1997), which may lead to the formation of indigestible residues. The availability of such starches therefore deserves consideration. This review therefore focuses on the availability of the major nutrient, that is, the starch, with special reference to RS. Starch and its classification Chemically, starches are polysaccharides, composed of a number of monosaccharides or sugar (glucose) molecules linked together with -D-(1-4) and/or -D-(1-6) linkages. The starch consists of 2 main structural components, the amylose, which is essentially a linear polymer in which glucose residues are -D-(1-4) linked typically constituting 15% to 20% of starch, and amylopectin, which is a larger branched molecule with -D-(1-4) and -D-(1-6) linkages and is a major component of starch (BNF 1990). Amylose is linear or slightly branched, has a degree of polymerization up to DP 6000, and has a molecular mass of 105 to 106 g/mol. The chains can easily form single or double helices (Takeda and Takeda 1989). On the basis of X-ray diffraction studies on oriented amylase fibers, the presence of type A and type B amyloe is indicated (Figure 1, Galliard 1987). The structural elements of type B are double helices, which are packed in an antiparallel, hexagonal mode. The central channel surrounded by 6 double helices is filled with water (36 H 2 O/unit cell). Type A is very similar to type B, except that the central channel is occupied by another double helix, making the packing closer. In this type, only 8 molecules of water per unit cell are inserted between the double helices. Amylopectin (107 to 109 g/mol) is highly branched and has an average DP of 2 million, making it one of the largest molecules in nature. Chain lengths of 20 to 25 glucose units between branch points are typical. Its structure is often described by a cluster model (Figure 2). The cluster model gained greater credence when Hizukuri postulated that amylopectin 2006 Institute of Food Technologists Vol. 5, 2006 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 1

2 CRFSFS: Comprehensive Reviews in Food Science and Food Safety chains are either located within a single cluster or serve to connect 2 or more clusters (Hizukuri 1986; Thompson 2000). Short chains (A) of DP that can form double helices are arranged in clusters. The clusters comprise 80% to 90% of the chains and are linked by longer chains (B) that form the other 10% to 20% of the chains. Most B-chains extend into 2 (DP about 40) or 3 clusters (DP about 70), but some extend into more clusters (DP about 110) (Figure 2 and 3) ( On the basis of X-ray diffraction experiments, starch granules are said to have a semicrystalline character, which indicates a high degree of orientation of the glucan molecules. About 70% of the mass of starch granule is regarded as amorphous and about 30% as crystalline. The amorphous regions contain the main amount of amylose but also a considerable part of the amylopectin. The crystalline region consists primarily of the amylopectin. Various ways to classify native starches X-ray diffraction. Three types of starches, designated as type A, type B, and type C, have been identified based on X-ray diffraction patterns. These depend partly on the chain lengths making up the amylopectin lattice, the density of packing within the granules, and the presence of water (Wu and Sarko 1978). Although type A and type B are real crystalline modifications, type C is a mixed form. The important features of the types of starches are as follows. Type A. The type A structure has amylopectin of chain lengths of 23 to 29 glucose units. The hydrogen bonding between the hydroxyl groups of the chains of amylopectin molecules results in the formation of outer double helical structure. In between these micelles, linear chains of amylose moieties are packed by forming hydrogen bonds with outer linear chains of amylopectin. This pattern is very common in cereals. Type B. The type B structure consists of amylopectin of chain lengths of 30 to 44 glucose molecules with water inter-spread. This is the usual pattern of starches in raw potato and banana. Type C. The type C structure is made up of amylopectin of chain lengths of 26 to 29 glucose molecules, a combination of type A and type B, which is typical of peas and beans. An additional form, called type V, occurs in swollen granules. X- ray diffraction diagrams of these starches are shown in Figure 4. Based on the action of enzymes According to Berry (1986), starches can be classified according to their behavior when incubated with enzymes without prior exposure to dispersing agents as follows. Rapidly digestible starch (RDS). RDS consists mainly of amorphous and dispersed starch and is found in high amounts in starchy foods cooked by moist heat, such as bread and potatoes. It is measured chemically as the starch, which is converted to the constituent glucose molecules in 20 min of enzyme digestion. Figure 1 Unit cells and arrangement of double helices (cross section) in A-amylose (top) and B-amylose (bottom) (Galliard 1987) Figure 2 Cluster model of amylopectin 2 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY Vol. 5, 2006

3 Resistant starch - a review Slowly digestible starch (SDS). Like RDS, SDS is expected to be completely digested in the small intestine, but for 1 reason or another, it is digested more slowly. This category consists of physically inaccessible amorphous starch and raw starch with a type A and type C crystalline structure, such as cereals and type B starch, either in granule form or retrograded form in cooked foods. It is measured chemically as starch converted to glucose after a further 100 min of enzyme digestion. Resistant starch. The term resistant starch was first coined by Englyst and others (1982) to describe a small fraction of starch that was resistant to hydrolysis by exhaustive -amylase and pullulanase treatment in vitro. RS is the starch not hydrolyzed after 120 min of incubation (Englyst and others 1992). However, because starch reaching the large intestine may be more or less fermented by the gut microflora, RS is now defined as that fraction of dietary starch, which escapes digestion in the small intestine. It is measured chemically as the difference between total starch (TS) obtained from homogenized and chemically treated sample and the sum of RDS and SDS, generated from non-homogenized food samples by enzyme digestion. RS = TS (RDS + SDS) Figure 3 (a) Shows the essential features of amylopectin. (b) Shows the organization of the amorphous and crystalline regions (or domains) of the structure generating the concentric layers that contribute to the growth rings that are visible by light microscopy. (c) Shows the orientation of the amylopectin molecules in a cross section of an idealized entire granule. (d) Shows the likely double helix structure taken up by neighboring chains and giving rise to the extensive degree of crystallinity in granule ( Based on the nutritional characteristics This classification is based on the extent of digestibility of the starch as follows. Digestible starches. These include the starches digestible by body enzymes, namely the rapidly digestible starches (RDS) and the slowly digestible starches (SDS). RDS consists mainly of amorphous and dispersed starch, found in high amounts in starchy foods cooked by moist heat. Like RDS, SDS is expected to be completely digested in the small intestine, but for 1 reason or another, it is digested more slowly. Resistant starch. RS is indigestible by body enzymes. It is subdivided into 4 fractions: RS 1, RS 2, RS 3, and RS 4. These are also called as type I, II, III, and IV starches. RS 1 represents starch that is resistant because it is in a physically inaccessible form such as partly milled grains and seeds and in some very dense types of processed starchy foods. It is measured chemically as the difference between the glucose released by the enzyme digestion of a homogenized food sample and that released from a nonhomogenized sample. RS 1 is heat stable in most normal cooking operations and enables its use as an ingredient in a wide variety of conventional foods. RS 2 represents starch that is in a certain granular form and resistant to enzyme digestion. It is measured chemically as the difference between the glucose released by the enzyme digestion of a boiled homogenized food sample and that from an unboiled, nonhomogenized food sample. In raw starch granules, starch is tightly packed in a radial pattern and is relatively dehydrated. This compact structure limits the accessibility of digestive enzymes, various amylases, and accounts for the resistant nature of RS 2 such as, ungelatinized starch. In the diet, raw starch is consumed in foods like banana. RS 1 and RS 2 represent residues of starch forms, which are digested very slowly and incompletely in the small intestine. RS 3 represents the most resistant starch fraction and is mainly retrograded amylose formed during cooling of gelatinized starch. Most moist-heated foods therefore contain some RS 3. It is measured chemically as the fraction, which resists both dispersion by boiling and enzyme digestion. It can only be dispersed with KOH or dimethyl sulphoxide (Asp and Bjorck 1992). RS 3 is entirely resistant to digestion by pancreatic amylases. Therefore, RS 1 = TS (RDS + SDS) RS 2 RS 3 RS 2 = TS (RDS + SDS) RS 1 RS 3 Figure 4 X-ray diffraction diagrams of starches: type A (cereals), type B (legumes), and type V (swollen starch, V a : water-free, V h : hydrated) (Galliard 1987) RS 3 = TS (RDS + SDS) - RS 2 RS 1 RS 4 is the RS where novel chemical bonds other than -(1-4) or Vol. 5, 2006 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 3

4 CRFSFS: Comprehensive Reviews in Food Science and Food Safety Table 1 Classification of types of resistant starch (RS), food sources, and factors affecting their resistance to digestion in the colon (Nugent 2005) Type of RS Description Food sources Resistance minimized by RS 1 Physically protected Whole- or partly milled grains and Milling, chewing seeds, legumes RS 2 Ungelatinized resistant granules Raw potatoes, green bananas, some Food processing and cooking with type B crystallinity, slowly legumes, high amylose corn hydrolyzed by -amylase RS 3 Retrograded starch Cooked and cooled potatoes, bread, Processing conditions cornflakes, food products with repeated moist heat treatment RS 4 Chemically modified starches Foods in which modified starches have Less susceptible to digestibility due to cross-linking with been used (for example, breads, cakes) in vitro chemical reagents Table 2 In vitro digestibility of starch in a variety of foods (BNF 1990) a Foods % RDS % SDS % RS 1 %RS 2 %RS 3 Flour, white Traces Short bread Traces Bread, white Bread, whole meal Spaghetti, white Biscuits made with Traces 50% raw banana flour Biscuits made with Traces 50% raw potato flour Peas, chick, canned Beans, dried, freshly Traces 6 cooked Beans, red kidney, canned a Values are expressed as % of the total starch present in the food. -(1-6) are formed. Modified starches obtained by various types of chemical treatments are included in this category. Table 1 outlines a summary of the different types of RS, their classification criteria, and food sources. In vitro digestibility of starch in a variety of foods (BNF 1990) is shown in Table 2. Structure of RS RS 1RS1 is the physically protected form of starch found in whole grains ( nutrition.com). Figure 5 shows microscopic view of the physically inaccessible RS 1 in cell or tissue structures of partly milled grains, seeds, and vegetables. RS 2 In raw starch granules, starch is tightly packed in a radial pattern and is relatively dehydrated. This compact structure limits the accessibility of digestive enzymes and accounts for the resistant nature of RS 2 such as, ungelatinized starch. Figure 6 shows the RS granules, that is, raw potato, banana, and high-amylose starch ( nutrition.com). RS 3RS3 represents retrograded starch. Thus, in the formation of RS 3, the starch granule is completely hydrated. Amylose leaches from the granules into the solution as a random coil polymer. Upon cooling, the polymer chains begin to reassociate as double helices, stabilized by hydrogen bonds (Wu and Sarko 1978). The individual strands in the helix contain 6 glucose units per turn in a 20.8 A repeat. The models for the double helices are left-handed, parallelstranded helices. A type A crystalline structure can be obtained if RS is formed in gelatinized starch stored at high temperature (that is, 100 C) for several hours (Eerlingen and others 1993a). It has a dense structure and only few water molecules in the monoclinic unit cell. Upon further retrogradation, the double helices pack in a hexagonal unit cell. The B form with hexagonal symmetry is more open. Water molecules (36 to 42 molecules per unit cell) in the B structure are located in fixed positions within a central channel formed by 6 double helices. The degree of polymerization (DP) of amylose also affects the yield of RS 3; it rises with DP up to 100 and thereafter remains constant (Eerlingen and others 1993b). A minimum DP of 10 and a maximum of 100 seems to be necessary to form the double helix (Gidley and others 1995). Schematic presentation of RS 3 formed in aqueous amylose solutions depicted as miscelle and lamella model is shown in Figures 7 and 8. Structural features of in vivo RS (ingestion of retrograded highamylose maize starch, complexed high amylose maize starch, bean flakes, or potato flakes) were assessed using the ileal contents of 4 humans (Faisant and others 1993). For all samples, starch fractions, which escaped digestion in the small intestine, were composed of 3 populations of -glucans with proportions differing according to the substrate. Small quantities of oligosaccharides made up the 1st population, illustrating a limitation of absorption in the small intestine. The 2nd population, the main RS, consisted of retrograded amylose of mean degree of polymerization (DPn) of about 35 glucose units with a melting temperature of 150 C and exhibiting a type B pattern. Finally high-molecular-weight semicrystalline -glucans were attributed to fragments of starch. This study showed that some potentially digestible starch could reach the colon and that crystalline fractions constituted only part of the starch that escaped digestion in the human small intestine. RS 4 Structure of RS4 includes structures of modified starches obtained by chemical treatments like distarch phosphate ester (Figure 9). Figure 5 Structure of resistant starch type I (RS 1 ) Figure 6 Structure of resistant starch type II (RS 2 ) 4 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY Vol. 5, 2006

5 Resistant starch - a review Functionality of RS RS has a small particle size, white appearance, and bland flavor. RS also has a low water-holding capacity. It has desirable physicochemical properties (Fausto and others 1997) such as swelling, viscosity increase, gel formation, and water-binding capacity, making it useful in a variety of foods. These properties make it possible to use most resistant starches to replace flour on a 1-for- 1 basis without significantly affecting dough handling or rheology. RS not only fortifies fiber but also imparts special characteristics not otherwise attainable in high-fiber foods (Tharanathan and Mahadevamma 2003). The functional properties and advantages of commercial sources of RS 2 and RS 3 (Nugent 2005) have been summarized as follows. They are natural sources, bland in flavor, white in color, with fine particle size (which causes less interference with texture). They have high gelatinization temperature, good extrusion and film-forming qualities, and lower water-holding properties than traditional fiber products. They allow the formation of low-bulk high-fiber products with improved texture, appearance, and mouth feel (such as better organoleptic qualities) Figure 7 Schematic presentation of enzyme resistant starch type III (RS 3 ) formed in aqueous amylose solutions. Micelle model. Double helices are ordered into a crystalline structure (C) over a particular region of the chain, interspersed with amorphous, enzyme degradable regions. compared with traditional high-fiber products; they increase coating crispness of products and the bowl life of breakfast cereals. They are functional food ingredients lowering the calorific value of foods and useful in products for coeliacs, as bulk laxatives and in products for oral rehydration therapy. Some of these properties of RS have been successfully used in a range of baked and extruded products as described subsequently. RS in bread-making for DF fortification ( The physical properties of RS, particularly its low water-holding capacity, allow it to be a functional ingredient that provides good handling in processing and crispness, expansion, and improved texture in the final product. Bread is commonly fortified with dietary fiber. However, dark color, reduced loaf volume, poor mouthfeel, and masking of flavor are all negative attributes that are often associated with high-fiber breads. A study was conducted at the American Inst. of Baking (AIB) to evaluate the effect of RS on bread characteristics and to compare their performance to traditional fibers. The study included cellulose, oat fiber, wheat fiber, and 2 commercially available RS with 23% (Hylon VII starch) and 40% TDF (Novelose 240 starch) and a blend of oat fiber with Novelose 240 starch in a 50/50 ratio based on TDF contribution. Compared with oat fiber, cellulose, and wheat fiber, both RS had a lower water-holding capacity, which is similar to that of flour. Although the water-holding capacity of the RS was lower than that of the other fiber sources, the quantity required to obtain the same level of TDF was greater. This raised the total water requirement of the RS dough. However, absorption of the RS doughs was less compared with the fiber doughs, despite a larger quantity of water used. The lower dough absorption consequently had less impact on dough rheology and was closest to the white pan bread dough. Bread containing 40% TDF RS had greater loaf volume and better cell structure compared with traditional fibers tested (Baghurst and others 1996). Figure 8 Schematic presentation of enzyme-resistant starch type III formed in aqueous amylose solutions. Lamella model. Lamellar structures are formed by folding of the polymer chains. The fold zones are amorphous (A), while the center of the lamella is crystalline (C). Figure 9 Preparation of cross-bonded starch Figure 10 Behavior of amylose molecules during cooling of a concentrated aqueous solution Vol. 5, 2006 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 5

6 CRFSFS: Comprehensive Reviews in Food Science and Food Safety RS as a texture modifier in baked goods One way to ensure that the general population receives adequate amounts of fiber in the diet is to fortify good-tasting foods that normally do not come to mind with fiber fortification but are often eaten as breakfast items or snacks. RS were incorporated in a variety of baked goods, many of which include batter systems, such as in cakes, cake-like muffins, or brownies. In general, application tests showed that RS acts as texture modifier, imparting a favorable tenderness to the crumb. A low-fat, loaf cake was formulated with RS and various fibers to obtain approximately 3% TDF or 2.5 g of fiber per 80 g serving. These included a 40% TDF RS (Novelose 240 starch), oat fiber, a blend of oat fiber with Novelose 240 starch in a 50/50 ratio based on TDF contribution, and a 23% TDF RS (Hylon VII starch). The baked cakes made with RS were similar to that containing oat fiber and the control in the amount of moisture loss after baking, height, specific volume, and density. A panel rated the 40% TDF RS loaf cakes as the best for flavor, grittiness, moisture perception, and tenderness 24 h after baking. RS as a crisping agent Among other functional properties, RS can be used as an ingredient that improves crispness in foods where high heat is applied to a product s surface during processing. French toast and waffles, especially frozen reheated types, represent foods in which surface crispness is desired. Tests were conducted to compare the functionality of RS and various fibers in a buttermilk waffle formulation. Based on the evaluation of the toasted waffles for initial crispness, crispness after 3 min, moistness, and overall texture by a trained sensory panel RS waffle indicated greater crispness than control or traditional fiber. RS as a functional ingredient in other foods Along with textural enhancement, RS can improve expansion in extruded cereals and snacks. Various cereals were formulated to contain 40% TDF RS (Novelose 240 starch) alone and in combination with oat fiber in ratios of 50/50 and 25/75 based on weight. The cereal with RS and no oat fiber had greater volumetric expansion than the control. In blends with oat fiber, the cereal containing 75% of RS had better expansion than the one containing only 50%. Dried pasta products containing up to 15% RS can be made with little or no effect on dough rheology during extrusion. Although the resultant pasta was lighter in color, a firm al dente texture was obtained in the same cooking time as a control that had no added fiber. RS may also be used in thickened, opaque health beverages in which insoluble fiber is desired. Insoluble fibers generally require suspension and add opacity to beverages. Compared with insoluble fibers, RS imparts a less gritty mouthfeel and masks flavors less. Factors influencing the formation of RS Several factors influence the formation of RS. Inherent properties of starch Crystallinity of starch. One of the causes of resistance to enzymes is the crystallinity of native type B starch granules as observed in the case of amylomaize starch and also the encapsulation of starch within plant cell or tissue structures. X-ray diffraction and differential scanning calorimetry studies on crystalline residues from amylomaize starch samples have suggested that chain fragments packed in a type B crystalline structure with a slightly enlarged crystal lattice contribute to formation of RS from amylomaize starch. Any treatment that eliminates starch crystallinity (that is, gelatinization) or the integrity of the plant cell or tissue structure (that is, milling) increases enzyme availability and reduces the content of RS, whereas recrystallization and chemical modifications tend to increase the RS. The modified food starches are partially resistant to enzymes as a result of chemical modifications induced intentionally (Englyst and Cummings 1986; Bjorck and others 1989; Schweizer and others 1990). Besides these, the cellular structure of plant foods influences the digestibility of starch in the small intestine as well as the intrinsic digestibility of a particular physical form of starch. Granular structure. A large variability in susceptibility to amylases shown by raw starch granules also influences RS formation. Potato starch and high amylose maize starch are known to be very resistant in vitro and incompletely absorbed in vivo, whereas most cereal starches are slowly but virtually completely digested and absorbed in vivo (Holm and others 1987). The smaller surface-to-volume ratio of the large potato granules is probably important. The nature of the granule surface also needs to be considered; an adsorbed layer of non-starch material would effectively impede the action of the enzyme (Ring and others 1988). Raw tepary starch is found to be more resistant to hydrolysis than maize starch, perhaps due to differences in granule structure and amylose content (Abbas and others 1987). Amylose:amylopectin ratio. A higher content of amylose lowers the digestibility of starch due to positive correlation between amylose content and formation of RS (Berry 1986; Sievert and Pomeranz 1989b). The importance of the amylose:amylopectin ratio in the postprandial glycaemic and insulinaemic responses to corn was studied in commonly consumed corn products (Granfeldt and others 1995). The meals containing high amylose (70%) corn flour had an RS of 20 g/100 g DM than that containing ordinary corn flour (25% amylose) that had RS of 3 g /100 g DM. Retrogradation of amylose. When heated to about 50 C, in the presence of water, the amylose in the granule swells; the crystalline structure of the amylopectin disintegrates and the granule ruptures. The polysaccharide chains take up a random configuration, causing swelling of the starch and thickening of the surrounding matrix such as, gelatinization a process that renders the starch easily digestible. On cooling/drying, recrystallization (retrogradation) occurs. This takes place very fast for the amylose moiety as the linear structure facilitates cross linkages by means of hydrogen bonds. Figure 10 shows the formation of gel and micelle on cooling of a concentrated solution of amylose (Belitz and Grosch 1999). The branched nature of amylopectin inhibits its recrystallization to some extent and it takes place over several days. Retrograded amylose in peas, maize, wheat, and potatoes was found to be highly resistant to amylolysis (Ring and others 1988). The rate and extent to which a starch may retrograde after gelatinization essentially depends on the amount of amylose present. Repeated autoclaving of wheat starch may generate up to 10% RS. The level obtained appeared to be strongly related to the amylose content, and the retrogradation of amylose was identified as the main mechanism for the formation of RS that can be generated in larger amounts by repeated autoclaving (Berry 1986; Bjorck and others 1990). During storage, the dispersed polymers of gelatinized starch are said to undergo retrogradation to semicrystalline forms that resist digestion by pancreatic -amylase. It forms a major portion of RS in wheat bread and corn flakes (Englyst and Cummings 1985), whereas only 25% of the RS in cooked, cooled potatoes can be accounted for as retrograded amylose. The digestibility of legume starch is much lower than that of cereal starch, which is attributable to higher content of amylose in the former. The digestibility of high amylose cereal starch is reported to be significantly lower (Tharanathan and Mahadevamma 2003). Native high-amylose starch is known to be high in type II RS (RS 2 ) (Berry 1986), which is defined as starch in its native granular state that is resistant to digestion in the small intestine. This after cooking and cooling gives high yields of type III RS (Berry 1986; Sievert and Pomeranz 1989a) or retrograded starch (Englyst and 6 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY Vol. 5, 2006

7 Resistant starch - a review others 1992). Heating of RS preparation from amylomaize VII resulted in broad endothermic transition, which is ascribed to melting of amylose crystallites (Sievert and Pomeranz 1989, 1990). Exothermic transitions during controlled cooling of isolated potato amylose fractions have been attributed to amylose chain association. The formation of RS likewise has been attributed to the ordering of amylose chains (Sievert and others 1991). Based on previous studies of amylose behavior, it has been suggested that the exotherms observed during the cooling of either amylose or a thermally treated RS preparation reflect chain association, which may involve amylose aggregation and gelation dominated by formation and subsequent lateral aggregation of type B double helices in crystalline arrays (Gidley 1989; Gidley and Bulpin 1989; Sievert and Wursch 1993). Gelatinized waxy corn starch stored at varying temperatures from 6 C to 60 C for 1 to 29 d also showed reduced enzyme susceptibility to pancreatic -amylase and amyloglucosidase (Eerlingen and others 1994). Influence of amylose chain length. Influence of amylose chain length on enzyme RS formation was studied by Eerlingen and others (1993b) by hydrolyzing potato starch amylose to varying degrees by incubation with barley -amylase for different periods, and monitored by measuring the number of average chain lengths or degree of polymerization (DP n ). The DP n of RS varied between 19 and 26 and was independent of the chain length of the amylose (DP n 40 to 610) from which it was formed. Results suggested that RS might be formed by aggregation of amylose helices in a crystalline -type structure over a particular region of the chain (about 24 glucose units). Linearization of amylopectin. Linearization of amylopectin occurs during the long low-temperature baking process due to the prolonged activity of intrinsic amylases in the dough, and is prominent in the presence of certain organic acids that is, in bread products baked with added lactic acid (Liljeberg and others 1996). It has been reported to significantly increase RS formation during wet-autoclaving (Berry 1986). Heat and moisture Water content is an important factor that affects formation of RS. Repeated heat/moisture treatment is associated with a decrease in the hydrolysis limit of pancreatic -amylase and increased formation of RS. Maximum RS yield was obtained at a starch:water ratio of 1: 3.5 (w/w) (Sievert and Pomeranz 1989b) and a heat treatment at 18% moisture gave increased levels of the degree of crystallinity of normal and waxy starches and thus reduced enzyme susceptibility. However, at 27% moisture, starch degradation to some extent made areas of starch more accessible to enzyme attack. Thus, proper heat treatment could be used as a method of preparation of RS (Franco and others 1995). In addition, higher temperature and less water results in type A configuration, whereas lower temperature and high water content results in type B configuration (Wu and Sarko 1978). Malshick-Shin and others (2003) determined solubility, water vapor sorption, and swelling characteristics for RS prepared from wheat starch and linterized wheat starch by autoclaving and cooling and by cross-linking The experimental RS made from wheat starch contained 10% to 73% RS versus 58% and 40% in commercial sources, Novelose 240 and 330 respectively, produced from high-amylose maize (corn) starch. In excess water, the experimental RS starches (except for the cross-linked wheat starch) gained 3 to 6 times more water than the commercial RS starches at 25 C, and 2 to 4 times more at 95 C. All starches showed similar water vapor sorption and desorption isotherms at 25 C and a w < 0.8. At a w 0.84 to 0.97, the RS made from wheat starch (except cross-linked wheat starch) showed approximately 10% higher water sorption than the commercial RS. RS determined in several selected cereals, legumes, and tubers subjected to dry and wet heat treatment brought out higher RS contents in foods subjected to dry heat treatment compared with wet processed ones. Sorghum, green gram dhal, and green plantain showed highest RS content (5.51%, 5.81%, and 10.7%, respectively) (Platel and Shurpalekar 1994). Interaction of starch with other components Interactions of starch with different components present in the food system are known to influence the formation of RS as follows. Protein. Starch-protein interaction has been believed to reduce RS contents as observed in case of potato starch and added albumin when autoclaved and subsequently cooled at 20 C (Escarpa and others1997). Dietary fiber. Insoluble dietary fiber constituents such as cellulose and lignin have been shown to have minimal effects on RS yields compared with other constituents such as potassium and calcium ions and catechin (Escarpa and others 1997). Enzyme inhibitors. Polyphenols, phytic acid, and lectins present mainly in leguminous seeds, have been reported to inhibit in vitro starch hydrolysis and to lower the glycemic index (Thompson and Yoon 1984). Tannic acid significantly inhibits both amylases and intestinal maltase activity (Bjorck and Nyman 1987). Indigestible residues from black beans (Phaseolus vulgaris cv. Tacari gua), green beans (P. vulgaris), carrots (Daucus carota), and rice bran (Oryza sativa) are all reported to inhibit pancreatic amylase in vitro (Moron and others 1989). Since amylolysis is inhibited by phytic acid, a decrease in phytate content increases starch digestibility (Thompson and Yoon 1984). Contradictory information exists in the literature on this aspect. The autoclaving and subsequent cooling of potato starch and catechin was found to significantly reduce the yields of RS, whereas the addition of phytic acid to potato starch reduced the RS contents to a minor extent (Escarpa and others 1997) compared with the RS formed from potato starch with no added constituent. The reasons for the same are still not clear. Ions. The yields of RS in potato starch gels decrease in the presence of calcium and potassium ions compared with those with no added constituent (Escarpa and others 1997), presumably due to the prevention of formation of hydrogen bonds between amylose and amylopectin chains caused by adsorption of these ions. Sugars. The addition of soluble sugars such as glucose, maltose, sucrose, and ribose has been found to reduce the level of crystallization and subsequently reduce the yields of RS (Buch and Walker 1988; I Anson and others 1990; Kohyama and Nishinari 1991). The mechanism of retrogradation inhibition was considered as the interaction between sugar molecules and the starch molecular chains, which change the matrix of gelatinized starch (the sugars act as anti-plasticizers and increase the glass transition temperature). The role of sugars on the formation of RS in starch gels (RS type III) was studied by Eerlingen and others (1994). Sugars influenced the RS levels in starch gels only when added in high concentration (final starch-water-sugar ratio of 1:10:5 w/w). In wheat starch gels, the RS yields decreased from approximately 3.4% to 2.8% in the presence of sucrose or glucose, and to 2.5% in the presence of ribose or maltose. An increase in RS yield was observed with high-amylose corn starch. The experiments showed that the differences in gelatinization temperature, lipid content, and apparent amylose content of the 2 starches were not the main causes of the different impact of sugars on RS yields. Lipids, emulsifiers. In a study, amylomaize VII starch, autoclaved at 125 C, was reacted during cooling below 100 C with lysophosphatidyl choline (LPC), sodium stearoyl lactylate (SSL), and hydroxylated lecithin (OHL) (Czuchajowska and others 1991). Differential scanning calorimetry (DSC) peaks at around Vol. 5, 2006 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 7

8 CRFSFS: Comprehensive Reviews in Food Science and Food Safety 95 C to 110 C indicated formation of amylose-lipid complexes, and at about 155 C indicated the presence of enzyme-resistant starch (RS). Yields of RS from complexed samples isolated by thermostable bacterial -amylase or amyloglucosidase were lower than yields of RS from the autoclaved and cooled control. Formation of complexes competes with amylose chains involved in generation of RS. Amylose-lipid complexes are enzyme-degradable, and an increase in complexed amylose reduced yields of RS. Amylose recrystallization in RS formation is competitively affected by complexation of amylose with LPC and SSL. Results of X-ray diffraction powder crystallography were in agreement with DSC measurements. Complexes of amylose with LPC, SSL, and OHL gave type V patterns; enzymic hydrolysis of the complexes yielded type B RS structures. However, the viewpoint differs among scientists working in this area. While some workers believe amyloselipid complex to reduce the formation of RS, others believe the amylose-lipid complex itself to be a form of RS. From studies on isolated barley starch autoclaved with sodium stearoyl lactylate (SSL), distilled monoglycerides, diacetyl tartaric acid esters of mono-diglycerides (DATEM), and ethoxylated monoglycerides (bakery additives), it is postulated that amylose crystallization (as measured by enthalpies of the 158 C endotherm) that is involved in the formation of RS is competitively affected by its complexation with lipids (Szczodrak and Pomeranz 1992). Effect of citric acid and 2 emulsifiers SSL and DATEM on formation and structure of RS during extrusion of cornstarch and guar gum were studied (Adamu 2001). X-ray diffraction of the extruded starches gave a V-diffraction pattern indicating the formation of amylose-lipid complexes. Purification of the isolated RS by size exclusion high-performance liquid chromatography (HPLC) indicated the additives to have a substantial effect on molecular weight; DATEM and SSL increased molecular weight of RS, while citric acid decreased it. The influence of endogenous lipids on the formation of RS from wheat starch showed defatting to decrease the RS content (Eerlingen and others 1994). When SDS was added to defatted wheat or amylomaize VII starch, RS yields decreased substantially. X-ray diffraction and DSC showed that amylose-lipid complexes were formed in the presence of both endogenous and added lipids (SDS). A similar behavior has been reported on addition of lipids (Eliasoson and others 1988) such as olive oil (Escarpa and others 1997). Thus, less amylose was available for interactions leading to the formation of double helices and RS. Adding SDS to the starch also caused a difference in RS quality. Amylose-lipid complexes can also be formed during food processing (autoclaving and cooling). Lecithin, palmitic acid, oleic acid, and soya bean oil affect retrogradation to a lower extent than monoglycerides. Nevertheless, these authors found that pure potato amylose and oleic acid formed complexes highly resistant to amylolysis (Mercier 1980). Processing conditions Processing techniques may affect both the gelatinization and retrogradation processes, influencing RS formation. This fact is of great importance for the food industry since it offers the possibility of increasing the RS content of processed foods and foodstuffs. Baking, pasta production, extrusion cooking, autoclaving, and so forth are known to influence the yield of RS in foods (Siljestrom and Asp 1985; Bjorck and Nyman 1987; Siljestrom and others 1989; Muir and O Dea 1992; Rabe and Sievert 1992). Highly processed cereal flours and foods made from the flours, such as pasta, contain much lower levels of RS, averaging only about 1.5% to 8% RS on a dry basis. Since the crystalline structure of starch in legumes (type C) is more stable compared with the crystal structure in cereal grains (type A) (Ring and others 1988), processing cereal grains results in a large decrease in RS content, while legumes are excellent sources of RS. Cooking under conditions of high moisture and temperature can significantly lower the RS content by disrupting crystalline structure. Increasing the levels of RS can be done in other conditions, such as extrusion followed by cooling to induce crystallization (Haralampu 2000). The RS contents in various processed food samples have been reported (Siljestrom and Bjorck 1990; Parchure and Kulkarni 1997; Kavita and others 1998). Thermal processing Steam cooking. Steam cooking helps in production of RS. Starches isolated from several steam-heated legumes were rich in indigestible RS (19% to 31%, DM basis), which was not observed in raw beans (Tovar and Melito 1996). Similarly, RS measured directly in conventionally and high-pressure steamed beans were 3 to 5 times higher than in the raw pulses, suggesting retrogradation to be mainly responsible for the reduction in digestibility. Prolonged steaming as well as short dry pressure heating decreased the enzymically assessed total starch content of whole beans by 2% to 3% (DM basis), indicating that these treatments may induce formation of other types of indigestible starch (Tovar and Melito 1996). Autoclaving. Autoclaving results in increase in RS. Autoclaved wheat starch has 9% RS compared with less than 1% in uncooked wheat starch (Siljestrom and Asp 1985). Autoclaved wheat starch contained 6.2% RS (of dm); this increased to 7.8% after 3 further reboiling/cooling cycles (Bjorck and others 1987). Quantitative and qualitative influence of incubation time and temperature of autoclaved starch on RS formation was studied by Eerlingen and others (1993a). In another study, white flour subjected to repeated autoclaving and cooling cycles showed an increase in total dietary fiber >3 times that of bread flours and 4 times that of pastry flours (Ranhotra and others 1991b). The increase was primarily due to the formation of RS. Investigations on the formation of enzyme-resistant starch (RS) during autoclaving and cooling by Sievert and Pomeranz (1989a) showed highest yield (21.3%) to be obtained from amylomaize VII starch (70% amylose). Formation of RS in amylomaize VII starch was affected by the starch/water ratio, autoclaving temperature and number of autoclaving-cooling cycles. The number of cycles exerted the most pronounced effect on RS; increasing the number of cycles to 20 raised RS level to >40%. Furthermore, the thermoanalytical data suggested that amylose-lipid complexes were not involved in the formation of RS. Yields in excess of 20% RS can be obtained from autoclaved amylomaize starch containing 70% amylose. They can be raised to levels of 40% by increasing the number of autoclaving-cooling cycles up to 20 (Eerlingen and Delcour 1995). The extent of RS formation in commercially available autoclaved corn, potato, and leguminous products and in autoclaved purees intended for consumption by infants aged 3 to 8 mo was investigated by Siljestrom and Bjorck (1990). RS levels found (g/ 100 g DM) were as follows: 0.8 to 2.4 in purees, 0.2 to 3.2 in canned legumes, 1.9 in canned potatoes, 0.5 and 0.9 in reconstituted dried potatoes, <0.1 in canned corn, and 1.1 in cornflakes. Native starch (NS) extracted from wheat and subjected to 5 autoclaving and cooling cycles was found to contain 11.5% RS, which was measured as insoluble fiber; NS contained 0.5% RS (Ranhotra and others 1991a). Heat-moisture treatment (autoclaving at 121 C) with subsequent cooling was used to produce amylase-resistant starch (RS) from purified high-amylose starch samples. The formation of RS in barley starch was strongly affected by the number of autoclaving-cooling cycles; increasing the number of cycles from 1 to 20 raised the RS yield from 6% to 26% (Szczodrak and Pomeranz 1991). Parboiling. Parboiling increases RS production. In studies on 5 rice varieties, differing in amylose content, the in vitro and in vivo RS levels were low and positively correlated with amylose content (Eggum and others 1993). Higher RS starch levels were found in 8 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY Vol. 5, 2006

9 Resistant starch - a review cooked and parboiled-cooked rice than in raw rice; waxy rice had very low values. Higher contents of RS have been reported in parboiled rice than raw white rice, which also increased by cooling or freezing (Marsono and Topping 1999). Baking. Baking increases RS content. In a study to evaluate the effect of baking on RS formation, white bread was baked and divided into 3 fractions (crumb, inner crust, and outer crust) (Westerlund and others 1989). Starch levels were found to be highest in dough and lowest in outer crust after baking for 35 min. RS levels were lowest in dough and highest in crumb after baking for 35 min. A low-temperature, long-time baked product contained significantly higher amounts of RS than bread baked under ordinary conditions (Liljeberg and others 1996). Addition of lactic acid increased RS recovery further whereas malt had no impact on RS yield. The highest level of RS was noted in long-time baked bread based on highamylose barley flour. RS isolated from wheat-based foods such as chapatti and phulka was structurally characterized as a linear 1, 4- linked -D-glucan essentially derived from retrograded amylose fraction, which was dependent on the severity of the processing treatments as well as the levels of gluten and damaged starch in the wheat flour (Tharanathan and Tharanathan 2001). Extrusion cooking. Effect of extrusion cooking, at different temperatures (90, 100, 120, 140, or 160 C), moisture contents (20%, 25%, 30%, 35%, or 40%) and screw speeds (60, 80, or 100 rpm), was investigated on the formation of RS of type 3 (RS 3 ) in hull-less barley flours from CDC-Candle (waxy) and Phoenix (regular). The RS 3 content of the native flours, in general, decreased by extrusion cooking, but not significantly. Storage of extruded flour samples at 4 C for 24 h before oven drying slightly increased RS 3 content (Faraj and others 2004). With pearl barley used as the primary material in tests designed to optimize the production of RS by extrusion an extrusion temperature of 150 C and a barley moisture content from 17.5% and 22.5% moisture, followed by cold storage at 18 C gave the best results (Gebhardt and others 2001). Corn starches with and without guar gum [10% (w/w)] and 2% (w/w) of diacetyl tartaric acid ester of monoglyceride, sodium stearoyl-2-lactylate or citric acid, respectively, were extrusioncooked in a twin-screw extruder at 18% moisture, 150 C, and 180 rpm screw speed (Adamu 2001). The formation of RS in extruded corn starch was found to be strongly affected by the addition of gum and the different food additives. X-ray diffraction of the extruded starches gave a V diffraction pattern indicating the effect of extrusion cooking and amylose-lipid complexes. Enzymatic digestion did not affect the V structure, which could apparently be attributed to extrusion cooking. Purification of the isolated RS by size exclusion-hplc showed a dependence of molecular weight on the added additives. Results of differential scanning calorimetry and X-ray diffraction suggest that amylose-lipid complexes could also be involved in the formation of RS in extruded cornstarch. Pyroconversion. Pyroconversion of starch increases RS content. Lima bean (Phaseolus lunatus) starch was modified using pyroconversion, the optimum product being recovered from native starch treated with a 160:1 starch/hcl ratio, at 90 C for 1 h, resulting in starches containing 49.5% indigestible starch (Tester and others 2004). Starch pyrodextrinization decreased the amount of enzymically available starch through formation of atypical glycosidic bonds that are not digested by the amylases and maltooligosaccharidases in the small intestine of humans. Microwave irradiation. Microwave irradiation improves the digestibility of tuber starches, which could be accompanied by physicochemical and structure changes ( Microwave cooking of legumes such as chickpeas and common beans produced a redistribution of the insoluble nonstarch polysaccharides to soluble fraction, although the total nonstarch polysaccharides were not affected. This was evaluated by assessing the physicochemical, nutritional, and microstructural modifications in starch and nonstarch polysaccharides (Marconi and others 2000). The RS level decreased from 32.5% of total starch in raw chickpeas and beans, respectively, to about 10% in cooked samples with a concomitant increase in the level of rapidly digestible starch from 35.6% and 27.5% to about 80%. Studies on effects of different heat treatments (cooking, microwave cooking, pressure cooking) on the rate of hydrolysis, hydrolysis index, and glycaemic index values of kudzu starch and cornstarch showed increase in digestible starch and decrease in RS following heat treatment. The rate of hydrolysis of kudzu starch and cornstarch increased following heat treatment, especially after microwaving (Geng and others 2003). Miscellaneous treatments. Milling. Leguminous seeds, in which cell structures are preserved after cooking (that is, bread with whole seeds); bean flour with intact cells (Schweizer and others 1990); foods containing large particles such as bread with whole seeds have lower physical accessibility of starch to amylase action, and thereby contribute to higher RS contents. In some foods, physically inaccessible starch is likely to be an important fraction of the total starch that is resistant to digestion in vivo. Schweizer and others (1990) found evidence of 20% starch malabsorption from a diet containing bean flour with intact cells. About half of the malabsorbed starch was retrograded amylose. The precooked flours (PCF) prepared from dried lentils and beans, rich in intact cells filled with starch granules, indicated that they contained important quantities of RS, such as retrograded amylose (3% to 9%, DM). Germination. Germination is shown to decrease the RS content in bengal gram, field beans, cow pea, and green gram (Kavita and others 1998). Fermentation. Fermentation reduces RS content. Flour from sorghum cv. Tabat was mixed with water and previously fermented dough starter, and fermented at 37 C for a maximum of 36 h showed an increase in the in vitro starch digestibility and a decrease in the content of RS and total starch (Abd-Elmoneim and others 2004). RS formation has also been shown to decrease in the fermented products, idlis and dhoklas (Kavita and others 1998). Storage conditions Generally, RS increases on storage, especially low-temperature storage. Cold storage seems to support an increase in RS content. Whole corn bread and corn bread crumb, when stored at different temperatures ( 20 C, 4 C, or 20 C) for 7 d showed RS contents to reach a maximum between 2 and 4 d at all storage temperatures, after which they decreased (Niba 2003). Lowest RS levels in whole corn bread were found after storage at 20 C (2.18 g/100 g) for 7 d. A comparison of masa and fresh and stored tortillas from common and Costeno corn varieties showed that Costeno had higher digestible starch and total dietary fiber contents than its common counterpart. During storage of both types of tortilla, digestible starch contents decreased, whereas those of RS starch increased (Mora-Escobedo and others 2004). Studies on the influence of cold storage on in vitro starch digestibility of tortillas showed a decrease in available starch content in tortillas after 48 h of cold storage, which was concomitant with increased total RS levels (Agama-Acevedo and others 2004). These changes were due mainly to retrogradation, as indicated by increased retrograded resistant starch (RRS) levels which accounted for the major portion of the total RS. Although amylolysis patterns for fresh and 72 h stored tortillas were similar, lower digestion rates were observed for stored samples. RS contents of gelatinized samples of corn, ragi, rice, sago, and potato flours increased on low-temperature storage and de- Vol. 5, 2006 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 9