Do not duplicate

Advances in Chemical and Physical Modifications of Starch

James N. BeMiller Do not duplicate Do not duplicate

Chemical Modifications of Granular Starch Do not duplicate Premises 1. No new chemical reagents or derivatives will be allowed/used 2. Allowable levels of treatment will remain the same

Reasons . Consumer safety . Worker safety . Environmental concerns . Economics Do not duplicate Hypotheses

• Control of location of reactions represents one avenue for different and better products. • Granule architecture influences accessibility of reagents and reaction patterns. • Location of reaction sites can be controlled for preparation of different products. Factors AffectingDo Starch not duplicate Derivatization • Intrinsic Factors • Extrinsic Factors – Starch granule natures – Reaction medium • Shape and conditions • Size • Temperature • Organization • Time • Structural features • pH – Presence of pores and • Reagent (type and channels conc.) – Nature of the granule surface • Swelling inhibiting salts – Composition (type and conc.) • Fine structures of AM and AP • AM:AP ratio • Molecular weights of AM and AP • Non-starch components Do not duplicate

Surface Pores Do not duplicate Corn/maize, sorghum, and millet starches have pores randomly distributed over their surfaces. Pores are also found in the equatorial groove of large granules of wheat, rye, and barley starches. ______Fannon et al. (Cereal Chemistry, 69 (1992) 284) Do not duplicate Do not duplicate Do not duplicate Evidence was obtained that pores are not artifacts of either processing or specimen preparation for SEM.

______Fannon et al. (Cereal Chemistry, 69 (1992) 284) Do not duplicate Surface pores were hypothesized to be openings to channels that provide access to the granule interior.

Fannon et al. (Cereal Chemistry, 70 (1993) 611) Do not duplicate

Proof of Channels

Huber & BeMiller (Cereal Chemistry, 74 (1997) 537; Carbohydrate Polymers, 41 (2000) 269) Do not duplicate Do not duplicate Do not duplicate Do not duplicate Do not duplicate Do not duplicate Do not duplicate

Hypothesis Channels influence the penetration of reagent solutions into granules

______Huber and BeMiller (Carbohydrate Polymers, 41 (2000) 333) Do not duplicate To determine the pattern of dye entry into the granule matrix, granules were treated with aqueous merbromin solution for 15, 30, and 60 sec. and optically and serially sectioned by CSLM. Heterogeneity in dye penetration was evident. Much of the observed penetration was from the central cavity outward; some dye penetrated laterally from channels, and there may have been some diffusion from the outside in. Huber & BeMiller (Carbohydrate Polymers, 41 (2000) 333) Do not duplicate Do not duplicate Do not duplicate Location of Reaction Sites

• Knowledge of and, therefore, control of reactions in starch granules requires reliable methods to locate reactions.

Huber & BeMiller, Cereal Chemistry, 78 (2001) 173) Do not duplicate Do not duplicate Do not duplicate Objective To develop a method to locate sites of reactions in starch granules that is simpler and faster than BSE-SEM

Gray & BeMiller (Cereal Chemistry, 81 (2004) 278) Do not duplicate Do not duplicate Then the specimen was washed to remove excess silver ions, the remaining silver ions were reduced to silver atoms, and reflectance confocal laser scanning microscopy (RCLSM) was used to locate clusters of silver atoms. Do not duplicate

POCl3 conc. (based on starch CCL-0 0 %

CCL-2 0.1 %

CCL-4 10 % Do not duplicate

POCl3 Cross-linked Potato Starch

PCL-0 (0% POCl3) PCL-4 (10% POCl3) Do not duplicate POCl3-Crosslinked Starches: Summary • Normal corn starch – Intensity positively correlated with reagent concentration – Reactions limited to surfaces, including those of channels and cavities – Control granules contained Ag in channels (proteins/lipids) – When high reagent concentrations were used, Ag was also located in granule matrix Do not duplicate POCl3-Cross-linked Starches: Summary (2)

• Potato starch – Reactions limited to outer surface – Control granules contained Ag in concentric rings (protein and/or phosphate monoester groups) Do not duplicate Hydroxypropylation

Hypothesis: Propylene oxide would react throughout granules, but – nothing to attach a metal ion to. Do not duplicate

Location of Hydroxypropylation Reaction Sites

Huber & BeMiller (Carbohydrate Chemistry, 78 (2001) 173); Gray & BeMiller (Carbohydrate Polymers, 60 (2005) 147) Do not duplicate PO-Analog Waxy DoMaize not duplicate

WPOA 1 WPOA 2 (1.7% POA) (4.3% POA)

WPOA 3 WPOA 4 (12.7% POA) (17% POA) Do not duplicate Do not duplicate Do not duplicate Conclusions

• Valid, simple method developed to locate reactions in starch granules • Reagent type influences reaction pattern

– POCl3 – limited to surfaces, incl. channels/cavities – PO-analog – pattern more uniform • Presence of channels influences reaction patterns • Anionic substances are located in granule channels Kim & Huber (CarbohydrateDo Research, not duplicate 95 (2013) 492) improved the method by using RCLSM plus 3D anaglyphic imaging and found that, with wheat starch granules, the POA reagent reached the hilum region of granules through channels and primarily entered the granule matrix via lateral diffusion from channels, producing a relatively homogeneous reaction pattern. The homogeneous nature of the POA reaction pattern was again attributed to the low reactivity of the reagent, i.e., rate of diffusion > rate of reaction. Do not duplicate

Effects of the Nature of the Reaction Medium on Derivatization Do not duplicate Na2SO4

“Least swollen “Most swollen conditions” conditions”

44 ºC, 10.7 pH, 49 ºC, 11.2 pH, 54 ºC, 11.7 pH,

0.527 m Na2SO4 0.527 m Na2SO4 0.395 m Na2SO4 MS Determination of HPDo Starches not duplicate Reacted Under Different Conditions

_MS_ 44 °C, pH 10.7, 0.527m Na₂SO₄ 0.029 49 °C, pH 11.2, 0.527m Na₂SO₄ 0.061 54 °C, pH 11.7, 0.395m Na₂SO₄ 0.068 In reactions of waxyDo notmaize duplicate starch with propylene oxide

• MS values increased as the pH was raised from 10.7 to 11.7 • Samples reacted in the presence of NaCl had lower or equal MS values as compared to those reacted in the

presence of Na2SO4.

Han & BeMiller (Carbohydrate Polymers, 64 (2006) 158) • Reactions carried out in the Dopresence not duplicate of NaCl were more sensitive to changes in pH than were those conducted in the

presence of Na2SO4.

• Temperature had less effect on reaction efficiency than did pH or the nature of the salt.

• The concentration of Na2SO4 only slightly affected MS values. The Nature of the ReactionDo not duplicate Medium Influences Starch Derivatization Swelling-inhibiting salts influence both the extent (1) and granular patterns of reaction (2).

(1) Shi & BeMiller (Carbohydrate Polymers, 43 (2000) 333); Villwock & BeMiller (Starch/Stärke, 57 (2005) 281) (2) Gray & BeMiller (Carbohydrate Polymers, 60 (2005) 147) Do not duplicate

• Sodium and potassium citrates (salts exhibiting a strong lyotropic effect) are extremely effective as gelatinization inhibitors but result in low reaction efficiency (with propylene oxide).

• Sodium and potassium chlorides have a weak protective effect, probably at least in part based upon generation of a Donnan potential. Do not duplicate

Sodium sulfate allows restricted swelling and good reaction efficiency, most likely as a result of a moderate lyotropic effect and some generation of a Donnan potential.

Villwock & BeMiller (Starch/Stärke, 57 (2005) 281) Some swelling is requiredDo not duplicate for good reaction efficiency Approximately 1.8 times as much reagent (PO) was needed to achieve the same MS level (on normal corn starch) when a highly effective granule swelling inhibitor (potassium citrate) was used in the reaction medium in place of the usual sodium sulfate.

Shi & BeMiller (Carbohydrate Polymers, 43 (2000) 333) Do not duplicate The extent of reaction was enhanced as the proportion of water in an aqueous ethanol system was increased.

Kweon et al. (Starch/Stärke, 48 (1996) 214; 49 (1997) 59) Relationship of the averageDo not duplicate number of channels per granule of normal maize starch to the properties of its modified starch products. ______Sui & BeMiller (Carbohydrate Polymers, 92 (2013) 894) Do not duplicate • Starches from 5 inbred lines of normal maize with different relative average degrees of channelization (RADC) that could be divided into two groups (2 with RADC values of 1.49-1.52 and 3 with RADC values of 0.10-0.17) were reacted with 4 highly reactive reagents. • No consistent correlations between RADC and the effects of derivatization with the 4 reagents on physical properties, either without or after surface protein removal, were found. Do not duplicate • The results indicate that there are inherent granular and molecular differences in the maize starches that control reactivity that are more influential than RADC (at least with the degrees of modification used), that the differences carry through chemical derivatization, and that different reagents react differently with different starches. Do not duplicate

Reaction Patterns Affect Starch Properties and --- The order of reagentDo addition not duplicate during preparation of dual- modified starches impacts overall DS/MS levels and reaction patterns.

• Initial cross-linking may reduce the efficiency of subsequent substitution.

• Initial substitution decreases the apparent extent of subsequent cross-linking. Effects of the order of Doaddition not duplicate of reagents and catalyst on modification of maize starches Sui, Huber, & BeMiller (Carbohydrate Polymers, 96 (2013) 118)

Normal and waxy maize starches were each reacted with acetic-adipic mixed anhydride (AAMA), phosphorus oxychloride (POCl₃), STMP, acetic anhydride (AA), succinic anhydride (SA), and octenylsuccinic anhydride (OSA). Do not duplicate Results

• No or almost no modification occurred when the reagent was added before the alkali and AAMA or AA were used. • Less than half as much reaction occurred when SA was added before the alkali. • About the same amount of reaction occurred when STMP and OSA were the reagents used. Do not duplicate Conclusions

• Most AAMA, AA, SA and reacted with surface protein molecules when the reagent was added before the alkali. • OSA was driven into the structured internal water of granules. Do not duplicate The internal water of polysaccharide gels (also called vicinal water when discussing cells) is more ordered than the bulk-phase water. For thermodynamic reasons, rather hydrophobic substances are more soluble in ordered water (than they are in bulk water) because the change in free energy for their dissolution in the ordered water is negative. We believe that the same is true for hydrated granules so that the OSA reagent is driven into them. Wheat StarchesDo not duplicate • Although there were differences between normal and waxy wheat starches and between the wheat starches and the maize starches, the results were quite similar (to what was found with maize starches) when wheat starches were reacted with the same reagents using the two methods. ______Sui, Huber, & BeMiller (Carbohydrate Polymers, 125 (2015) 180) Do not duplicate

• An important finding of both studies is that both maize and wheat starch granules reacted with POCl₃ before the pH was raised and the granules swelled and that they even reacted with the POCl₃ when they were in a pH 7 buffer. Do not duplicate In another study of this type, Hong, BeMiller, & Huber (Carbohydrate Polymers, 151 (2016) 851) allowed DTAF (a large fluorescent reagent) to infiltrate maize and wheat starch granules for 0, 5,10, 30, and 60 min, then initiated the reaction by raising the pH to 11.5, and allowed it to proceed for 3 h. • A gradual inhibition of starchDo pasting, not duplicate i.e., some cross-linking, and an increase in the homogeneity of reaction within both maize and wheat starch granules was found. • The more homogeneous reaction pattern generally appeared to provide a greater degree of inhibition of granule swelling during pasting. • Wheat starch appeared to have both a greater degree of substitution homogeneity and viscosity inhibition (than did maize starch). Do not duplicate

• Also, the AM:AP reactivity ratio for wheat starch steadily increased as reagent infiltration time increased, suggesting a greater reaction of AM molecules as reaction progressed further into the granule matrix. (This phenomenon was not apparent with maize starch.) • In summary, increasing lengthsDo notof reagent duplicate infiltration time appeared to be associated with changes in both granular and molecular reaction patterns (much more so with wheat starch than with maize starch), indicating that both granular and molecular reaction patterns might be altered by manipulating reaction system conditions, and that specific combinations of granular and molecular reaction patterns might have the potential to produce modified starches with different properties and functionalities. Do not duplicate

Other Factors Affecting Reaction Patterns Do not duplicate Heterogeneity of granule structure and composition of starches from different botanical sources and within a given population of granules results in different granule reactivities and reaction patterns.

Mussulman & Wagoner (Cereal Chem., 45 (1968) 162); Allen et al. (J. Food Technol., 11 (1976) 537); Singh et al. (Starch/Stärke, 45 (1993) 59); Azemi & Wooton (Starch/Stärke, 47 (1995) 465); Bhattacharya et al. (Carbohydr. Polym., 27 (1995) 247); Huber & BeMiller (Cereal Chem., 78 (2001) 173); Bertolini et al. (Cereal Chem., 80 (2003) 544); Gray & BeMiller (Carbohydr. Polym., 60 (2005) 147); Ji et al. (Carbohydr. Polym., 57 (2004) 177) Do not duplicate

While small granules (relative to large granules) have greater surface area (on an equal weight basis), the extent of reaction is not always related to surface area, i.e., the effect of granule size is not always clear and can be reagent specific.

Bertolini et al. (Cereal Chemistry, 80 (2003) 544) Some Actual DifferencesDo not duplicate in Derivatization of Starch Polysaccharide Molecules

Differences in and Location of Substitution on AM and AP Molecules Within Granules When Slowly Reacting Reagents Are Used Do not duplicate There was some previous evidence that differences in granule structure and composition also influence derivatization patterns on the starch polymer molecules (for slowly reacting reagents).

Steeneken & Smith (Carbohydr. Res., 209 (1991) 239); Jane et al. (Cereal Chem., 69 (1992) 405); Steeneken & Woortman (Carbohydr. Res., 258 (1994) 207); Wilke & Mischnik (Starch/Stärke, 49 (1997) 453); Richardson et al. (Anal. Chem., 75 (2003) 6499). Do not duplicate It was believed that reactions were preferentially (perhaps, almost exclusively) confined to amorphous regions in granules, i.e., on AM molecules, in branching regions of AP molecules, and near some non-reducing ends of AP branch chains.

Hood & Mercier (Carbohydr. Res., 247 (1978) 279); Steeneken & Woortman (Carbohydr. Res., 258 (1994) 207); Kavitha & BeMiller (Carbohydr. Polym., 37 (1998) 115) For hydroxypropylation, AM becomesDo notmore duplicate highly derivatized than does AP. (For a hydroxypropylated potato starch of MS 0.099, the relative MS values were for AP 0.096 and for AM 0.113, i.e., the AM contained 18% more HP groups.

Kavitha & BeMiller (Carbohydr. Polym., 37 (1998) 115) See also, Steeneken & Woortman (Carbohydr. Res., 258 (1994) 207); Shi & BeMiller (Carbohydr. Polym., 43 (2000) 333); Chen et al. (Carbohydr. Polym., 56 (2004) 219); Kaur et al. (Carbohydr. Polym., 55 (2004) 211) Do not duplicate

Up to 40% of AP chains of DP ~15 are completely unreacted when the starch is hydroxypropylated.

Hood & Mercier (Carbohydr. Res., 247 (1978) 279); Kavitha & BeMiller (Carbohydr. Polym., 37 (1998) 115) Do not duplicate Do not duplicate

E. Bertoff (Carbohydrate Polymers, 68 (2007) 433) • Therefore, granule structureDo influences not duplicate both granular (crystalline vs. amorphous regions) and molecular (AM vs. AP) reaction patterns, and • Structural and reactivity variation occurs not only between starches of different botanical origin, but also from granule to granule in a population of granules from a single botanical source.

Allen et al. (J. Food Technol., 11 (1976) 537); Ji et al. (Carbohydr. Polym., 57 (2004) 177) Do not duplicate

In two papers utilizing reaction of wheat starch with a fluorescent probe (DTAF at pH 11.6), Kerry Huber and his students reported the results of studies of reaction rates and the location of substituents on starch polymer molecules. Do not duplicate • Hong and Huber (Carbohydrate Polymers, 122 (2015) 437) found that the initial rate of derivatization (0-0.5 h) was relatively rapid, but decreased thereafter. Reaction initially occurred at granule surfaces, then gradually shifted into the granule matrix (CSLM). [This could be the result of reaction with protein molecules on the surfaces.] • Starch chain reactivities were in the general order: AP long chains >> AM, AP medium chains > AP short chains. Do not duplicate • During the late stages of reaction (12-24 h), the amount of reaction with AP medium and short chains (crystalline regions), relative to AM and AP long chains (amorphous regions) continued to increase, providing evidence that crystalline regions were gradually opened up and became more available for reaction as the extent of derivitization increased. Do not duplicate

• These results indicated that chain reactivities were related to their location within the granule and that reaction patterns of starch chains are impacted by granule architecture. Do not duplicate Then, Hong and Huber (2015) (Carbohydrate Polymers, 122 (2015) 446), in another analysis of the same wheat starch that had been reacted with DTAF determined that the overall reactivity of starch chains followed the general order AP- LC > AM ≥ AP-MC > AP-SC. • Overall, DTAF reactions involved a minority of starch chains, with 11-12% of the total AM molecules and AP branch chains accounting for 63-75% of the fluorescence intensity regardless of the Do not duplicate duration of reaction. This result is consistent with the previous observation that reaction occurred predominately at or near granule surfaces (particularly in the early stages of reaction). • They also identified an intermediate material (IM) that co-eluted with AM, but reacted more like AP-LC. Both the extent and uniformity of reaction for IM chains appeared to exceed those of all other starch chains. Do not duplicate • Again, they concluded that initial reaction favored chains comprising granule amorphous regions (containing IM, AP-LC, and exposed segments of AM molecules). • They state that heavy derivatization of IM chains occurred in the very early stages of reaction, which could imply that these chains are located near granule surfaces including those of channels, but subsequently Dotapered not duplicate off as these chains likely became saturated with substitutions. • In contrast, respective reactions of AM and AP-LC generally increased or remained constant throughout the reaction period. • Again, they reported that, in the latter reaction stages, reaction gradually shifted to include greater proportions of AP-MC and AP-SC. Do not duplicate

Octenylsuccinylation Do not duplicate Octenylsuccinic Anhydride

Using NMR, Bai, Shi, Herrera, and Prakash (Carbohydrate Polymers, 83 (2011) 407) identified the OSA reagent as being primarily trans-(2-octenyl)succinic anhydride with a minor amount of the cis isomer.

Qui, Bai, and Shi (Food Chemistry, 135 (2012) 665) separated the isomers using HPLC. Do not duplicate Do not duplicate

O O C _ H C ( ) CH CH CH Starch OH + 3 CH2 4 2 CH Starch O C HC CH CH CH (CH ) CH O 2 2 4 3 CH H2C C 2 O C O - + O Na

O

Starch O C CH2 HC CH2 CH CH (CH2)4 CH3 C O - + O Na Molecular LocationDo notof duplicate Octenylsuccinate Groups

Bai, Shi, Herrera, and Prakash (Carbohydrate Polymers, 83 (2011) 407) and Bai and Shi (Carbohydrate Polymers, 83 (2011) 520) found that, when granular waxy maize starch was reacted with OSA, most, if not all, of the OS groups were attached at the O-2 and O-3 positions. Do not duplicate Do not duplicate • Wang, He, Fu, Huang, and Zhang (Carbohydrate Polymers, 135 (2016) 64) reported that OS groups were located near branch points and non-reducing ends of amylopectin molecules (in octenylsuccinylated waxy maize starch). Do not duplicate Do not duplicate

E. Bertoff (Carbohydrate Polymers, 68 (2007) 433) Granular LocationDo of not duplicate Octenylsuccinate Groups

After granular starches were esterified with OSA, • OS groups were found at the outer surface of granules [via confocal Raman spectroscopy (1)], via FT-IR confocal microscopy (2)] • OS groups were enriched at the outer surface of granules by a factor of 2 [via x-ray photoelectron spectroscopy (3)] • OS groups were uniformly distributed over cross- sections of granules [via back-scattered electron imaging (4)] Do not duplicate • OS groups appeared to be distributed throughout granules, but were somewhat concentrated at the surface [via CLSM (5)]

______(1)Wetzel et al. (Vibrational Spectroscopy, 53 (2010) 173) (2) Wetzel et al. (Applied Spectroscopy, 64 (2010) 282) (3) Huang et al. (Food Hydrocolloids, 24 (2010) 60) (4) Shogren et al. (Starch/Stärke, 52 (2000) 196) (5) Zhang et al. (Carbohydrate Polymers, 84 (2011) 1276) Do not duplicate

Digestion of Intact Granules by Amylases Do not duplicate Enzymic digestion of uncooked corn starch granules begins at the hilum and proceeds from the inside out.

Schwimmer (J. Biol. Chem., 161 (1945) 219); Nikuni (J. Agri. Chem. Soc. Japan, 30 (1956) A131); Nikuni & Whistler (J. Biochem. (Tokyo), 44 (1957) 227); Leach & Schoch (Cereal Chem., 38 (1961) 34); Fuwa et al. (Starch/Stärke, 30 (1978) 186); Helbert et al. (Int. J. Biol. Macromol., 19 (1996) 165) Do not duplicate Do not duplicate Do not duplicate Do not duplicate

Modification in Aqueous Slurries Do not duplicate

Standard Process The great majority of all starch is modified in aqueous slurry. • ca. 35% solids • alkaline pH (ca. 8.5-11.5) • ca. 118°F (48°C) • In the presence of a swelling-inhibiting salt (usually sodium sulfate, 7.5% anhydrous basis) Do not duplicate

Derivatization in Aqueous Alcohols

(i.e., using an alcohol as a swelling inhibitor, rather than using a salt, such as sodium sulfate) Cationization of normalDo not and duplicate waxy corn and barley starches and pea starch • Effectiveness of alcohols: ethanol > 2- propanol > methanol • Alcohol concentrations could vary between 35% and 65%, with an optimum of 65% ethanol. • Optimum starch-water ratio: 1:1 w/w (critical) • Optimum temp.: 50-55 °C Do not duplicate • Optimum time: 6 h • DS: linear to reagent concentration up to DS ca. 0.1. • Reaction efficiency: 60-70% ______Kweon et al. (Starch/Stärke, 48 (1996) 214) Do not duplicate Preparation of Amphoteric Starches (simultaneous cationization and phosphorylation) • Reagents: CHPTAC, STPP • Catalyst: NaOH • Optimum starch-water ratio: 1:1 w/w • Ethanol conc.: 65% • Optimum temp.: 50 °C • Optimum time: 190 min. ______Kweon et al. (Starch/Stärke, 49 (1997) 419) Do not duplicate “Highly” substituted carboxymethylstarch is made by reacting starch in alkaline aqueous alcohol with sodium chloroacetate.

• Kittipongpatana et al. (Carbohydr. Polym., 63 (2006) 105) made a series of carboxymethyl mung bean starches (of DS 0.06 to 0.66) of different properties using different aqueous alcohols and conditions. Do not duplicate Hydroxypropylstarch

It has been reported that hydroxypropyl starch (?) (MS 0.03~0.08) can be made using aqueous ethanol (≤50%) with a reaction efficiency of at least 80% and that, by altering the conditions, MS values of 0.10~0.14 can be achieved. ______Shao (Taiwan Nongye Huaxue Yu Shipin Kexue, 39 (2001) 223) Do not duplicate Modification of Starches by Means Other Than in Aqueous Slurries Do not duplicate “Dry”/Semi-dry Reactions

• Dry reactions (etherifications) can provide “high” levels of substitution without concern for swelling and without effluents.

• However, catalyst (alkali), salts (if used), unreacted reagents, and any by-products remain in the product. Classic Semi-Dodry not duplicate Preparations

• Monostarch phosphates: Impregnate granules with orthophosphate salts, dry, heat

• Distarch phosphates: Impregnate granules with STMP or STMP + STPP, dry, heat Many patents issued for “dry” Docationization not duplicate (up to 25% moisture) and hydroxyalkylation.

More recently, starches have been heated “dry” or semi-dry with – • Acrylamide (makes carbamoylethyl starch) (60 °C, 2h) • Sodium chloroacetate • Citric acid • Dimethyl carbonate • Epichlorohydrin Do not duplicate • Fatty acids • Hydrogen peroxide (ferrous sulfate, 30% moisture)

• H2O2 (NaOH, 27% moisture, 65 °C • Maleic acid • Monomers for grafting • OSA (up to 18% moisture) • Peroxysulfate • Mixtures of primary and secondary phosphates Do not duplicate • Polyacrylamide • Tartaric acid

Microwave heating was used in some cases. Do not duplicate Some Papers on Cationization by Dry Processes

• Khalil & Farag (Starch/Stärke, 50 (1998) 267) • Zhang et al. (Carbohydr. Polym., 69 (2007) 123) • Jiang et al. (Carbohydr. Polym., 80 (2010) 467) Do not duplicate US Patents 4,785,087 (1988) and 4,812,257 (1989) describe an ‘activator’ consisting of spray-dried, precipitated silica with a surface area of 190 m²/g that contains an alkaline agent, such as calcium oxide or calcium hydroxide, and silicates. When using 0.5% 3-chloro-2- hydroxypropyldimethyl-ethanolamine or the epoxide, reaction efficiencies of 90- 95% and DS values of 0.5 are claimed (Starch/Stärke, 44 (1992) 69). US Patent 5,116,967 (1992) extendsDo not duplicatethis process by incorporating up to 1% of a mixture of sodium peroxydisulfate and sodium peroxocarbonate (1:2-1:4 w/w) to thin the product.

US Patent 5,492,567 (1996) describes equipment for continuously preheating the mixture of starch and the cationizing reagent while maintaining the moisture content to acieve faster reaction. Preparation of a NewDo Anionic not duplicate Starch by a Dry Process

• Aly (Starch/Stärke, 58 (2006) 391) reacted corn starch with 3-chloro-2- hydroxypropylcitric acid in a dry process. Do not duplicate Carboxymethylation • Zhou et al. (J. Appl. Polym. Sci., 116 (2010) 2893) made carboxymethylstarch (with DS values up to 1.2 and a reaction efficiency of up to 97%) by a dry process.

Corn starch + NaOH + a small amount of MeOH blended together. Added powdered chloroacetic acid. Cooled (generates heat). Reacted at 50 °C. Washed with 85% MeOH. Do not duplicate Microwave Heating • More rapid reactions (1-10 min) • Cationics • Acetates and succinates [Koroskenyi & McCarthy (J. Polym. Environ., 10 (2002) 93)] • Methyl ethers [Singh & Tiwari (Carbohydr. Res., 343 (2008) 151)] • Succinate esters (DS up to 3.1; RE up to 93.5%; high paste clarity) [Joythi et al. (Starch/Stärke, 57 (2005) 556)] Do not duplicate

• Corn starch maleate ester was made in a dry process using maleic anhydride and microwave heating (Xing et al. (Starch/Stärke, 58 (2006) 464) Do not duplicate Reactions During Milling

• Some “dry” starch reactions have been carried out in stirring-type and other ball mills. These processes are often referred to as being done by the mechanical activation method. • Some reactions that have been reported are cross-linking with epichlorohydrin, acetylation, and cationization. Do not duplicate OS-Starch via Dry Milling

• Chen et al., Starch/Stärke, 66 (2014) 985.

• In a ball mill for 20 h. Called “dry media milling” and “mechanical activation”. Do not duplicate

Reactive Extrusion

a Combination of Chemical and Physical Modification Do not duplicate • Reactive extrusion involves reaction during extrusion processing, i.e., uses an extruder as a continuous reactor. Starch polymer molecules are derivatized and depolymerized while in a molten state.

• Warning: In many papers and patents, what is called reactive extrusion is simply polymer blending, i.e., melt blending of thermoplastic starch with a thermoplastic synthetic polymer. Do not duplicate • Starch granules are “destructurized” in the process – whether they are pregelatinized or melted depends on the moisture content, temperature, pressure, shear, and additives. • Problems encountered include reaction control under conditions of high viscosities, high temperatures, and short residence times. • Because reactions must be fairly rapid, more esters (using acid anhydrides) and graft co-polymers (via free-radical reactions) than ethers have been made. Do not duplicate Advantages of Reactive Extrusion

• Continuous process • Wide range of possible processing conditions • Enhanced contact between reactants (“homogeneous” reaction medium) • Lack of granule structure on molecular reaction patterns Do not duplicate

• Good heat transfer • Short reaction times • High throughput • Simultaneous partial depolymerization of starch polymers • Lack of process effluents Do not duplicate More than 50 different reactions of starch by reactive extrusion have been reported.

Some examples of are -- • Tara et al. (J. Appl. Polym. Sci., 93 (2004) 201) made a cationic wheat starch. • Bhandari and Hanna (Starch/Stärke, 63 (2011) 771) made carboxymethyl corn starch (DS 1.5, RE 42%). Do not duplicate • Bhandari et al. (Industrial Crops and Products, 41 (2013) 324) studied the carboxymethylation of cross-linked potato starch (preparation of sodium starch glycolate). • Dai et al. (Starch/Stärke, 64 (2012) 374) made oxidized (NaOCl) corn starch. • A novel cross-linked starch was made by Nossa et al. (Polymer International, 64 (2015) 1366) Do not duplicate • Tian et al. (Carbohydrate Polymers, 133 (2015) 90) esterified starch with dodecenyl succinic anhydride. • Heebthong et al. (Starch/Stärke, 68 (2016) 528) cross-linked tapioca starch with STMP. • Xie et al. (Starch/Stärke, 68 (2016)) esterified carboxymethyl starch with cetyl bromide. Do not duplicate • Maleated starches, i.e., starches esterified with maleic anhydride, were made by Raquez et al. (Journal of Applied Polymer Science, 122 (2011) 639), Stagner et al. (Journal of Polymers and the Environment, 19 (2011) 589), Hablot et al. (European Polymer Journal, 49 (2013) 873), and Zuo et al. (Journal of Thermoplastic Composite Materials, 29 (2016) 397). Do not duplicate

• Starch-graft copolymers were made by Kugler et al. (Journal of Applied Polymer Science, 127 (2013) 2847), Spychaj et al. (Polimery, 57 (2012) 95), Xu & Finkenstadt (Starch/Stärke, 65 (2013) 984), and Willett & Finkenstadt (Journal of Applied Polymer Science, 132 (2015) 42405). Do not duplicate

Some Other Process and Reagent Developments Ultrahigh Pressure-DoAssisted not duplicate Reactions • Also called high hydrostatic pressure • Pressures up to 400 Mpa (58,000 psi) • Room temperature Reviews • Kim et al. (Crit. Rev. Food Sci. Nutr., 52 (2012) 123) • Chotipratoom et al. (Journal of Applied Glycoscience, 61 (2014) 31) Do not duplicate Acetylation of Corn Starch • Lower reaction efficiencies • At an equivalent DS, UHP/HHP-assisted reactions (relative to conventionally made products) exhibited decreased swelling power, gelatinization temperatures, and peak viscosities. ______Kim et al. (Carbohydr. Polym., 78 ( 2009) 862; J. Agric. Food Chem., 58 (2010) 3573) HydroxypropylationDo of not Corn duplicate Starch • Reaction accelerated under UHP/HHP conditions, but lower reaction efficiency • Lower gelatinization temperature as compared to controls (starch treated under the same conditions without propylene oxide) ______Kim et al. (Carbohydr. Polym., 83 (2011) 755); Chun et al. (Carbohydr. Polym., 146 (2016) 328) Do not duplicate Cationic Starch

• Chang et al. (Carbohydrate Polymers, 99 (2014) 385) found that DS values were lower (compared to products made by the conventional method) for non-granular products, but were the same for granular products. • Baik et al. (US Patent Appl. Publ. (2014) US 20140350236) Do not duplicate Cross-linking with POCl3

• UHP-assisted reactions gave products with pasting profiles similar to products obtained using the conventional procedure. ______Kim et al. (Food Chemistry, 130 (2012) 977) Do not duplicate

Reactions Using Microwave Heating and Iodine as a Catalyst Do not duplicate

• These reactions are done with “dry” starch. • Reaction times are quite short (minutes). • High DS values (up to 3) can be achieved. • Depolymerization (to various degrees) and loss of crystallinity occurs. Do not duplicate • To date, only starch acetates via reaction with acetic anhydride have been made [Biswas et al. (Carbohydrate Polymers, 74 (2008) 137; Sanchez-Rivera et al. (Starch/Stärke, 62 (2010) 62; Diop et al. (Industrial Crops and Products, 33 (2011) 302); Shi et al. (Asian Journal of Chemistry, 26 (2014) 7931)]. Do not duplicate

Starch Organic Esters via Transesterification Do not duplicate • Amylomaize (80% AM) hexanoate, octanoate, decanoate, laurate, and palmitate via transesterification using vinyl esters and M2CO3 (Winkler et al., Carbohydr. Polym., 98 (2013) 208)

• Acetylation using vinyl acetate, K2CO3, and microwave heating (Bushra et al. (Starch/Stärke, 65 (2013) 236)

• No solvents were used. Do not duplicate • Starch laurate and other starch fatty acid esters (Winkler et al., 98 (2013) 208; Winkler et al.,102 (2014) 941) were prepared by homogeneous transesterification of fatty acid vinyl esters in DMSO. Do not duplicate Esterification Using Lipases

• All lipase-catalyzed esterifications must be done in the virtual absence of water, but the reaction times are often rather short. Do not duplicate • Qiao et al. (Carbohydr. Polym., 66 (2006) 135) made hydrophobic products by reacting starch and amylose with alkyl and alkenyl ketene dimers in the presence of a lipase. • Rajan et al. (Intern. J. Biol. Macromol., 39 (2006) 265; Ind. Crops. Prod., 27 (2008) 50) made fatty acid esters of corn and tapioca starches using coconut oil, a lipase, and microwave heating. Do not duplicate • Xu et al. (Carbohydr. Polym., 87 (2012) 2137) optimized conditions for esterification of waxy maize starch with OSA using a lipase (DS 0.02, RE 84%). • Lukasiewicz and Kowalski (Starch/Stärke, 64 (2012) 188) made esters using acetic, lauric, and stearic acids, a lipase, and microwave heating. • Xu et al. (Carbohydrate Polymers, 87 (2012) 2137) effected lipase-catalyzed esterification with OSA. • Lu et al. (Journal of AgriculturalDo notand duplicate Food Chemistry, 60 (2012) 9273) esterified starch with palmitic acid by a lipase- catalyzed reaction in mixed ionic liquids. • Luo et al. (Faming Zhuanli Shenqing, CN 102732582) claimed hydrophobic starch fatty acid esters by using a lipase and fatty acid methyl esters in an ionic liquid. • Wang et al. (Zhongguo Liangyou Xuebao, 27 (2012) 39) esterified starch with oleic acid in a solvent-free system using an immobilized lipase. Do not duplicate

Oxidation with Ozone, Chlorine Dioxide, and Sodium Chlorate Oxidation with OzoneDo not duplicate • Often done in a “dry” state • Chan et al. (J. Ag. Food Chem., 57 (2009) 5965) studied the oxidation (10 min) of corn, tapioca, and sago starches and found increases in both carboxyl and carbonyl contents, with more of the latter. All products showed a decrease in viscosity, indicating depolymerization. The swelling power of tapioca and sago starches decreased; that of corn starch increased. The solubility of tapioca starch↓, sago starch↑, corn st. unchanged. • Sandhu et al. (Carbohydr. PolymDo not., 87duplicate (2012) 1261) oxidized wheat starch (30 min) with ozone and found depolymerization of amylopectin and the formation of glucuronic acid units and C2 keto groups. Peak and final viscosities were reduced; breakdown increased. • Klein et al. (Food Chem., 155 (2014) 167) oxidized tapioca starch in aqueous suspension (60 min) and found that the carboxyl and carbonyl contents and degrees of depolymerization and cross- linking were pH dependent. Oxidation with ChlorineDo not duplicate Dioxide and Sodium Chlorate • Peng et al. (Zhongguo Liangyou Xuebao, 27 (2012) 39) used the two oxidants with “dry” starch and microwave heating and found that sodium chlorate gave a much higher carboxyl group content. Gels of the starch were reported to have good freeze- thaw stability and better alkali and acid stability than the chlorine dioxide oxidized starch. Do not duplicate

Use of Enzymes to Change the Structures of Starch Molecules Do not duplicate Amylomaltases

• Amylomaltases (α-1,4-α-1,4- glucanotransferases) catalyze the transfer of a segment of an α-1,4-D-glucan (amylose) to a new O4 position of an acceptor, which is usually another α-1,4 D- glucan chain. • As a result, some starch molecule chains are lengthened and some are shortened. • Modification is done with pasted starch. Do not duplicate Do not duplicate • Amylose is removed or its content reduced. • Average molecular weights and percentage of branching are unchanged. • Amylomaltases from different organisms have different activities. • Different starches give different products. Do not duplicate • Products from amylomaltase treatment of tapioca starch had a reduced tendency to retrograde. • Gels of tapioca starch products had increased freeze-thaw stability. • Some starches (potato) modified in this way produced thermoreversible gels that could be used as a gelatin substitute. • Some products increased creaminess in low-fat yoghurt. Do not duplicate

• Amylomaltase and transglucosidase were combined to make long-chain isomaltooligosaccharides [Rudeekulthamrong et al. (Biotechnology and Bioprocess Engineering, 18 (2013) 778)]. • Amylomaltase and branchingDo enzyme not duplicate were combined in a step-by-step process to produce extreme branch-on-branch, soluble α-glucans of low digestibility [Sorndech et al. (Carbohydrate Polymers, 132 (2015) 409]. • A higher amylose content in the substrate efficiently produced α-1,6 linkages and a higher-molecular-weight and a less- digestible product [Sorndech et al. (Carbohydrate Polymers, 152 (2016) 51)]. Do not duplicate Other References • Kaper et al. (Biochem. Soc. Trans., 32 (2004) 279) • van der Maarel et al. (Starch/Stärke, 57 (2005) 465) • Hansen et al. (Carbohydr. Polym., 78 (2009) 72) • Alting et al. (Food Hydrocoll., 23 (2009) 980) • Suriyakul et al. (J. Food Sci., 81 (2016) C1363) Treatment with GlycogenDo not duplicate- branching Enzyme (GBE or BE) • Done with pasted starch. • Branches amylose (mostly with G6 and G7 chains) • With amylopectin, it reduces the length of longer branches, favoring branches of G5, G6, and G7, decreasing the mid-point DP from 12 to 6. • Overall, treatment with the enzyme results in shorter branch chains and more branch chains. ______Do not duplicate Kim et al. (Food Chem., 110 (2008) 979)

• Ciric et al. (Analytical Chemistry, 84 (2012) 10463) also concluded that GBE prefers to transfer short oligosaccharides. Do not duplicate • Fan et al. (Starch/Stärke, 68 (2016) 355) concluded that GBE had the greatest specificity towards amylose and that it could be used to generate a maize starch with a low amylose content and a more branched amylopectin of low MW and richer in short and intermediate chains. Amylosucrase (AS)-Domodified not duplicate Starch • Amylosucrase transfers α-D- glucopyranosyl units from sucrose to the non-reducing ends of amylopectin branch chains, considerably increasing the branch chain length. • Kim et al. (Journal of Agricultural and Food Chemistry, 64 (2016) 5045) heat-moisture treated AS-modified normal and waxy rice starches. Do not duplicate

Published in: Ji Hyung Kim; Ha Ram Kim; Seung Jun Choi; Cheon-Seok Park; Tae Wha Moon; J. Agric. Food Chem. 2016, 64, 5045-5052. DOI: 10.1021/acs.jafc.6b01055 Copyright © 2016 American Chemical Society Do not duplicate

Amylopectin branched chain length distribution of amylosucrase-modified normal and waxy rice starches.

Published in: Ji Hyung Kim; Ha Ram Kim; Seung Jun Choi; Cheon-Seok Park; Tae Wha Moon; J. Agric. Food Chem. 2016, 64, 5045-5052. DOI: 10.1021/acs.jafc.6b01055 Copyright © 2016 American Chemical Society Do not duplicate

• The modification produces some insoluble RS. • The HMT treatment increased the degree of crystallinity. • The main attribute of AS-modified starches seems to be their substantially increased SDS and RS contents (RS > SDS). Do not duplicate

• In one study [Kim et al. (Food Chemistry, 138 (2013) 966)] the thermostable RS content of a rice starch increased from 0.1% to 43% after AS treatment. • The branch chain length is correlated with the RS content. • All starches give a B-type x-ray diffraction pattern after AS treatment. Do not duplicate Other References • Ryu et al. (Starch/Stärke, 62 (2010) 221) • Shin et al. (Carbohydr. Polym., 82 (2010) 489) • Kim et al. (Food Chem., 152 (2014) 113) • Kim et al. (Carbohydr. Polym., 125 (2015) 61) • Jo et al. (Carbohydr. Polym., 143 (2016) 164) • Kim et al. (Food Sci. Biotechnol., 25 (2016) 457) Do not duplicate

Physical Modifications of Granular Starch Do not duplicate

Material taken from – Physical Modifications of Food Starch Functionalities, J.N. BeMiller and K.C. Huber (Ann. Rev. Food Sci. Technol., 6 (2015) 19-69) Do not duplicate Why Study Physical Modifications?

• Physically modified starches are “clean label” (“label friendly”, “functional native”) starches. • If sufficient functionality can be realized, physical modifications are generally easier to do, less expensive, and produce no effluents containing salts, reagents, or reagent byproducts. Do not duplicate • Some treatments increase amounts of thermostable SDS and RS. • Some physical treatments have been studied because the treatment had been proposed as a non-thermal means of food processing, so it was of interest to determine what effect the treatment had on the starch (or flour). Do not duplicate Thermal Treatments

• Those that produce pregelatinized starch • Those that produce granular cold-water- swelling starch • Heat-moisture treatments • Annealing • “Osmotic pressure” treatments • Heating of “dry” starch Do not duplicate Non-thermal Treatments

• Ultrahigh pressure • Instantaneous controlled pressure drop • High-pressure homogenizers • High-speed jet • Dynamic pulsed pressure • Pulsed electric field • Freezing and thawing • Freeze-drying Granular Cold-waterDo-swelling not duplicate (GCWS) Starches

• Also referred to as cold-water-soluble starches. • Three general processes are most often used – 1. Heating an amylose-containing starch in an aqueous solution of an alcohol. 2. Rapidly heating a starch dispersion in a special spray-drying nozzle and drying the droplets in a spray dryer. 3. Treating the starch with an alkaline, aqueous solution of an alcohol at room temperature. Do not duplicate • In all 3 processes, granules containing amylose swell to the point of losing their crystalline order while maintaining their integrity when added to an aqueous system with low shear. Products made from a single starchDo not using duplicate different method conditions and by using the same method conditions with different starches may vary in characteristics, but they have the common property that their granules rapidly hydrate and swell without granule disintegration (when little or no shear is applied) when placed in an aqueous system at room temperature. In the end, they produce the functionalities of the cook- up starches from which they are made without application of heat. Do not duplicate Heat-Moisture Treatment (HMT)

• A hydrothermal process consisting of heating starch granules above their glass transition temperature (at the moisture level employed)

• Variables: type of starch, moisture content, duration/length of treatment, temperature, heat source Do not duplicate Usual HMT Conditions

• Moisture content: 10-40% (sealed container) • Temperature: 84-140 °C (183-284 °F) (usually above 94 °C [201 °F]) • Treatment times: 1 to more than 24 h • Heat source: oven, microwave, other Do not duplicate Common Structural Changes Believed to be Effected By HMT

• Due to thermal energy and the plasticizing effect of water molecules, increased mobility of both starch polysaccharide chain segments and helical structures in both crystalline and amorphous regions of granules occur. • The greatest changes may occur in the amorphous regions. Do not duplicate • In amorphous regions, HMT disrupts helical structures, leading to formation of new ordered structures and/or crystallites. • In B-type starches, lamellar structures are disturbed and double helices move laterally and along their helix axes to effect the B→A transition. • (Fact) There is an increase in crystallinity in A-type starches, but no change in the type of crystallinity. Do not duplicate • (Fact) Starches with B-type crystallinity have their crystallinity changed to a (A+B)- type (i.e., C-type) crystallinity or completely to an A-type crystallinity with an accompanying loss of crystallinity.

• In A-type starches, double helices move within crystallites and form more ordered and closely packed structures, i.e., HMT disrupts the least stable structures and allows growth and/or more perfect alignment of the more stable crystallites. Do not duplicate • Amylose-lipid complexes may form in cereal starches.

• Overall, HMT is believed to change the nature of, i.e., perfect, crystallites and to produce new crystallites. Common Property DoChanges not duplicate Effected By HMT • Due to the diverse combinations of starches and conditions employed for HMT (moisture content, temperature, length of heating, heat source), it is not possible to define the properties of HMT starches in a consistent and unified way. • For every change reported in the literature, there are also reports of no change and of changes in the opposite direction. Majority findings are given here. Do not duplicate • Granules remain intact. (Should not be gelatinized.)

• Increases in To, Tp, and Tc and a broadening of Tc - To (probably as a result of changes in amorphous regions) • Also increases in pasting temperature (RVA) and hot-paste viscosity • Decreases in swelling power, leaching of amylose from swollen granules, peak viscosity, and breakdown. Do not duplicate

• All changes differ with starch source and are often increased by increases in moisture content and/or temperature. • Changes may be in one direction at a given temperature and in the opposite direction at a different temperature. Do not duplicate • But together the changes often give the HMT starch an increase in granule stability similar to those of a lightly cross-linked starch.

• Slightly to moderately increased amounts of thermostable SDS and RS. Do not duplicate • For example, Kim and Huber (Carbohydrate Polymers, 98 (2013) 1245) found that uncooked, native potato starch had SDS and RS contents of 2.0% and 93%, respectively. • HMT of the same starch resulted in maximum respective SDS and RS contents of 22-23% and 83%. • Cooking the HMT potato starch resulted in respective SDS and RS contents of 1.6% and 5.9%. Do not duplicate Enhancement of Effects of HMT • Conduct HMT under slightly acidic conditions (pH 4 and above) • Treat with acid before HMT • Add lauric acid before HMT Do not duplicate

HMT has been done – • In dual processes prior to and following annealing, • before, after, and simultaneously with chemical and other modifications, • with whole-tissue flours, and • with microwave heating. Do not duplicate Annealing (ANN)

• Annealing involves heating and holding starch granules in an excess of water at a temperature that is above the starch’s glass transition temperature and below its gelatinization temperature.

• Variables: starch type, temperature, duration of treatment Do not duplicate Usual Conditions for Annealing

• Starch concentration: generally >39% w/w

• Temperature: generally 5-15 °C below To Common Structural DoChanges not duplicate Believed to be Effected by Annealing • (Granules remain intact.) • It is believed that, because of the high degree of plasticization by water, the increased temperature increases the mobility of double-helical chain segments, allowing them to improve their alignment. Do not duplicate • In other words, the least stable structures within both amorphous and crystalline regions are disrupted and crystallization, perfection of crystallites, and/or increased molecular ordering forms more stable and more homogeneous structures, without increasing the number of double helices (in normal and waxy starch granules). • Changes are believed to be determined by the amylose content of the starch and the fine structure of the amylopectin. Common Property DoChanges not duplicate Effected by Annealing

• Decreases in swelling power, solubility, and viscosity (in AM-containing starches)

• Increases in To and Tp • Decreases in peak viscosity and breakdown • Increased gel firmness Do not duplicate • Together these changes give the annealed starch an increase in granule stability similar to that of a lightly cross-linked starch.

• No change in the crystalline packing arrangement • Little or no increase in thermostable SDS and RS Do not duplicate

Annealing has been done – • both as single and multi-step processes • In dual processes, prior to and following HMT, • with whole-tissue flours, • prior to and following chemical modification, and • following acid-catalyzed hydrolysis. Do not duplicate Heating of Dry Starch Starches with acid-, shear-, and temperature-tolerance profiles similar to those of chemically cross-linked starches have been prepared by heating a starch with a low (<15%) moisture content at a temperature above 100˚C, but below that which effects thermal degradation. Drying to <1% moisture before heating and alkalinity (pH 8.0-9.5) facilitates the alterations of physical properties. Do not duplicate

Starches with insufficient water content to effect gelatinization have been subjected to microwave irradiation. • Not usually done in closed containers, so moisture contents are reduced. • Both increases and decreases in degrees of crystallinity have been reported. • Changes in properties have also varied. Do not duplicate

Non-thermal Treatments Do not duplicate Milling

• Has been termed “micronization” in recent literature, but -- • The effects of milling on flour production have mostly been studied. • Starches have been milled with a variety of types of mills, including different types of ball mills. Do not duplicate

• Variables: type of mill, duration of milling, type of starch, moisture content of starch

• Mechanical force can produce relatively high temperatures at the point of impact, so changes to granular and molecular structures are likely caused by both thermal and mechanical energies. Common StructuralDo Changes not duplicate Imparted by Milling • Products contain various ratios of undamaged granules, damaged granules, and fragments of damaged granules. • Small fragments are likely to agglomerate. • Amylopectin may become depolymerized. (An increase in fragmented AP molecules increases the stickiness of food products.) • Decreases in double-helix content, crystallinity, and crystallite perfection. Common Property DoChanges not duplicate Imparted by Milling

• Increases in water vapor sorption, granule swelling, solubility, gel clarity, and susceptibility to amylases.

• Decreases in paste and gel viscosity, To, Tp, and Tc Ultrasonic TreatmentDo not ofduplicate Granules

• Ultrasound may be used in food processing

• Variables: ultrasound input (frequency, power, amplitude, duration of treatment), nature of the medium, natures and concentrations of dissolved gases, temperature, nature and concentration of starch Do not duplicate Mechanism

• Ultrasonic treatment probably brings about changes in starch granular and molecular structures, properties, and functionalities via both mechanical effects and the generation of OH radicals. • Ultrasound generates micrometer-size bubbles, followed by their growth and subsequent collapse. Common StructuralDo Changes not duplicate Effected by Ultrasound • Erosion, pitting, and cracking of granule surfaces • Ultrasound energy is generally insufficient to disrupt, or even distort, the crystalline lamellae of granules, but it can impact amorphous regions, perhaps disrupting double helices, with B-type organizations being more susceptible to disruption. • May depolymerize amylopectin molecules Common Property DoChanges not duplicate Effected by Sonication

• Differences in results are due to different combinations of treatment conditions and the different natures of the starch. Common changes are --

• Increases in water sorption, swelling power, and solubility Do not duplicate

• Increases in gel clarity, hardness, and adhesiveness • Decreases in paste and gel viscosities and the consistency coefficient (k) of pastes due to depolymerization. Do not duplicate

High-pressure Treatments Do not duplicate Ultrahigh Pressure (UHP)

• Also called high hydrostatic pressure (HHP) because aqueous slurries are pressurized • Usually defined as pressures above 400 MPa (58,000 psi) • Variables: pressure, temperature, duration of treatment, type of starch, concentration of starch Do not duplicate Common Structural Changes Effected by UHP Treatment

• UHP treatments effect partial (or complete) gelatinization of the starch with maintenance of the granular form. • It is possible to obtain different degrees of gelatinization of a given starch by manipulating the combination of slurry concentration, treatment duration, temperature, and pressure. Do not duplicate • A-type granules are more susceptible to UHP than are B-type granules. • Waxy maize and waxy rice starch granules are more susceptible to UHP than are normal maize and rice starch granules. • As the temperature increases, the pressure required for gelatinization decreases, and vice versa. • At constant pressure, the degree of gelatinization is time dependent. Do not duplicate

• The degree of gelatinization decreases as the starch content of the slurry increases. • Little or no leaching of AM from starches gelatinized by UHP treatment. Do not duplicate Common Structural Changes Believed to be Effected by UHP

• UHP treatment is believed to force water into amorphous regions without inducing granule swelling. • There is evidence that water molecules are also forced into crystalline regions, effecting gelatinization. Property Changes EffectedDo not duplicate by UHP • There are reports of both increases and decreases in various attributes. • Discrepancies may arise because the less structurally sound granules lose their crystallinity first, leaving ungelatinized granules in a gel of swollen granules when the product is added to water. (There are reports that Gʹ and Gʹʹ values first increase, then decrease as the pressure or treatment time is increased.) Use of High-pressureDo not duplicate Homogenizers

• Equipment, such as the Microfluidizer, produce high shear, turbulence, cavitation, and heat (although usually not much heat).

• To, Tp, and ΔH decrease as the homogenizer pressure increases. Do not duplicate Structural Changes That May Occur During Homogenization

• Granules may deform and fragment, with the extent of both increasing as the homogenizer pressure increases. • Granules first lose crystallinity and then birefringence (gelatinize) as the homogenizer pressure increases. Do not duplicate Common Property Changes Effected by Homogenization

• Increases in To and Tp. Do not duplicate High-speed Jet

• In the high-speed jet, a starch slurry is forced through a small orifice producing an ultra-high-velocity jet that impinges a target. Changes EffectedDo by not the duplicate High-speed Jet

• Granule surface gelatinization and damage • Loss of crystallinity • Depolymerization of amylopectin • Increased solubility • Rheological properties of gels changed, particularly was there a large decrease in gel elasticity. Do not duplicate

Pulsed Electric Field (PEF) • Has been used for non-thermal pasteurization of liquid foods • Variables: electric field strength, duration of treatment Do not duplicate Common Changes Effected by PEF

• Loss of crystallinity • Depolymerization of amylopectin Do not duplicate Freezing and Thawing

• Repeated freezing and thawing of potato starch (13% moisture) resulted in surface erosion and increased total micro- and mesopore volume and mean pore diameter. Do not duplicate Effects of Freeze-drying

• Freeze-drying of starch granules disrupted and reduced amounts of both short- and long-range molecular order of potato starch granular amylopectin more than that of granular maize starch amylopectin. • Freeze-drying damaged the surfaces and increased the susceptibility to amylases of potato starch granules, but not of maize starch granules. Do not duplicate

Resistant Starch Do not duplicate • Up to August 31, 2017, (according to SciFinder) there had been 4,679 publications specifically on resistant starch and 15,552 publications containing the concept resistant starch. • In 2016, there were 321 publications specifically on RS and 1,218 containing the concept RS. • It has only been rather recently that RS has been considered to be a form of dietary fiber. Do not duplicate

Humans and their ancestors have been consuming resistant starch since before recorded history. Consumption of much of any other kind of starch could not have occurred until they learned to control fire, which occurred about a million years ago. (Estimates range from 0.2 to 1.7 million years ago.) Do not duplicate • Now, not only is resistant starch consumed unknowingly, it is widely used as an ingredient, especially in functional foods. • Its attributes include its availability in different forms, its relatively low cost, its known positive health benefits, and perhaps its functionality. • Resistant starch is sometimes referred to as amylase-resistant starch or enzyme- resistant starch. Do not duplicate What is Resistant Starch?

• Resistant starch is that portion of a starch or starch product that is not converted into glucose in the small intestine of healthy individuals and passes through it to the colon, where it is fermented by the colon’s microbiota. Starch Digestion/ConversionDo not duplicate into D-Glucose • A total of 6 enzyme activities (2 α- amylases and 4 small-intestinal, brush- border/mucosal α-glucosidases) are required to convert starch polysaccharides into D-glucose (glucogenesis). ______Diaz-Sotomayor, et al., J. Pediatric Gastroenterology and Nutrition, 57 (2013) 704. Do not duplicate

1. α-Amylases (salivary and pancreatic) catalyze hydrolysis of internal linkages of starch polysaccharide molecules (amylose and amylopectin), forming small linear oligosaccharides (mainly maltose and maltotriose) and branched oligosaccharides (α-limit dextrins). Do not duplicate

2. The combined action of mucosal-bound α- glucosidases (maltase-glucoamylase and sucrase-isomaltase complexes) in the small intestine catalyze hydrolysis of the products produced by the action of α- amylase, producing D-glucose. Do not duplicate • The focus of this section is the production of RS as an ingredient. • RS is not a specific entity. Rather it is that portion of a starch product that is not converted into glucose in the small intestine. • There are different types/forms of RS with quite different physical properties. • First these different types will be reviewed briefly. Do not duplicate Types of RS

• RS 1 – A form of starch that is physically inaccessible to α-amylase because it is surrounded by cell-wall materials and/or a protein matrix or is otherwise trapped within a food matrix. Do not duplicate • RS 2 – Native uncooked granules, such as native potato starch granules.

(Native potato starch granules contain (by in vitro analysis) about 46% RS, but baking or boiling a potato reduces the RS content to about 3-5%.) High-amylose maize starches fall into this category because most of their granules are not gelatinized during normal food processing or preparation techniques. Amylomaize starches are commercial RS products. Do not duplicate

• RS 3 – Retrograded starch. Primarily retrograded amylose.

(Commercial products exist.)

• RS 4 – Chemically modified starch.

(Commercial products exist.) Do not duplicate

• RS5 – Complexes of starch molecules (primarily amylose molecules) with fatty acids) and alcohols and some monoglycerides. Analysis for RSDo not duplicate • Most analyses done are in vitro analyses. • In my opinion, in most cases, analysis should be done with an analysis for dietary fiber such as AOAC Official Method 991.43 (AACCI Approved Method 32- 07.01) which contains a heating step (30 minute boiling) and concurrent treatment with a thermostable α-amylase because, when RS is included as an ingredient, the product almost always undergoes heating during processing and often during preparation before consumption. Do not duplicate

• The method of Goni et al. (Food Chemistry, 56 (1996) 445-449) gelatinizes the starch with 2M KOH. Do not duplicate RS3

• Review by S.G. Haralampu, Carbohydrate Polymers, 41 (2000) 285.

• Retrograded amylose • Relatively highly crystalline • Very stable thermally (Can only be rehydrated at temperatures of 80-150 °C – primarily at temperatures above 120 °C) Do not duplicate Processes For Making RS3

• RS3 is made by pasting an amylose- containing starch and letting it retrograde. • Ways to improve the yield of RS3, i.e., to reduce the percentages of RDS (and perhaps SDS) and increasing the percentage of RS have been found. All involve the production of shorter and more linear chains. Autoclaving FollowedDo not by duplicate Retrogradation

Reviews: J.H. Dupuis, Q. Liu, & R.Y. Yada, Comprehensive Reviews in Food Science and Food Safety, 13 (2014) 1219-1234. B.A. Ashwar, A. Gani, A. Shah, I.A. Wani, & F.A. Masoodi, Starch/Stärke, 68 (2016) 287- 301. • Different starches have been used, but most procedures start with a normal or high-amylose starch. Do not duplicate

• Autoclaving has been done at different temperatures and for different times. • Different conditions (times and temperatures of storage) have been used for the retrogradation step. • In general, samples receiving more autoclaving/retrogradation cycles had higher RS contents. • What is going on? Do not duplicate

• Although there are only a few papers on the subject, there are good indications that polysaccharides, including starch polysaccharides, undergo thermal depolymerization (chain cleavage). • It has also been established that the rate and degree of amylose retrogradation increases as its chain length is shortened. Do not duplicate • B. Pfannemüller, H. Mayerhöfer, & R.C. Schulz (Biopolymers, 10 (1971) 243-261) reported that the association rate of amylose in water had a sharp maximum at DP 80. • R.C. Eerlingen, M. Deceuninck, & J.A. Delcour (Cereal Chem., 70 (1993) 345- 350) reported that the yield of RS increased with DP to plateau values of 23- 28% and that the isolated RS from these samples had DP values between 19 and 26. • B.E. Hickman, S. Janaswamy, &Do Y. Yaonot duplicate(J. Agricultural Food Chem., 57 (2009) 7005-7012) subjected normal maize and wheat starches to 3 cycles of autoclaving (30 min each at 121 °C). • These materials were treated with β-amylase. • Some of these products were autoclaved a fourth time. • The highest content of thermostable RS (30%) from normal maize starch was obtained both with and without the fourth autoclaving. • The highest content of thermostable RS (28%) from wheat starch required a fourth autoclaving. Do not duplicate

Other ways of depolymerization to promote retrogradation (the production of RS3) are more effective (and less costly). Do not duplicate Treatment with a Debranching Enzyme • Start with a normal or high-amylose starch. • Most often, pullulanase has been used as the debranching enzyme. • Recently, C.K. Reddy, M. Suriya, & S. Haripriya (Carbohydrate Polymers, 95 (2013) 220-226) increased the percent of thermostable RS* in red kidney bean starch from 21% (native starch) to 31% (debranched) and 42% (pasted before debranching). (*Slurries of all samples were autoclaved, then stored at 4 °C for 24 h before analysis for RS.) Do not duplicate

• Also recently, K.S. Trinh, S.J. Choi, & T.W. Moon (Starch/Stärke, 65 (2013) 679-685) treated pasted water yam starch with isoamylase, recovered the product, then heated a suspension of it to boiling, and finally dried the product in an oven. The thermostable RS content was 35-40%. • Subsequent HMT treatment greatly reduced the RS content. Do not duplicate • H. Zhang & Z. Jin (Carbohydrate Polymers, 86 (2011) 1610-1614) heated NMS to 80 °C (20 min); treated the gel first with α-amylase, then with pullulanase; allowed the product to retrograde at 4 °C (24 h); again treated the mixture with α- amylase (presumably to remove some of the non-retrograded starch); and recovered the product by centrifugation. • The product contained 59% RS using the method of Goni et al. Do not duplicate Partial Acid Hydrolysis (PAH) • J.O. Brumovsky & D.B. Thompson (Cereal Chemistry, 78 (2001) 680-689) treated amylomaize starch (ca. 70% apparent AM) with 1% HCl at 25 °C for various times up to 78 h, then treated the neutralized products by HMT or annealing. They were able to make products with as much as 33% “boiling stable” RS by ANN of a product (6 h PAH) and 63% “boiling stable” RS by HMT of a product (78 h PAH). Do not duplicate • S.I. Shin, C.J. Lee., D.-I. Kim, H.A. Lee, J.- J. Cheong, K.M. Chung, M.-Y. Baik, C.S. Park, C.H. Kim, & T.W. Moon (J. Cereal Science, 45 (2007) 24-33) acid-modified rice starch using citric acid, and were able to achieve a product with 30% thermostable RS. (control 13%) Do not duplicate • J. Hasjim & J.-L. Jane (J. Food Science, 74 (2009) C556-C562) reported that, when normal maize starch was treated with 1 M HCl at 25 °C for 40 h (and then washed to remove the acid), the percentages of crystallinity and RS were increased slightly due to removal of amorphous material. • A subsequent HMT treatment considerably increased the thermostable RS content to 31%, but there was no statistical difference between that value and that of the control (no PAH). Do not duplicate • S. Ozturk, H. Koksel, & P.K.W. Ng (J. Food Engineering, 103 (2011) 156-164) first treated amylomaize starches by autoclaving a suspension of them (30 min at 135 °C), then storing the paste/gels at 4 or 95 °C for 24 h. This treatment was followed by twice autoclaving the samples (30 min at 133 °C) and again storing the paste/gels at 4 or 95 °C for 24 h. Some samples were first treated with 1.64 M HCl at 40 °C for various times. • First, it was determined that Dothe not proportion duplicate of the highest MW fraction (intact amylopectin) decreased and the proportions of lower MW fractions increased with times of the acid treatments. • The largest amount of RS from ca. 55%- AM amylomaize starch was 28% (after 3 h of hydrolysis and storage at 95 °C). • The largest amount of RS from ca. 70%- AM amylomaize starch was 39.5% (after 1 h hydrolysis and storage at 4 °C). Do not duplicate

• S.-H. Mun & M. Shin (Food Chemistry, 96 (2006) 115-121) treated normal maize starch and RS3 made from that starch (by autoclaving at 121 °C for 1 h and storing the gel at 4 °C overnight) with 0.1 M HCl at 30 °C for up to 30 d. The thermostable RS content increased during the 30 d period. The RS content of the native starch increased from 6% to 9%, while that of the RS3 increased from 14% to 26% - both probably as a result of removal of some of the amorphous starch, i.e., as a result of a different mechanism. Do not duplicate • Normally, HMT changes the RS contents very little, but P.V. Hung & N.T.L. Phi (Food Chemistry, 191 (2016) 67-73) added 0.2 M citric, lactic, and acetic acids to rice starches to obtain 30% moisture and then heated the starches at 110 °C for 8 h. After determining that the treatments reduced the average DPs of the starches (with effectiveness of the acids being in the order given), the products were subjected to HMT. Thermostable RS in amounts of 37% (normal rice starch, citric acid), 35% (waxy rice starch, citric acid), and 35% (high-amylose rice starch, lactic acid) were obtained. Do not duplicate Treatment with Amylosucrase • Using this method to obtain RS was presented earlier. • In a scaled-up production, B.-S. Kim, H.-S. Kim, & S.-H. Yoo (Carbohydrate Polymers, 125 (2015) 61-68) obtained thermostable RS contents of 30-51% from 4 rice starches and of 43 and 47% from 2 barley starches. RS4 Do not duplicate • C.C. Maningat and P.A. Seib, Chapter 3 in Y-C. Shi and C.C. Maningat (eds.), Resistant Starch: Sources, Applications and Health Benefits, John Wiley & Sons, 2013. • RS4 products are chemically modified starches. • With both substituted and cross-linked starches, the amount of RS in the product is a function of the degree of modification. Do not duplicate Substituted/Stabilized Starches

• Substituent groups (such as acetate and hydroxypropyl groups) along the linear chains impart steric hindrances that obstruct binding of α-amylase. Do not duplicate • As mentioned earlier, the conversion of starch into D-glucose requires the combined action of the endo-enzyme α-amylase and small-intestinal α-glucosidases, with an α-amylase acting first to produce oligosaccharides. • Humans have two forms of α-amylase: salivary and pancreatic. • The two α-amylases are highly homologous (97%) and have a high degree of structural similarity. • Both have 5 substrate glucosyl unit binding sites. Do not duplicate

Binding Sites of α-Amylases Do not duplicate Do not duplicate • S. Simek, M. Ovando-Martínez, K. Whitney, & L.A. Bello-Pérez (Food Chemistry, 134 (2102) 1796-1803) modified bean starches via acetylation (DS not given), ozonation, and annealing. • All three processes increased the thermostable RS content from 36% to 43- 44% for black bean. • For pinto bean, the thermostable RS contents were 42% (native), 43% (acetylated), 45% (ozonated), and 58% (annealed). Do not duplicate Octenylsuccinylation

• Octenylsuccinylation decreases both the rate and extent of starch digestion (several studies). • In some studies, the OS-starch product was also heat-moisture treated. • Indications from both kinds of studies were that the amount of SDS was increased considerably more than was the amount of RS. Do not duplicate

• B. Zhang, Q. Huang, F.-x. Luo, X. Fu, H. Jiang, & J.-l. Jane (Carbohydrate Polymers, 84 (2011) 1276-1281) found that, before cooking, the RS content of DS 0.04 OS ca. 55%-AM amylomaize starch was 86%, while after cooking, the same starch had a RS content of 7.5%. • This is a good example of why a dietary fiber analysis needs to be used. Do not duplicate • J. He, J. Liu, & G. Zhang (Biomacromolecules, 9 (2008) 175-184) suggested (based on their data) that OS waxy maize starch molecules (after HMT and subsequent cooking) fed to human subjects might act as uncompetive inhibitors of one or more glucogenic enzymes. Do not duplicate • J.-A Han & J.N. BeMiller (Carbohydrate Polymers, 67 (2007) 366-374) reported that reaction of tapioca, normal maize, and potato starches with 3% OSA produced, respectively, thermostable RS contents of 28%, 29%, and 33%. (The unmodified starches had thermostable RS contents of 0.1%, 1.1%, and 3.9%, respectively.) • But, subsequent HMT of the products reduced the thermostable RS contents. Do not duplicate

• But reaction of waxy maize starch with both propylene oxide and OSA followed by HMT resulted in a product with 41% thermostable RS. (control 0%) Do not duplicate

• J. Juansang, C. Puttanlek, V. Rungsardthong, & S. Puncha-arnon (Food Chemistry, 131 (2012) 500-507) obtained 45% thermostable RS from canna starch by octenylsuccinylation (control 12%). • Acetylation, hydroxypropylation, and STMP cross-linking produced, respectively, only 27%, 32%, and 20% thermostable RS. Cross-linkingDo not duplicate • K.S. Woo, C.C. Maningat, & P.A. Seib, Cereal Foods World, 54 (2009) 217-223. • “Commercially available cross-linked resistant wheat starch typically exhibits ≥90% dietary fiber”.

• Sufficient cross-linking inhibits granule swelling and access of amylolytic enzymes/amylases to required binding sites. It may also provide steric hindrance. Do not duplicate

• Studied cross-linking reagents include

POCl3, STMP, STMP + STPP, acetic- adipic mixed anhydride, citric acid, and epichlorohydrin. Do not duplicate

• K.S. Woo & P.A. Seib (Cereal Chemistry, 79 (2002) 819-825) conducted 3 types of cross-linking of wheat starch. With the conditions and amounts of reagent used, they obtained the following thermostable RS contents: 1. Using STMP/STPP – 76% 2. Using epichlorohydrin – 76%

3. Using POCl3 – 86% Do not duplicate

• K. Kahraman, H. Koksel, & P.K.W. Ng (Food Chemistry, 174 (2015) 173-179) optimized the conditions (pH and temperature) for cross-linking normal maize and wheat starches with 99:1 w/w STMP-STPP (plus sodium sulfate) to produce thermostable RS. • They produced NMS with 78% thermostable RS and wheat starch with 96% thermostable RS. Do not duplicate • Y. Sang & P.A. Seib (Carbohydrate Polymers, 63 (2006) 167-175) simultaneously cross-linked and HMT amylomaize starches (ca. 55% and ca. 70% AM). • They heated (at 110 °C for 4 h) the starches at 45% moisture content and an initial pH of 11.5 with a 99:1 w/w STMP-STPP mixture (plus 5% based on the starch). • Thermostable RS contents reached 32% for the ca. 55%-AM starch (control 13%) and 43% for the ca. 70%-AM starch (control 25%). Do not duplicate

• Z. Sui, A. Shah, & J.N. BeMiller (Carbohydrate Polymers, 86 (2011) 1461-1467) subjected normal maize kernels to HMT followed by a temperature cycling regime, then isolated the starch, and subjected it to a low level of cross-

linking (POCl3) and/or hydroxypropylation. • Hydroxypropylation alone produced 35% thermostable RS, while the low degree of cross- linking followed by hyroxypropylation produced 33% RS (control 16% RS). Cross-linking with CitricDo not Acid duplicate • The pH of a citric acid acid solution is adjusted to pH 3.0 (with NaOH), then mixed with a starch. • The moisture content of the starch is reduced to 5 -10% at 50 °C. • The starch is then heated to 130 °C for 5 h. • J.-Q. Mei, D.-N. Zhou, Z.-Y. Jin, X.-M. Xu, & H.-Q. Chen (Food Chemistry, 187 (2015) 378-384) produced tapioca starch with 70% thermostable RS in this way. Do not duplicate Pyrodextrins as RS

• C.C. Maningat & P.A. Seib, Chapter 3 in Y-C. Shi and C.C. Maningat (eds.), Resistant Starch: Sources, Applications and Health Benefits, John Wiley & Sons, 2013. Do not duplicate Pyrodextrin Formation • These products are made by heating “dry” acidified starch. • Three types of reaction may occur: (1) hydrolysis of glycosidic bonds, (2) transglycosylation (transglycosidation), and (3) reversion. • Transglycosylation introduces more branches and new linkages, e.g., α-(1→3), β-(1→6). Such bonds arte neither recognized or cleaved by amylases. Do not duplicate

• Unlike RS1, RS2, RS3, and the cross- linked RS4 products, pyrodextrins are water-soluble, so they would be classified as soluble fiber. That makes them different than other forms of RS in that cooking is not required in their analysis. • Reports of their RS contents are not available. Do not duplicate • Ones that have been treated with an α- amylase to remove the digestible portion are commercially available and result in production of significant amounts of butyric and propionic acids in the colon. • Perhaps, such pyrodextrins should be considered to be prebiotics rather than RS. Do not duplicate RS5

• J. Hasjim, Y. Ai, & J.-l. Jane, Chapter 4 in Y-C. Shi and C.C. Maningat (eds.), Resistant Starch: Sources, Applications and Health Benefits, John Wiley & Sons, 2013. Do not duplicate Preparation of RS5

• Lipids (especially unsaturated fatty acids, natural lysolecithins, and monoglycerides and related emulsifiers) form single helical complexes with amylose molecules. • RS5 is easier to make than RS3 or RS4. • RS5 is more heat stable than most starch granules. Do not duplicate • In the laboratory, AM-lipid complexes disassociate when heated to temperatures above 100 °C, but spontaneously and immediately reform during cooling. (It is not known what happens in a food formulation.) Do not duplicate • F. Tufvesson, M. Wahlgren, & A.-C. Elliasson (Starch/Stärke, 55 (2003) 138- 149) and S. Yotsawimonwat, K. Siroth, S. Kaewvichit, K. Piyachomkwan, J.-L. Jane, & J. Sirithunyalug (Intl. J. Biol. Macromol., 43 (2008) 94-99) obtained RS contents as high as 75% from complexation of palmitic and stearic acids with isoamylase debranched ca. 70%-AM amylomaize starch. Do not duplicate • J. Hasjim, S.-O. Lee, S. Hendrich, S. Setiawan, Y. Ai, & J.-l. Jane (Cereal Chemistry, 87 (2010) 257-262) also debranched pasted ca. 70%-AM amylomaize starch (using isoamylase at pH 3.5), then heated the suspension 1 h at 95 °C with palmitic acid (PA). RS content was determined as TDF. • They found the following RS contents: native starch that had undergone the same treatments without addition of enzymes or PA (35%), native starch + PA (39%), native starch (41.5%), debranched starch (47%), debranched starch + PA (53%). • They attributed the 53% value to the presence of both RS3 and RS5. Do not duplicate

The resistance of AM-lipid complexes to hydrolysis by amylolytic enzymes (amylases) depends on –

The structure of the “amylose” molecules, including whether they have been debranched. The nature of the lipid molecules Do not duplicate 1. The size of the fatty acid moiety. (In general, the dissociation temperature of the AM-lipid complex increases with the hydrocarbon chain length.) 2. The degree of saturation. (The dissociation temperature of the complex decreases with the number of cis double bonds in the hydrocarbon chain.) Do not duplicate Digestion of Complexes • J. Holm, I. Björck, S. Ostrowska, A.-C. Eliasson, N.-G. Asp, K. Larsson, & I. Lundquist (Starch/Stärke, 35 (1983) 294- 297) found that – • a potato AM-lysolecithin complex disappeared from the GI tract within 120 min and • that the complexed AM was hydrolyzed and the released glucose absorbed to the same extent as free AM in vivo, but somewhat slower. Do not duplicate • Then, T.C. Crowe, S.A. Seligman, & L. Copeland (The Journal of Nutrition, 130 (2000) 2006-2008) reported that complexed lauric, myristic, palmitic, and oleic acids and lysolecithin reduced hydrolysis of potato AM by α-amylase + amyloglucosidase (37 °C, 120 min) from 77% (uncomplexed) to 48-71%, with myristic acid being the most effective and stearic acid being the least effective. • The lipids had no effect on the hydrolysis of AP. Country Origins of PatentsDo not duplicate for RS preparation Over the Past 5 Years • China – 50 • South Korea – 5 • PCT Intl. – 5 • USA – 2 • Czech Republic, India, Japan, Mexico, Poland, & Taiwan - 1 Subjects of Patents andDo Patentnot duplicate Applications for RS Preparations Over the Past 5 Years • Rice genetic engineering or breeding – 10 • Treatment with α-amylase and a debranching enzyme – 7 • Production of green banana starch – 5 • Debranching alone – 4 • Cross-linking – 4 • HMT – 4 Do not duplicate • Retrogradation - 3 • α-Amylase alone – 3 • Treatment with amylosucrase – 3 • Acetylation – 2 • Treatment with α-amylase and glucoamylase – 2 • Treatment with acidic alcohol – 2 • UHP – 2 • Fatty acid complexes – 1 • Cross-linking with citric acid – 1 Do not duplicate • Octenylsuccinylation - 1 • “Enzymatic” – 1 • Wheat genetic engineering or breeding – 1 • Dry heating with acid (pyrodextrinization) – 1 • Dry heating with carrageenan – 1 • Cross-linked pasted starch with pectin – 1 • Retrograded starch with pectin – 1 • Ferrmentation and debranching – 1 • Ultrasonication – 1 • Other - 5 Do not duplicate

The End