Progress 07/01/05 to 06/30/11
Outputs
OUTPUTS: Nine separate research activities conducted over the duation of this project are briefly described. 1) Granule microstructures (i.e., the nature of cavities/channels) within normal and waxy wheat starches were elucidated using fluorescent dyes in conjunction with confocal laser scanning microscopy (CLSM). 2) Roles of channels and cavities in wheat starch modification reactions were established by derivatizing granules with 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF, a fluorescent probe used to simulate a reagent) and viewing granular reaction patterns by CLSM. 3) To investigate the effect of granule surface protein removal on starch chain reactivity, waxy and normal wheat starches (A- type granules) were treated as follows: a) no treatment (control), b) treatment with Tris-HCl buffer (pH 7.5), c) treatment with Proteinase K in Tris-HCl buffer, or d) treatment with protease XIV cocktail in Tris-HCl buffer. Treated starches were derivatized with DTAF (fluorescent reagent) under non-hydrated reaction conditions, after which granular reaction patterns were visualized by CLSM, and molecular reaction patterns of debranched starch derivatives were assessed via high performance size exclusion chromatography (HPSEC) equipped with refractive index (RI) and fluorescence (FL) detection. 4) Wheat starch A- and B-type granule reactivities were compared by first segregating the two wheat starch granule types and modifying them independently with various commercial reagents: propylene oxide analogue (POA), sodium trimetaphosphate (STMP), and phosphorous oxychloride (POCl3). Extents of reaction and granular reaction patterns (CLSM - reflectance mode) of modified granules were contrasted. 5) To understand the differential reactivities of the two wheat starch granule types at the molecular level, normal and waxy A- and B-type granules were derivatized with DTAF (fluorescent reagent), and molecular reaction patterns were assessed via HPSEC (as previously noted) to contrast molecular reaction densities on amylose (AM) and amylopectin (AP) branch chains. 6) Wheat starch (A-type granule) molecular reaction kinetics were monitored in reactions with DTAF (fluorescent reagent) over a 24 hour period. Derivatized starches were analyzed via HPSEC (as previously noted) to assess the relative reaction densities of AM and AP branch chains for the various reaction times. 7) Research efforts were devoted to generation of potato whole-tissue materials with enhanced resistant starch (RS) contents and moderated rates of starch digestibility. 8) Sodium acid pyrophosphate (SAPP) is commonly used as a process aid in potato processing operations to minimize the occurrence of after-cooking darkening (ACD). Research was conducted to identify possible alternatives for sodium acid pyrophosphate due to wastewater concerns. 9) To streamline molecular characterization of wheat starch, a rapid method for achieving starch solubilization was researched to expedite analysis by size exclusion chromatography (SEC). PARTICIPANTS: Kerry C. Huber (Project Director), James Nelson (Technical Support), Kathy Hendrix (Technical Support), Juan Pablo Hernandez-Uribe (Visiting Research Scientist - Potato Resistant Starch Products), Hyun-Seok Kim (Graduate Research Assistant - confocal laser scanning microscope and high performance size-exclusion chromatography methods; starch reactions with commercial reagents), Jung Sun Hong (Graduate Research Assistant - Starch Modification Reaction Kinetics), Chao-Feng Hsieh (Graduate Research Assistant - Protease Treatment of Starch Granules), Arkkrapan Anantachote (Potato Resistant Starch Products), Wei-Chen Yu (Graduate Research Assistant - Potato Resistant Starch Products), Melissa Leija (Graduate Research Assistant - Potato Resistant Starch Products), Jeremy Higley (Graduate Research Assistant - Molecular Reaction Patterns), Vinoth Thirugnanasambantham (Graduate Research Assistant - Wheat Starch A- and B-type Granule Modification), Caroline Campbell (Undergraduate Research Assistant - Starch Modification) TARGET AUDIENCES: The target audiences for this project include scientists and researchers working within the cereal/starch chemistry research area and industrial corporations comprising cereal, starch, biopolymer, and potato processing industries. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Research outcomes/impacts for nine research efforts are provided. 1) A-type starch granules exhibited radially-oriented, channel structures (only occasionally observed in B-type granules), which were lined/plugged with protein (were more easily visualized after protease treatment). 2) In hydrated reactions, reagent likely entered A-type granules via channels and connected cavities, and diffused into the granule matrix (akin to the corn starch model), despite being lined/plugged with protein. 3) Removal of protein via either buffer and/or protease generally enhanced reagent access to the granule matrix, and increased reaction density of AM and AP branch chains (non-hydrated reactions). 4) No reactivity differences were observed between wheat starch granule types in reactions with POA, while B-type type granules were generally more reacted (6-21%) than A-type granules in cross-linking reactions (STMP, POCl3). For granular reaction patterns, the extent to which reagent diffused into the granule matrix followed the order: POA>STMP>POCl3 (coincided to reagent reaction rate). Thus, reagent reactivity influences granular locale of reaction. 5) For wheat starch, B-type granules were generally reacted to a greater extent than A-type granules, though this result was limited primarily to waxy wheat starch (no such reactivity difference was observed for normal starch). Where differing extents of reaction were observed between starch derivatives, differences could be effectively explained on the basis of reaction densities on starch AM and AP branch (long, medium, and short) chains. 6) In assessing starch reaction kinetics with DTAF over the 24 hour reaction period, AM was more densely reacted than AP. Extent of derivatization for AP branch chains followed the order: long>medium>short, with AP long chains being even more densely reacted than AM. These findings represent the first-ever report of relative reactivities at the AM and AP branch chain level, and provide insight needed to understand and manipulate starch reactivity at the molecular level for enhancement of modified starch properties. 7) Potato whole-tissue resistant starch (RS) materials representing the range of possible RS types (RS1-RS4) have been developed within our laboratory, achieving RS levels as high as 50%. This technology has led to the filing of a U.S./PTC patent, and has generated interest from an industrial partner regarding commercialization. Products with enhanced RS content and moderated glycemic response provide potato producers entry into new market areas not accessible to traditional potato products. 8) Our research efforts identified both citric acid and ascorbic acid as viable alternatives to SAPP for minimizing ACD in processed potato strips. The alternative process aides additionally minimize waste water concerns relative to SAPP. 9) Various solvents (KOH, DMSO, etc.) in conjunction with short-time (35 sec) microwave heating were used to achieve solubilization of starch, while minimizing molecular degradation. The procedure represents a major improvement over tedious traditional methods and facilitates more rapid analysis of starch molecular structure.
Publications
- Huber and W.C. Yu. 2010. Potato products with enhanced resistant starch content and moderated glycemic response and methods thereof. Filed October 2, 2010 (pending U.S. Application No: 12896542; International Application No: PTC/US10/51164).
- Huber, K.C., and M. Leija. 2011. Methods of preparing potato food products with enhanced resistant starch content, Provisional Patent.
- BeMiller, J.N., and Huber,K.C. 2011. Starch, In Ulmann's Encyclopedia of Industrial Chemistry (7th Edition), B. Elvers (ed.), Wiley-VCH, Weinheim.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Activities: Four sets of research activities (designated 1, 2, 3, and 4) are reported. 1) Waxy and normal laboratory-isolated starches of both wheat (almost exclusively A- type granules) and corn were treated as follows: a) no treatment (control), b) treatment with Tris-HCl buffer (pH 7.5), c) treatment with Proteinase K in Tris-HCl buffer, or d) treatment with protease XIV cocktail in Tris-HCl buffer (wheat starches only) to investigate the effect of granule surface protein removal on starch chemical reactivity. All treated starches were derivatized with 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF, fluorescent probe) under non-hydrated reaction conditions. Granular reaction patterns of starch derivatives were visualized by confocal laser scanning microscopy (CLSM), while molecular reaction patterns were assessed via high performance size exclusion chromatography (HPSEC) equipped with refractive index (RI) and fluorescence (FL) detection. Prior to HPSEC analysis, derivatized starch fractions were debranched with isoamylase to facilitate determination of the relative reaction densities of amylose (AM) and amylopectin (AP) branch chains. 2) Potato-based resistant starch research efforts were focused on generating type 3 resistant starch (RS3) and type 2 (RS2) resistant starch in specific food applications. 3) Research in the area of potato processing (after-cooking darkening) was conducted for a regional potato processor. 4) Reaction kinetics of AM and AP branch chains continue to be elucidated. Products/Services/Dissemination: A M.S. thesis describing methods for producing RS within whole-tissue potato products was completed (one M.S. student was graduated in Food Science). Based on the research described within this thesis, one patent application was filed in 2010. Potato resistant starch research efforts have attracted industrial partners/collaborators. Research describing methods for elucidating granular reaction patterns within chemically modified wheat starch granules (reacted with propylene oxide analogue, POCl3, and sodium trimetaphosphate reagents) was presented at the SPIE (Scanning Microscopy) annual meeting, while two sets of research findings investigating: 1) the role of A:B-type granule ratio on cross-linked wheat starch reactivity and properties, and 2) the reaction kinetics of AM and AP branch chains within a model reaction system, were presented at the AACC International annual meeting. Research findings related to: 1) the role of the A:B-type granule ratio on hydroxypropylated wheat starch reactivity and properties, and 2) the composition, properties and AP fine structures of wheat starch A- and B-type granule, were published in quality journals. A book chapter (currently in press) on starch production, structure, properties, utilization, and applications was submitted for publication. Consulting activities were conducted for two industrial clients in the area potato processing and enhancing food applications of potatoes. PARTICIPANTS: Kerry C. Huber (Project Director), James Nelson (Technical Support), Kathy Hendrix (Technical Support), Juan Pablo Hernandez-Uribe (Visiting Research Scientist - Potato Resistant Starch Products), Jung Sun Hong (Graduate Research Assistant - Starch Modification Reaction Kinetics), Chao-Feng Hsieh (Graduate Research Assistant - Protease Treatment of Starch Granules), Wei-Chen Yu (Graduate Research Assistant - Potato Resistant Starch Products), Melissa Leija (Graduate Research Assistant - Potato Resistant Starch Products), Vinoth Thirugnanasambantham (Graduate Research Assistant - Wheat Starch Modification), Caroline Campbell (Undergraduate Research Assistant - Starch Modification) TARGET AUDIENCES: The target audiences for this project include scientists and researchers working within the cereal/starch chemistry research area and industrial corporations comprising cereal, starch, biopolymer, and potato processing industries. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Changes in Knowledge: 1) Granular reaction patterns revealed that reaction locale in non-hydrated reactions was confined to external granule surfaces (i.e., surface-reacted), though removal of protein via either buffer and/or protease generally enhanced reagent access to the granule matrix (exception: normal wheat starch). At the molecular level, protease treatment (and in some cases buffer alone) enhanced reaction density of AM (normal starch starches) and AP branch chains (especially for waxy starches). Removal of starch granule associated proteins has potential to enhance starch reactivity at both the granular and molecular levels, which could be useful for generating modified starch products with altered properties. 2) Using laboratory techniques, it was possible to generate resistant starch (RS2/RS3) levels of 15-20% in potato-based food products. It is anticipated that these emerging methods for producing resistant starch will be patented in the future. 3) Sodium acid pyrophosphate (SAPP) is commonly used as a process aid in potato processing operations to minimize the occurrence of dark color development following heating or cooking (after-cookening darkening). Our research findings have shown that there are other process aids that can function equally well as SAPP, providing potential alternatives to the use of SAPP in potato processing operations. 4) Our starch reaction kinetic data has provided evidence in support of a recently proposed alternative fine structure for amylopectin.
Publications
- Kim, H.S., and Huber K.C. 2010. Physicochemical properties and amylopectin fine structures of A- and B-type granules of waxy and normal wheat starch. J. Cereal Sci. 51-256-264.
- Kim, H.S., and Huber, K.C. 2010. Impact of A/B-type granule ratio on reactivity, swelling, gelatinization, and pasting properties of modified wheat starch. Part I: Hydroxypropylation. Carbohydr. Polym. 80:94-104.
- Huber, K.C., and BeMiller, J.N. 2010. Modified starch: Chemistry and properties. In Starch Characterization, Properties, and Applications, (ed.) A. Bertolini. Boca Raton: Taylor and Francis Group, LLC.
- BeMiller, J.N., and Huber, K.C. 2010. Starch. In Ulmann's Encyclopedia of Industrial Chemistry, (ed.) B. Elvers. Wiley-VCH: Weinheim (in press).
- Hong, H.J., and Huber, K.C. 2010. Reaction kinetics of amylose and amylopectin branch chains in a model derivatization system. Cereal Foods World Supplement 55(4):A8.
- Kim, H.S., and Huber, K.C. 2010. Influence of A/B-type granule ratio on cross-linked wheat starch starch properties. Cereal Foods World Supplement 55(4):A54.
- Yu, W.C. 2010. Development of a Multifunctional Potato Ingredient with Enhanced Resistant Starch Content and a Moderated Glycemic Response, M.S. Thesis, University of Idaho.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Activities: Three sets of research findings (designated 1, 2, and 3) are reported. 1) A preliminary investigation was conducted to assess the differential reactivities of the two wheat starch granule types at the molecular level. Two wheat starch genotypes (normal, waxy) and granule types (A-, B-types), as well as the influence of granule proteins in starch reactions, were tested. Isolated waxy and normal wheat starches were fractionated into their respective A- and B-type granule populations, after which portions of starch representing each isolated fraction were subjected to treatment with protease (24 hr) to facilitate removal of granule surface proteins. Isolated normal and waxy wheat starch A- and B-type granule fractions (both native and protease-treated) were derivatized with 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF, fluorescent probe) under both non-hydrated and hydrated reaction conditions. Granular reaction patterns of starch derivatives were visualized by confocal laser scanning microscopy (CLSM), while molecular reaction patterns were assessed via high performance size exclusion chromatography (HPSEC) equipped with refractive index (RI) and fluorescence (FL) detection. Prior to HPSEC analysis, derivatized starch fractions were debranched with isoamylase to facilitate determination of the relative reaction densities of amylopectin branch chains. 2) Using commercial wheat starch (comprised of A-type granules), the reaction kinetics of DTAF were investigated over the course of an extended reaction period (0, 0.5, 3, 8,16, and 24 hr). Reacted starch representing each reaction time interval was solubilized, debranched and analyzed on an HPSEC system equipped with RI and FL detection to assess the relative reaction densities of amylose and amylopectin branch chains for the various time intervals. 3) Starch of potatoes was chemically modified (substituted and cross-linked) to produce novel resistant starch products. Structure, composition, and properties of these novel products were assessed. Products/Services/Dissemination: A Ph.D. dissertation reporting findings on wheat starch A- and B-type granule composition, properties, reactivity, and granular reaction patterns was completed. Two manuscripts from this dissertation were submitted to quality journals in 2009 (one in press; one accepted). Three papers describing wheat starch reactivity and molecular reaction patterns of wheat starch A- and B-type granule populations were presented at the 2009 AACC International and Starch Round Table meetings. Findings from wheat research projects were included within a book chapter on modified starches. Potato research work was formally presented at the AACC International annual meeting. An invention disclosure was filed regarding a novel potato-based resistant starch product. Results from potato research were presented to two regional potato processing companies. Analytical and consulting services based on research interests were performed for multiple potato processors within the region. PARTICIPANTS: Kerry C. Huber (Project Director), James Nelson (Technical Support), Kathy Hendrix (Technical Support), Jung Sun Hong (Graduate Research Assistant - DTAF reaction kinetics), Hyun-Seok Kim (Graduate Research Assistant/Post-Doc - confocal laser scanning microscope and high performance size-exclusion chromatography methods), Vinoth Thirugnanasambantham (Graduate Research Assistant - reactivity experiment with wheat starch granule types), Chao-Feng Hsieh (Graduate Research Assistant - protease treatment of starch granules), Weichen Yu (Graduate Research Assistant - potato resistant starch products) TARGET AUDIENCES: The target audiences for this project include scientists and researchers working within the cereal/starch chemistry research area and industrial corporations comprising cereal, starch, biopolymer, and potato processing industries. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Changes in Knowledge: 1) In preliminary investigations of wheat starch granule reactivity with DTAF (fluorescent probe), granular reaction patterns visualized by CLSM revealed that reaction locale in non-hydrated reactions was confined to external granule surfaces (i.e., surface-reacted), while hydrated starch reactions permitted reagent to react throughout the granule matrix (i.e., matrix-reacted). For all surface- and matrix-reacted granule derivatives, starch material eluting in the amylose (AM) region of IPSEC chromatograms was consistently more densely reacted than that associated with amylopectin (AP). Protease treatment had no impact on the extent of reaction in surface-reacted derivatives, while A-type (relative to B-type) granule reactivity in matrix-reacted derivatives was enhanced after protease treatment. For both surface- and matrix-reacted starch derivatives, B-type granules were more heavily reacted than A-type granules. In those cases where differing extents of reaction were observed between starch granule derivatives, reactivity differences could be attributed to differential reaction densities on starch AM and AP branch (long, medium, and short) chains. 2) In studying the reaction kinetics of DTAF (wheat starch), overall extent of reaction increased over the 24 hr reaction period, exhibiting a two-phase reaction rate (rapid/0-3 hr; slow/3-24 hr). For all time intervals of reaction, AM was more heavily reacted than AP. Of the various chain fractions, AP long chains were most densely reacted, followed by AM, AP medium branch chains, and AP short branch chains. The relative reaction densities noted between the various starch chains (i.e., AM and AP long, medium, and short branch chains) were maintained at all time intervals of reaction, implying that the same types of chains were reacted in similar proportions at all time stages of reaction. 3) Resistant starch content of modified potato starch products approached 50%, and modified potato products exhibited a slowed rate of digestion by amylolytic enaymes. Impacts: The findings from DTAF reactions represent the first-ever report capable of accounting for differences in starch reactivity at the AM and AP branch chain level, and provide insight to understand, and potentially manipulate, starch reactivity at the molecular level to diversify modified starch properties. Advances in potato-based resistant starch ingredients offer new opportunities for potato products (due to the fact that traditional potato products generally possess very low levels of resistant starch and a high rate of starch digestibility ~ considered high glycemic foods). This emerging technology has potential to provide potato growers and processors access into product markets that have been previously inaccessible.
Publications
- Kim, H.S. 2009. Wheat Starch A- and B-type Granule Microstructure and Reactivity, Ph.D. Dissertation, University of Idaho.
- Kim, H.S., and Huber, K.C. 2009. Physicochemical properties and amylopectin fine structures of A- and B-type granules of waxy and normal wheat starch. J. Cereal Sci. (accepted).
- Kim, H.S., and Huber, K.C. 2009. Impact of A/B-type granule ratio on reactivity, swelling, gelatinization, and pasting properties of modified wheat starch. Part I: Hydroxypropylation. Carbohydrate Polymers (in press), doi:10.1016/j.carbpol.2009.10.070.
- Huber, K.C., and BeMiller, J.N. 2009. Modified starch: Chemistry and properties, In 'Starches: Applications, Characterization, and Properties', A.C. Bertolini (Ed.), Taylor and Francis Group, LLC, Boca Raton, FL.
- Thirugnanasambantham, V., Kim, H.S., and Huber K.C. 2009. Relating wheat starch A- and B-type granule reactivity to molecular reaction patterns on derivatized starch chains. Cereal Foods World Supplement 54(4):A69
- Kim, H.S., and Huber, K.C. 2008. Impact of A- and B-type granule ratio on swelling, gelatinization, and pasting properties of hydroxypropylated wheat starch. Cereal Foods World Supplement 54(4):A50.
- Anantachote, A., Kim, H.S., and Huber K.C. 2009. Separation and isolation of intact parenchyma cells from raw (uncooked) potato (Solanum tubersosum) tissue. Cereal Foods World Supplement 54(4):A33
- Huber, K.C., and Yu, W. 2009. Chemically modified potato products, Invention disclosure.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Activities: Research was focused on continued elucidation of wheat starch A- and B-type granule reactivity in modification reactions with substitution (propylene oxide analog/POA) or cross-linking (phosphorus oxychloride/POCl3, sodium trimetaphosphate/STMP) reagents. Substitution reactions were conducted in both aqueous (containing 1 molal sodium sulfate) or aqueous ethanol (65% v/v) reaction media under basic conditions (pH 11.5). Cross-linking reactions were conducted in aqueous reaction system (containing 15% sodium sulfate on a starch basis) at pH 11.5. Purified A- and B-type granules of both waxy and normal wheat starch were the substrates of all reactions, and each reaction was investigated at three reagent addition levels to allow observation of derivatization trends. Degree of substitution (DS) or molar substitution (MS) values were determined for all modified starch A- and B-type granule fractions. Leaching of starch molecules from granules was also monitored over the course of reaction. Granular reaction patterns of starch derivatives were further studied by reflectance confocal laser scanning microscopy (R-CLSM). Anionic starch derivatives were converted to silver(I) salts, after which silver(I) cations bound at reaction sites were reduced to elemental silver by exposure to UV light. Serial optical sections of modified starch granules were obtained via CLSM. To visualize reaction patterns within the inner regions of modified starch granules, select numbers of optical sections representing just the inner regions of granules were used to reconstruct 3-dimensional (3D) stereoimages of modified granules. In a related study, molecular patterns of reaction within A- and B-type wheat starch granules, modified with a fluorescent probe, were investigated via high performance size-exclusion chromatography (HPSEC) equipped with fluorescence detection. Modified starches were analyzed both as intact polymers and debranched (amylopectin branch) chains. A collaborative project investigating the molecular degradation pattern of starch within flaked waxy barley grain during digestion within the cow rumen was also conducted. Molecular degradation patterns of starch polymers were tracked via HPSEC. Services/Events/Dissemination: Research findings on channels within wheat starch granules were published in a quality journal. Research depicting reaction patterns within modified wheat starch A- and B-type granules was presented at the 2008 AACC International annual meeting. A method for tracking molecular reaction patterns on amylopectin branch chains was presented within a symposium at the 2008 ACS meeting in Philadelphia. An invention disclosure was filed regarding a novel potato-based resistant starch product. Analytical and consulting services based on research interests were performed for multiple potato processors within the region. PARTICIPANTS: Kerry C. Huber (Project Director): Directed the research efforts of all personnel in support of project objectives. James Nelson (Research Support Scientist): Provided technical support, training of lab personnel, and day to day supervision of the laboratory in support of project objectives. James N. BeMiller (Collaborator): Purdue University, Alex Hristov (Collaborator) University of Idaho/Penn. St. University, Hyun-Seok Kim (Graduate Student): University of Idaho, Wei Chen Yu (Graduate Student): University of Idaho, Arkkrapan Anantachote (Graduate Student): University of Idaho, Vinoth Thirugnanasambantham (Graduate Student): University of Idaho, Jung Sun Hong (Graduate Student): University of Idaho, Chao-Feng Hsieh (Graduate Student): University of Idaho. TARGET AUDIENCES: The target audiences for this project include other scientists working within the cereal/starch research area and corporations comprising cereal, starch, and potato processing industries. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in Knowledge: No reactivity differences were observed between the two granule types in substitution reactions. POA-substituted A- and B-type granule fractions exhibited similar trends in swelling, gelatinization, and pasting properties. While chemical substitution slightly reduced the inherent physical property differences noted between native A- and B-type granules, modification at investigated MS levels (0.002-0.025) did not fully overcome inherent property differences between the two granule types. In cross-linking reactions, B-type granules were generally reacted to slightly greater levels (6-21%) than A-type granules. In further contrasting POCl3 and STMP cross-linked A- and B-type starch granule fractions (at comparable DS levels), the two cross-linking agents produced notably different effects on starch swelling and gelatinization properties. These differences were likely attributable to differential cross-linking efficiency (ratios of monostarch vs. distarch phosphate) and/or granular patterns of reaction for the two reagents. For granular patterns of reaction within modified (POA, STMP, and POCl3) A- and B-type starch granule fractions, heavy reaction was observed at external granule surfaces, regardless of the reagent type. However, the strong signal emanating from external granule surfaces (due to heavy reaction) prevented direct observation of reactions patterns within the granule matrix. This limitation was overcome by constructing 3D images from select R-CLSM optical sections representing just the inner regions of the granule matrix. Via this technique, distinct reaction patterns for the three reagent types were clearly visualized, providing the most detailed granular reaction patterns to date. The 3D stereoimages also contributed topographical information that clearly illustrated the role of channels in all reactions. In many cases, reaction within channels was not uniform, but appeared to be relatively heavier within the regions of the amorphous (as opposed to the semicrystalline) growth rings of granules. Within A- and B-type granules, channels were the primary means by which reagent entered the granule matrix. In general, the ability for reagent to diffuse into the granule matrix followed the sequence: POA>STMP>POCl3 (based on reagent rate of reaction with starch molecules). For molecular reaction patterns on starch molecules reacted a fluorescent probe, amylose was more densely reacted than amylopectin for both A- and B-type granules; among amylopectin branch chains, long chains (B3 and larger) were most highly reacted. Significant advances are being made to understand the relationship between granular and molecular reaction patterns, reaction conditions, and modified starch properties, all of which enhance capability to generate novel modified starch products. A method was developed to successfully track starch degradation patterns in flaked grain. Using this analytical method, processing conditions for flaked grains for animal feed can be optimized. Change in Actions: Analytical efforts for a potato processor led to a major capital investment in processing operations to enhance product quality.
Publications
- Kim, H.S., and Huber, K.C. 2008. Channels within soft wheat starch A- and B-type granules. J. Cereal Sci. 48:159-172.
- Anantachote, A. 2008 (December). Chemical-assisted Separation and Characterization of Parenchyma Cells from Potatoes (Solanum tuberosum), Master's Thesis, University of Idaho.
- Kim, H.S., and Huber, K.C. 2008. Granular reaction patterns within chemically modified wheat starch A-type granules, presented at the AACC International annual meeting, Honolulu, HI. Cereal Foods World Supplement 53(4):A65.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Activities: Research focused on continued elucidation of wheat starch A-and B-type granule microstructure to understand its impact on starch reactivity. While channels had been previously observed in wheat starch A-type granules, the microstructure of B-type granules has proven more difficult to visualize due to their smaller size. Both scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) techniques were utilized to investigate the possibility of granule surface pores and channels on/within B-type starch granules. As channels within A-type starch granules had been previously shown to be partially filled with protein, the approach to investigating B-type granule microstructure also involved strategic treatment of B-type starch granules with proteases and fluorescent probes to further assess the potential for and nature of possible channels. Further work was also directed to visualization of granular patterns of reaction of wheat starch A- and
B-type granules using industrial reagents, including relatively fast-reacting (phosphorus oxychloride) and slow-reacting (propylene oxide analog, POA; and sodium trimetaphosphate, STMP) reagents to generate anionic starch derivatives. Anionic derivatives were utilized to facilitate binding of silver cations to starch granule reaction sites. Silver cations bound at reaction sites were reduced to elemental silver by exposure to UV light, and the locale of elemental silver within starch granules was observed via CLSM to reveal granular reaction patterns for the various modified starch products. In another aspect of this research effort, a method for rapid microwave dissolution of granular starch (in the presence of KOH/urea) that was reported in the previous year was studied for use in conjunction with high performance size-exclusion chromatography (HPSEC) to investigate starch structural analysis. Starch solubilized by this method and analyzed by HPSEC displayed a significant
interfering peak (attributable to salt/urea) that coeluted with the amylose peak. The use of a desalting column for preparative treatment of the solubilized starch solution was investigated to remove contaminants responsible for the interfering peak to allow for rapid analysis by HPSEC. In regard potato-based resistant starch (RS) products, various products were evaluated for physical properties. Collaborative projects investigating the possibility of dual-purpose hard white wheat and value added animal feed processing were also conducted. Services/Events/Dissemination: Research relating to wheat starch granule channels and granular reaction patterns was reported at the AACC International annual meeting. Lastest findings involving development of a model system for tracking molecular reaction patterns on starch chains were reported at the Starch Round Table meeting, an international forum for starch research. In relation to potato research, consulting activities were performed for a
primary potato processor within the state. Research findings on starch channels, starch sample preparation for HPSEC analysis, and animal feed processing were published in appropriate journals.
PARTICIPANTS: Kerry C. Huber (Project Director): Directed the research efforts of all personnel in support of project objectives. James Nelson (Research Support Scientist): Provided technical support, training of lab personnel, and day to day supervision of the laboratory in support of project objectives. James N. BeMiller (Collaborator): Purdue University, Alex Hristov (Collaborator): University of Idaho, Kim Kidwell (Collaborator): Washington State University, Hyun-Seok Kim (Graduate Student): University of Idaho, Wei Chen Yu (Graduate Student): University of Idaho, Ark Anantachote (Graduate Student): University of Idaho, Vinoth Thirugnanasambantham (Graduate Student): University of Idaho,
TARGET AUDIENCES: The target audiences for this project include other scientists working within the cereal/starch research area and corporations comprising cereal, starch, and potato processing industries.
Impacts
Change in Knowledge: The presence of surface pores and the nature of channels associated with wheat starch B-type granules was revealed using SEM and CLSM. This work reports the first visualization of channels within wheat starch B-type granules. While A-type starch granules exhibited a network of radially-oriented, channel structures (typical of those observed within corn starch granules), B-type granule channels predominantly occurred as less-defined voids extending to granule surfaces. However, some discrete radially-oriented channels were also visualized within B-type granules, though these features were observed in a relative minority of cases. Channels of A- and B-type starch granules were demonstrated to facilitate transfer of fluorescent probes into the granule matrix, though this effect was aided by granule swelling (hydration) and/or removal of channel-associated protein. In assessing granular reaction patterns of wheat starch granules modified by industrial
reagents, it was demonstrated that surface pores and channels played significant roles in facilitating reagent entry into granules. Nevertheless, wheat starch granular reaction patterns varied according to reagent type. Fast-reacting reagents such as phosphorus oxychloride tended to react at granule surfaces, including those of channels and inner cavities within granules; highlighted channels within granules became less visible as reagent addition levels increased and reaction became relatively more homogeneous. In contrast, slower-reacting regents (propylene oxide analog and sodium trimetaphosphate) generally yielded more homogeneous reaction patterns, though none of the reaction levels investigated could be characterized as completely homogeneous. The wheat starch reactivity research findings, which were presented at the AACC International meeting, were awarded a Carbohydrate Division best paper award. Relating to starch structural analysis, a technique was established to remove
impurities from starch dissolved in strong alkali and neutralized with acid to accommodate starch structural analysis via HPSEC. Starch (corn and wheat) subjected to an alkaline-microwave dissolution scheme (35 s microwave heating in a mixture of 6 M urea and 1 M KOH) was passed through a desalting column, which process removed impurities (e.g., salts, urea) and eliminated the interfering chromatographic peak. This simple technique coupled with the rapid alkaline-microwave starch dissolution procedure greatly expedites structural investigation of starch by facilitating analysis by HPSEC. In other collaborative research efforts, inclusion of a surfactant in animal feed steam flaking operations resulted in a higher ruminal dry matter degradability, while evidence was provided that a partial waxy dual-purpose hard white wheat may be suitable for both bread and noodle applications. Change in Actions: Consulting activity based on potato research efforts resulted in troubleshooting of
processing limitations for a potato processor that are now being modified to achieve higher quality products.
Publications
- Kim, H.S., and Huber, K.C. 2007. A simple purification (desalting) procedure to facilitate structural analysis of alkali-solubilized/neutralized starch solution by intermediate-pressure size-exclusion chromatography. J. Agric. Food Chem. 55:4944-4948.
- Hristov, A.N., Zaman, S., Vanderpol, M., Szasz, P., Huber, K.C., and Greer, D. 2007. Effect of a saponin based surfactant and ageing time on ruminal degradability of flaked corn grain dry matter and starch. J. Animal Sci. 85:1459-1466.
- Kim, H.S., and Huber, K.C. 2007. Channels within soft wheat starch A- and B-type granules. J. Cereal Sci. (in press; doi: 10.1016/j.jcs.2007.09.002).
- Kim, H.S., and Huber, K.C. 2007. Reactivity of wheat starch A- and B-type granules, AACC International, San Antonio, TX, October, 2007.
- Kim, H.S., and Huber, K.C. 2007. Nature of channels within wheat starch A- and B-type granules, AACC International, San Antonio, TX, October, 2007.
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Progress 01/01/06 to 12/31/06
Outputs
Three sets of findings (designated 1, 2, and 3) are reported. Project 1) Progress has been made toward development of potato-based ingredients with both significant resistant starch (RS) content and moderated rates of starch digestibility (glycemic response). Both RS1/RS2 and RS4 products have been developed with emphasis on whole tissue potato ingredients, as opposed to those just based on pure starch. For the RS1/RS2 products, two potential processing approaches have been assessed for generating products, which will be characterized for properties over the course of the coming year. Project 2) Channel structures within waxy and normal wheat starch A- and B-type granules were investigated for their potential to influence wheat starch chemical modification. Channel structures in corn starch granules have been shown to facilitate reagent entry into starch granules and influence granular reaction patterns. As wheat starch granule channels were previously observed to
possess protein, it was not clear whether they would prove capable of facilitating reagent entry into starch granules. This aspect was explored by reacting starch with a reactive fluorescent dye in both the non-hydrated and hydrated states, and tracking granular reaction patterns with confocal laser scanning microscopy. In non-hydrated starch reactions, fluorescent dye was limited to external granule surfaces, highlighting also a few channels and cavities connected to the granule exterior (mainly A-type granules). In contrast, granules derivatized in the hydrated state exhibited reaction within the granule matrix (was not just limited to external surfaces). For A-type starch granules reacted in the hydrated state, relatively large channels (highlighted by fluorescent reagent) appeared to originate primarily from the equatorial groove region of granules, though some narrower channels originating from regions other than the equatorial groove were also apparent. For hydrated starch
reactions, it is hypothesized that reagent entered A-type granules via channels and was further relayed to granule central cavities, where it diffused outward into the granule matrix (akin to corn starch). Channels in wheat starch granules (particularly A-type) do facilitate reagent penetration into the granule matrix (similar to corn starch granules) in hydrated reaction systems, despite the fact that channels in wheat starch granules are filled to some degree with protein. Project 3) Waxy and normal barley grain processed as animal feed was analyzed with respect to starch relative crystallinity and gelatinization properties to provide insight and explanation of starch digestibility patterns in feeding trials with dairy cows. Normal processed barley grain, which exhibited greater in situ ruminal dry matter degradability compared to that of waxy barley, also exhibited a lesser starch relative percent crystallinity value and lower gelatinization onset, peak, and completion temperatures
than processed waxy barley grain. Both of these observations are consistent with a reduced dry matter and starch effective degradability observed for waxy (relative to normal) processed barley grain.
Impacts
Impact statements are identified according to the numbering scheme set forth in the previous section. 1) Resistant starch offers important physiological benefits including moderation of blood sugar levels and production of butyrate in decreasing risk for development of human disease (type II diabetes, heart disease, etc.). This approach is intended to diversify the assortment of potato-based ingredients available for food formulation, and expands opportunities for potato-based products into previously inaccessible market areas. 2) Starch represents the second greatest biomass on the planet and a superb source of functional biopolymers. However, prior to use, most granular starch that is to be used as a food ingredient is first chemically modified, while yet in the granular form, to alter and improve the physical properties of starch polymers in accordance with the intended end-use. Starch granule structure dictates the accessibility of individual starch molecules to
chemical reagents during reaction to impact reaction patterns and, ultimately, starch properties. Understanding of wheat starch granule microstructure provided by this study will yield insight needed for better control and manipulation of chemical modification practices for production of more competitive and/or novel value-added starch products. 3) A fundamental knowledge of the basis for dry matter/starch degradation and utilization in animal feeds will provide understanding necessary to enhance selection of raw materials and processing schemes for production of the most desirable products.
Publications
- Takhar, .P.S., Kulkarni, M.V., and Huber, K.C. 2006. Dynamic viscoelastic properties of pasta as a function of temperature and water content. J. Texture Studies 37:696-710.
- Geera, B.L., Nelson, J.E., Souza, E, and Huber, K.C. 2006. Composition and properties of A- and B-type granules of normal, partial waxy, and waxy soft wheat. Cereal Chem. 83:551-557.
- Geera, B.L., Nelson, J.E., Souza, E, and Huber, K.C. 2006. Starch factors influencing growing environment-induced fluctuation of flour pasting behavior in normal and partial waxy soft wheat. Cereal Chem. 83:558-564.
- Foley, A.E., Hristov, A.N., Melgar, A., Ropp, J.K., Etter, R.P., Zaman, S., Hunt, C.W., and Huber, K.C. 2006. Effect of barley and its amylopectin content on ruminal fermentation and nitrogen utilization in lactating dairy cows. J. Dairy Sci. 89:4321-4335.
- Kim, H.S., Higley, J.S, and Huber, K.C. 2006. Alkaline dissolution of starch facilitated by microwave heating for analysis by size-exclusion chromatography (SEC). J. Agric. Food Chem. 54:9664-9669.
- Geera, B.L., Nelson, J.E., Souza, E, and Huber, K.C. 2006. Granule bound starch synthase I (GBSSI) gene effects related to soft wheat flour/starch characteristics and properties. Cereal Chem. 83:544-550.
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Progress 01/01/05 to 12/31/05
Outputs
Four sets of findings (designated 1, 2, 3, and 4) are reported. 1) The nature of cavities and channels within granules of normal and waxy wheat starch was investigated using fluorescent dyes in conjunction with confocal laser scanning microscopy (CLSM). Starch stained with a fluorescent probe throughout the granule matrix revealed cavities within both A- and B-type starch granules. For channels, A-type starch granules stained with a protein-specific dye revealed a network of radially-oriented, channel-like, structures, which were not visualized in B-type granules. When subjected to treatment with fluorescent dye under non-swelling conditions (limiting dye to external granule surfaces including those of any channels extending to the granule exterior), channels were not directly observed within B-type granules. For A-type starch granules, very short channels were periodically visualized in a small percentage of granules. Channels in wheat starch A-type granules appeared
to be filled with protein, and were visualized in greater frequency following protease treatment, which enhanced access of dye to channels and cavities under non-swelling conditions. These microstructural findings are key to interpretation of granular reaction patterns of chemically modified starches. 2) To enhance molecular characterization of wheat starch, a new method utilizing alkali in conjunction with short-time microwave heating was pioneered for starch solubilization to facilitate analysis by size exclusion chromatography (SEC). Short-time microwave heating offered good SEC separation of starch fractions, while minimizing molecular degradation, and represents a novel experimental procedure for starch solubilization. 3) A fluorescent probe, confocal laser scanning microscopy (CLSM), and gel permeation chromatography (GPC) were used to probe amylose and amylopectin locale and structure within native starch granules. Starch granules were derivatized with a fluorescent probe to
produce both surface-derivatized (probe confined to granule surfaces) and internally-derivatized (probe reacted throughout granule matrix) derivatives. Native and isoamylase-debranched starch derivatives were fractionated by GPC, and analyzed for fluorescence intensity. Amylose was more heavily derivatized than amylopectin (AGU basis). For surface-derivatized starches, both amylose and amylopectin fractions were labeled by the fluorescent probe, indicating the presence of both molecules at granule surfaces. However, the proportion of fluorescent-labeled amylose at the granule surface was substantially lower than that of the entire granule. Amylopectin structural characteristics were also shown to differ at the granule surface compared to the overall granule matrix. 4) Experimental efforts have been successfully directed toward generation of potato-based ingredients with enhanced resistant starch (RS) contents and moderated rates of starch digestibility. Potato products, which are
traditionally considered high glycemic foods, have been processed with up to 50% RS content in comparison to traditional commercial controls that possess only negligible amounts of RS.
Impacts
Research summaries of the previous section are individually addressed coinciding with the numbering scheme previously set forth. 1) Granule microstructure and composition ultimately dictate starch reactivity to chemical modification agents for production of value-added starch products with enhanced functionality. Elucidation of the microstructural differences between A- and B-type granules provides key insight needed to manipulate and control granular reactions to achieve high quality modified wheat starch products for global commerce. 2) Molecular structure is essential to understanding function. The developed procedure provides a fast, simple, starch dissolution method that represents an improvement over tedious traditional methods for accurate and more rapid assessment of starch molecular structure. 3) This novel method utilizes a directed-derivatization scheme involving fluorescence imaging and size exclusion chromatography to bridge the gap between starch granule
microstructure and molecular structure, and represents a powerful tool for future elucidation of starch structural regimes. 4) Resistant starch offers important physiological benefits including moderation of blood sugar levels and production of butyrate in decreasing risk for development of human disease (type II diabetes, heart disease, etc.). This approach diversifies the assortment of potato-based ingredients available for food formulation, and expands opportunities for potato-based products into market areas that were previously inaccessible.
Publications
- Higley, J.S. 2005. Elucidation of Starch Granule Surface Composition and Reactivity aided by Directed-Derivatization and Gel Permeation Chromatography Coupled with Fluorescence Detection, M.S. Thesis. University of Idaho, Moscow, ID.
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