Enzymatic Approaches for Structuring Starch to Improve Functionality.

Starch is one of the most abundant renewable biopolymers in nature and is the main constituent in the human diet and a raw material for the food industry. Native starches are limited in most industrial applications and often tailored by structural modification to enhance desirable attributes, minimize undesirable attributes, or create new attributes. Enzymatic approaches for structuring starch have become of interest to the food industry precisely because the reactions minimize the formation of undesirable by-products and coproducts and are therefore considered environmentally friendly methods for producing clean-label starches with better behavioral characteristics. Starches with improved functionalities for various applications are produced via enzyme hydrolysis and transfer reactions. Use of novel, multifunctional, starch-active enzymes to alter the structures of amylose and/or amylopectin molecules, and thus alter the starch's physiochemical attributes in a predictable and controllable manner, has been explored. This review provides state-of-the-art information on exploiting glycosyl transferases and glycosyl hydrolases for structuring starch to improve its functionalities. The characteristics of starch-active enzymes (including branching enzymes, amylomaltases, GH70 α-transglycosylases, amylosucrases, maltogenic amylases, cyclomaltodextrinases, neopullulanases, and maltooligosaccharide-forming amylases), structure-functionality-driven processing strategies, novel conversion products, and potential industrial applications are discussed.

Microbial Starch-Converting Enzymes: Recent Insights and Perspectives.

Starch is an abundant, natural, renewable resource, and present as the major storage carbohydrate in the seeds, roots, or tubers of many important food crops, such as maize, wheat, rice, potato, and cassava. Uses of native starches in most industrial applications are limited by their inherent properties. Hence, they are often structurally modified after isolation to enhance desirable attributes, to minimize undesirable attributes, or to create new attributes. Enzymatic, rather than chemical, approaches are used in the production of starch syrups, maltodextrins, and cyclodextrins. However, the desire for starch-active enzymes working optimally at high temperatures and low pH conditions with superior stability and activity is still not satisfied and this stimulates interest in developing novel and improved starch-active enzymes through a variety of strategies. This review provides current information on enzymes belonging to GH13, 57, 70, and 77 that can be used in structural modifications of the starch polysaccharides or to produce starch-derived products from them. The characteristics and catalytic mechanisms of microbial enzymes are discussed (including 4-α-glucanotransferase, branching enzyme, maltogenic amylase, cyclomaltodextrinase, amylosucrase, and glucansucrase). Product diversity after starch-converting reaction and utilization in industrial applications are also dealt with.

Impact of reagent infiltration time on reaction patterns and pasting properties of modified maize and wheat starches.

The impact of granular and molecular reaction patterns on modified starch properties was investigated as a function of the length of time allowed for reagent to infiltrate starch granules. A fluorescent reagent [5-(4,6-dichlorotriazinyl)aminofluorescein] was dispersed in aqueous normal maize or wheat starch slurries (35%, w/v) for 0, 5, 10, 30, or 60min, after which reaction was initiated by increasing the pH to 11.5 and allowing reaction to proceed for 3h. With increasing lengths of infiltration, the reaction became increasingly homogeneous within the granule interior (matrix) and the AM:AP reactivity ratio increased (wheat starch), as assessed by confocal laser scanning microscopy (CLSM) and size-exclusion chromatography (refractive index and fluorescence detection), respectively. A longer reagent infiltration time also led to a more inhibited (i.e., cross-linked) pasting viscosity, suggesting that both granular and/or molecular reaction patterns were altered by varied reagent infiltration times to ultimately impact modified starch properties.

Effect of high-speed jet on flow behavior, retrogradation, and molecular weight of rice starch.

Effects of high-speed jet (HSJ) treatment on flow behavior, retrogradation, and degradation of the molecular structure of indica rice starch were investigated. Decreasing with the number of HSJ treatment passes were the turbidity of pastes (degree of retrogradation), the enthalpy of melting of retrograded rice starch, weight-average molecular weights and weight-average root-mean square radii of gyration of the starch polysaccharides, and the amylopectin peak areas of SEC profiles. The areas of lower-molecular-weight polymers increased. The chain-length distribution was not significantly changed. Pastes of all starch samples exhibited pseudoplastic, shear-thinning behavior. HSJ treatment increased the flow behavior index and decreased the consistency coefficient and viscosity. The data suggested that degradation of amylopectin was mainly involved and that breakdown preferentially occurred in chains between clusters.

Physical modification of food starch functionalities.

Because, in general, native starches do not have properties that make them ideally suited for applications in food products, most starch is modified by dervatization to improve its functionality before use in processed food formulations, and because food processors would prefer not to have to use the modified food starch label designation required when chemically modified starches are used, there is considerable interest in providing starches with desired functionalities that have not been chemically modified. One investigated approach is property modification via physical treatments, that is, modifications of starches imparted by physical treatments that do not result in any chemical modification of the starch. Physical treatments are divided into thermal and nonthermal treatments. Thermal treatments include those that produce pregelatinized and granular cold-water-swelling starches, heat-moisture treatments, annealing, microwave heating, so-called osmotic pressure treatment, and heating of dry starch. Nonthermal treatments include ultrahigh-pressure treatments, instantaneous controlled pressure drop, use of high-pressure homogenizers, dynamic pulsed pressure, pulsed electric field, and freezing and thawing.

Effects of the order of addition of reagents and alkali on modification of wheat starches.

The objective of this research was to determine if adding reactive reagents to wheat starch granules before addition of alkali (the TRF method) would produce products that are different than those obtained with the conventional procedure (adding alkali before addition of reagent). Laboratory-isolated (LI) and commercial (C) normal (NWS) and waxy (WWS) wheat starches were each reacted with 6 reagents (acetic-adipic mixed anhydride (AAMA), phosphoryl chloride (POCl3), sodium trimetaphosphate (STMP), acetic anhydride (AA), succinic anhydride (SA), octenylsuccinic anhydride (OSA)). Data obtained were similar to those previously obtained with maize starches (Sui, Huber, & BeMiller, 2013). Almost no starch polymer molecule modification occurred when the TRF method and AAMA or AA were used; less than a third as much reaction when SA was the reagent used, and about the same amount of reaction when POCl3, STMP, or OSA were the reagents used (for different reasons).

Preparation and characterization of octenylsuccinylated plantain starch.

Plantain starch was esterified with octenylsuccinic anhydride (OSA) at two concentrations (3 and 15% w/w) of OSA. The morphology, granule size distribution, pasting, gelatinization, swelling, and solubility of granules and structural features of the starch polymers were evaluated. Granules of the OSA-modified starches increased in size during cooking more than did the granules of the native starch, and the effect was greater at the higher OSA concentration. Pasting viscosities also increased, but gelatinization and pasting temperatures and enthalpy of gelatinization decreased in the OSA-modified starches. It was concluded that insertion of OS groups effected disorder in the granular structure. Solubility, weight average molar mass, Mw¯, and z-average radius of gyration, RGz, of the amylopectin decreased as the OSA concentration increased, indicating a decrease in molecular size.

Effects of the amylose-amylopectin ratio on starch-hydrocolloid interactions.

Combinations of 4 rice starches with amylose (AM) contents of 0%, 15%, 22%, and 28% and 8 hydrocolloids (xanthan, guar gum, CMC, sodium alginate, HPMC, κ-, ι-, λ-carrageenan) were used (4.75% starch and 0.25% hydrocolloid). With a few exceptions, addition of a hydrocolloid increased peak and final η, breakdown, setback, G″, and η(*), K, and ηa,100 values. It is concluded that the AM content of the starch was a greater determinant of pasting, paste, and gel properties than was the added hydrocolloid at the 19:1 (w/w) starch-hydrocolloid ratio used. Reinforced is the previous conclusion that the properties of a starch-hydrocolloid combination are determined by the specific combination.

Effects of the order of addition of reagents and catalyst on modification of maize starches.

The objective of this research was to determine if adding reactive reagents to starch granules before addition of alkali (TRF method) would produce products that are different than those obtained by adding alkali before addition of reagent. Normal (NMS) and waxy (WMS) maize starches were each reacted with acetic-adipic mixed anhydride (AAMA), phosphoryl chloride (POCl3), sodium trimetaphosphate (STMP), acetic anhydride (AA), succinic anhydride (SA), and octenylsuccinic anhydride (OSA). Almost no or no starch polymer molecule modification occurred when the TRF method and AAMA, AA, or POCl3 were used; less than half as much reaction when SA was the reagent used, and about the same amount of reaction when STMP or OSA were the reagents used (for different reasons). It was concluded that most AAMA, AA, SA, and POCl3 reacted with surface protein molecules when the TRF method was used and that OSA molecules were driven into the structured internal water of granules.

Relationship of the channels of normal maize starch to the properties of its modified products.

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. Reaction with propylene oxide, a slowly reacting reagent whose reaction should be independent of RADC, resulted in an inverse relationship between several physical properties and RADC. 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.

Confocal laser scanning microscopy of dextran-rice starch mixtures.

The microstructure of rice starch and dextran-rice starch mixtures was studied using confocal laser scanning microscopy (CSLM) and scanning electron microscopy (SEM). Surface pores and channels of rice starch were looked for. Channels could not be found in rice starch granules after reaction with 3-(4-carboxybenzoyl)-quinoline-2-carboxaldehyde (CBQCA). Fluorescein-labeled dextran was mixed with rice starch in order to locate hydrocolloid molecules in hydrocolloid-rice starch mixtures. The results showed that FITC-dextran (ave. Mw 4000; FD4) penetrated into raw rice starch granules, with the degree of penetration varying from granule to granule. FD4 could penetrate throughout cooked rice starch granules. FITC-dextran with an average Mw of more than 10,000 could not penetrate either raw or cooked rice starch granules.

Extraction of polysaccharides from a species of Chlorella.

The objective of this project was to devise a method to recover the total, water-soluble cell-wall polysaccharides of Chlorella. It was found that substantial quantities of polysaccharides could be extracted after treatment of the cells with a mildly acidic solution of sodium chlorite (yield of recovered polysaccharide, 19-22%). Water-soluble (13-19% yield) and 2% NaOH-soluble (3-6% yield) fractions were obtained. A second treatment gave a total yield of water-soluble polysaccharides of 24-25%, while reducing the amount of material soluble in 2% NaOH to 0.3%. Each polysaccharide fraction was composed of 6 different neutral sugars (rhamnose, arabinose, xylose, mannose, galactose, and glucose). There was evidence of the presence of uronic acid in all fractions. An alkaline hydrogen peroxide treatment of the cells resulted in a 19% yield of polysaccharides that were not further examined.

One hundred years of commercial food carbohydrates in the United States.

Initiation and development of the industries producing specialty starches, modified food starches, high-fructose sweeteners, and food gums (hydrocolloids) over the past century provided major ingredients for the rapid and extensive growth of the processed food and beverage industries. Introduction of waxy maize starch and high-amylose corn starch occurred in the 1940s and 1950s, respectively. Development and growth of the modified food starch industry to provide ingredients with the functionalities required for the fast-growing processed food industry were rapid during the 1940s and 1950s. The various reagents used today for making cross-linked and stabilized starch products were introduced between 1942 and 1961. The initial report of enzyme-catalyzed isomerization of glucose to fructose was made in 1957. Explosive growth of high-fructose syrup manufacture and use occurred between 1966 and 1984. Maltodextrins were introduced between 1967 and 1973. Production of methylcelluloses and carboxymethylcelluloses began in the 1940s. The carrageenan industry began in the 1930s and grew rapidly in the 1940s and 1950s; the same is true of the development and production of alginate products. The guar gum industry developed in the 1940s and 1950s. The xanthan industry came into being during the 1950s and 1960s. Microcrystalline cellulose was introduced in the 1960s. Therefore, most carbohydrate food ingredients were introduced in about a 25 year period between 1940 and 1965. Exceptions are the introduction of maltodextrins and major developments in the high-fructose syrup industry, which occurred in the 1970s.

Effects of protein on crosslinking of normal maize, waxy maize, and potato starches.

Channels of maize starch granules are lined with proteins and phospholipids. Therefore, when they are treated with reagents that react at or near the surfaces of channels, three types of crosslinks could be produced: protein-protein, protein-starch, starch-starch. To determine which of these may be occurring and the effect(s) of channel proteins (and their removal) on crosslinking, normal and waxy maize starches were treated with a proteinase (thermolysin, which is known to remove protein from channels) before and after crosslinking, and the properties of the products were compared to those of a control (crosslinking without proteinase treatment). After establishing that treatment of starch with thermolysin alone had no effect on the RVA trace, three reaction sequences were used: crosslinking alone (CL), proteinase treatment before crosslinking (Enz-CL), proteinase treatment after crosslinking (CL-Enz). Two crosslinking reagents were used: phosphoryl chloride (POCl3), which is known to react at or near channel surfaces; STMP, which is believed to react throughout the granule matrix. Three concentrations of POCl3 (based on the weight of starch) were used. For both normal maize starch (NMS) and waxy maize starch (WMS) reacted with POCl3, the trends were generally the same, with apparent relative degrees of crosslinking indicated to be CL-Enz=CL>Enz-CL, but the effects were greater with NMS and there were differences when different concentrations of reagent were used. The basic trends were the same when potato starch was used in the same experiments. Crosslinking with STMP was done both in the presence and the absence of sodium sulfate (SS). Both with and without SS and with both NMS and WMS, the order of indicated crosslinking was generally the same as found after reaction with POCl3, with the indicated swelling inhibition being greater when SS was present in the reaction mixture. Examination of the maize starches with a protein stain indicated that channel protein was removed by treatment with thermolysin when the proteinase treatment occurred before crosslinking with either POCl3 or STMP, but only incompletely or not at all if the treatment with the proteinase occurred after crosslinking. Because the crosslinking reactions were less effective when the protein was removed, the results are tentatively interpreted as indicating that they involved protein molecules, although there may not be a direct relationship.

The Ruff degradation: a review of previously proposed mechanisms with evidence that the reaction proceeds by a Hofer-Moest-type reaction.

The Ruff degradation reaction is critically reviewed. Based on available information, the Hofer-Moest decarboxylation mechanism is presented as the mechanism for it. Cu(III) is proposed as the active species of the copper variant of the Ruff degradation, which is the most efficient form of the reaction.

The Hofer-Moest decarboxylation of D-glucuronic acid and D-glucuronosides.

Research was undertaken to effect the oxidative decarboxylation of glycuronosides. Experiments with free D-glucuronic acid and aldonic acids were also executed. Both anodic decarboxylation and variants of the Ruff degradation reaction were investigated. Anodic decarboxylation was found to be the only successful method for the decarboxylation of glucuronosides. It was, therefore, proposed that glycuronosides can only undergo a one-electron oxidation to form an acyloxy radical, which decomposes to form carbon dioxide and a C-5 radical, that is, a Hofer-Moest decarboxylation. The radical is subsequently oxidized to a cation by means of a second one-electron oxidation. The cation undergoes nucleophilic attack from the solvent (water), whose product (a hemiacetal) undergoes a spontaneous hydrolysis to yield a dialdose (xylo-pentodialdose from D-glucuronosides).

Extension of the Nef reaction to C-glycosylnitromethanes.

Acid-catalysed methanolysis of 3,4,5,6-tetra-O-acetyl-1,2-dideoxy-l-arabino-hex-1-enitol proceeds via a cascade set of consecutive reactions resulting in its regiospecific conversion to a mixture of alpha- and beta-C-L-arabinofuranosylmethanal dimethyl acetals and a mixed internal methyl acetal. Structures of the final products of the overall process provide unique evidence that a kinetically controlled, five-membered-ring closure precedes a six-membered-ring closure in reversible systems capable of giving both five-membered and six-membered all-sp3-atom rings. Determination of the reaction intermediate enabled extension of the Nef reaction to C-glycosylnitromethanes. Protonated aci-nitro forms of C-glycosylnitromethanes that are resistant to the Nef reaction in aqueous acidic media undergo a modified Nef reaction in acidified methanol, and the corresponding C-glycosylmethanal dimethyl acetals with alpha-L-arabinopyranosyl, beta-D-glucopyranosyl, beta-D-galactopyranosyl, beta-D-mannopyranosyl and beta-L-rhamnopyranosyl configurations were obtained in moderate yields.