Dry‐Grind Processing of Corn with Endogenous Liquefaction Enzymes

An amylase corn has been developed that produces an α‐amylase enzyme that is activated in the presence of water at elevated temperatures (>70°C). Amylase corn in the dry‐grind process was evaluated and compared with the performance of exogenous amylases used in dry‐grind processing. Amylase corn (1–10% by weight) was added to dent corn (of the same genetic background as the amylase corn) as treatments and resulting samples were evaluated for dry‐grind ethanol fermentation using 150‐g and 3‐kg laboratory procedures. Ethanol concentrations during fermentation were compared with the control treatment (0% amylase corn addition or 100% dent corn) which was processed with a conventional amount of exogenous α‐amylase enzymes used in the dry‐grind corn process. The 1% amylase corn treatment (adding 1% amylase corn to dent corn) was sufficient to liquefy starch into dextrins. Following fermentation, ethanol concentrations from the 1% amylase corn treatment were similar to that of the control. Peak and breakdown viscosities of liquefied slurries for all amylase corn treatments were significantly higher than the control treatment. In contrast, final viscosities of liquefied slurries for all amylase corn treatments were lower than those of the control. Protein, fat, ash, and crude fiber contents of DDGS samples from the 3% amylase corn treatment and control were similar.     

Effect of Partial Gelatinization and Lipid Addition on α‐Amylolysis of Barley Starch Granules

The effect of partial gelatinization with and without lipid addition on the granular structure and on α‐amylolysis of large barley starch granules was studied. The extent of hydrolysis was monitored by measuring the amount of soluble carbohydrates and the amount of total and free amylose and lipids in the insoluble residue. Similarly to the α‐amylolysis of native large barley starch granules, lipid‐complexed amylose (LAM) appeared to be more resistant than free amylose and amylopectin. Partial gelatinization changed the hydrolysis pattern of large barley starch granules; the pinholes typical of α‐amylase‐treated large barley starch granules could not be seen. Lipid addition during partial gelatinization decreased the formation of soluble carbohydrates during α‐amylolysis. Also free amylose remained in the granule residues and mostly amylopectin hydrolyzed into soluble carbohydrates. These findings indicate that lysophospholipid (LPL) complexation with amylose occurred either during pretreatment or after hydrolysis, and free amylose was now part of otherwise complexed molecules instead of being separate molecules. Partial gelatinization caused the granules to swell somewhat less during heating 2% starch‐water suspensions up to 90°C, and lipid addition prevented the swelling completely. α‐Amylolysis changed the microstructure of heated suspensions. No typical twisting of the granules was seen, although the extent of swelling appeared to be similar to the reference starch. The granules with added LPL were partly fragmented after hydrolysis.     

Determining the Role of Starch in Flour Tortilla Staling Using α‐Amylase

Effects of α‐amylase modification on dough and tortilla properties were determined to establish the role of starch in tortilla staling and elucidate the antistaling mechanism of this enzyme. Control and amylase‐treated tortillas were prepared using a standard bake test procedure, stored at 22°C, and evaluated over four weeks. Amylase improved shelf‐stability of tortillas. The enzyme also produced a significant amount of dextrins and sugars, decreased loss of amylose solubility, and weakened starch granules. Amylopectin crystallinity increased with time, but was similar for the control and treated tortillas. Staling of tortillas appears to mainly involve the starch in the amorphous phase. As such, amylase activity does not significantly interfere with amylopectin crystallization. It is proposed that amylase partially hydrolyzed the dispersed starch (i.e., mostly amylose), starch bridging the crystalline region, and protruding amylopectin branches. Starch hydrolysis decreases the rigid structure and plasticized polymers during storage. The flexibility of tortillas results from the combined functionalities of the amylose gel and amylopectin solidifying the starch granules during storage. Protein functionality may also be involved in tortilla staling, but this needs further research.     

Development of an Ethanol Yield Procedure for Dry‐Grind Corn Processing

In 2008, the United States produced ethanol at a rate of 39.5 billion L/year; an additional 8.5 billion L/year capacity was under construction. Kernel composition and physical properties are not correlated with ethanol yield. A procedure that measured the potential of hybrids to produce ethanol would benefit corn seed companies, corn producers, and ethanol processors. The objective was to develop a laboratory procedure to measure ethanol yield from corn samples and evaluate the developed procedure for accuracy and precision. To determine parameters for routine analyses, effects of mill type, dry solids, and yeast addition were investigated separately followed by effects of fermentation time (T_f), glucoamylase dose, and yeast addition. Measurement of ethanol using HPLC and gravimetric (change in weight due to CO₂ loss) methods were compared. Using the procedure developed, ethanol yields for five diverse hybrids (dent, waxy, white, high oil, and high amylose) were measured. Effects of mill type, dry solids, T_f, glucoamylase dose, and yeast addition were significant (P < 0.05). The gravimetric method estimated higher yields (428 ± 10 L/tonne) than HPLC (405 ± 15 L/tonne) and had a higher level of precision. Both methods had coefficients of variations of <4% and gave similar conclusions. In the final procedure, we used corn (25 g/batch) liquefied with α‐amylase (60 min at 90°C) in 75 mL of distilled water. Simultaneous saccharification and fermentation was used (64 hr at 32°C) with glucoamylase and yeast. Gravimetric and HPLC methods measured differences in ethanol yield for the five hybrids (158–435 L/tonne). The method is suitable for routine testing of ethanol yield potential and as a reference method for verifying more rapid measurement techniques.     

Partial Characterization of Buckwheat (Fagopyrum esculentum) Starch

Laboratory-isolated buckwheat (Fagopyrum esculentum) starch was compared to commercial corn and wheat starches. Buckwheat starch granules (2.9–9.3 μm) were round and polygonal with some holes and pits on the surface. Buckwheat starch had higher amylose content, waterbinding capacity, and peak viscosity, and it had lower intrinsic viscosity when compared with corn and wheat starches. Buckwheat starch also showed restricted swelling power at 85–95°C and lower solubility in water at 55–95°C and was more susceptible to acid and enzymatic attack. Gelatinization temperatures, determined by differential scanning calorimetry, were 61.1–80.1°C for buckwheat starch compared to 64.7–79.2°C and 57.1–73.5°C for corn and wheat starches, respectively. A second endotherm observed at 84.5°C was an amylose-lipid complex attributed to the internal lipids in buckwheat starch, as evidenced by selective extraction. The retrogradation of buckwheat, corn, and wheat starch gels was examined after storage at 25, 4, and -12°C for 1–15 days. In general, buckwheat starch retrogradation was slower than that of corn and wheat starch, but it increased as storage time increased, as did that of the other starch pastes. When the values of the three storage temperatures were averaged for each storage period analyzed, buckwheat starch gels showed a lower percentage of retrogradation than did corn and wheat starch gels. Buckwheat starch also had a lower percentage of water syneresis when stored at 4°C for 3–10 days and had better stability to syneresis after three freeze-thaw cycles at -12 and 25°C.     

Enzymatic hydrolysis of chestnut purée: process optimization using mixtures of alpha-amylase and glucoamylase

The enzymatic hydrolysis of starch present in chestnut purée was performed through a one-step treatment with a mixture of a commercial thermostable alpha-amylase (Termamyl 120 L, type S) and glucoamylase (AMG 300 L) at 70 degrees C. The effect of the enzyme concentration and the ratio of both amylases in the reaction mixture was studied by means of a factorial second-order rotatable design, which allowed conditions to be set leading to the total conversion of starch to glucose after 15 min of incubation (60 total enzymatic units g(-1) of chestnut; ratio of alpha-amylase/glucoamylase enzymatic units, 0.35:0.65). At lower enzyme concentration, the delay in the addition of the glucoamylase with regard to the addition of the alpha-amylase allowed a slightly higher hydrolysis percentage to be reached when compared to the simultaneous addition of both amylases at the same low enzyme concentration. The kinetics of liberation of glucose supports the existence of a synergistic effect between these two enzymes only in the first moments of the reaction. Finally, a sequential one-step hydrolysis was assayed, and more concentrated glucose syrups were thus obtained.     

Extraction of β‐Glucan from Oat Bran in Laboratory Scale

Effects of various enzymes and extraction conditions on yield and molecular weight of β‐glucans extracted from two batches of commercial oat bran produced in Sweden are reported. Hot‐water extraction with a thermostable α‐amylase resulted in an extraction yield of ≈76% of the β‐glucans, while the high peak molecular weight was maintained (1.6 × 10⁶). A subsequent protein hydrolysis significantly reduced the peak molecular weight of β‐glucans (by pancreatin to 908 × 10³ and by papain to 56 × 10³). These results suggest that the protein hydrolyzing enzymes may not be pure enough for purifying β‐glucans. The isolation scheme consisted of removal of lipids with ethanol extraction, enzymatic digestion of starch with α‐amylase, enzymatic digestion of protein using protease, centrifugation to remove insoluble material, removal of low molecular weight components using dialysis, precipitation of β‐glucans with ethanol, and air‐drying.     

Effects of Mutant Thermostable α‐Amylases on Rheological Properties of Wheat Dough and Bread

Thermostable mutant α‐amylases (21B, M111, and M77) with various degrees of thermostability were purified from Bacillus amyloliquefaciens F and used as improvers for breadmaking. Test baking with the mutant enzymes was conducted using the long fermentation sponge‐dough method. Addition of an appropriate amount of mutant α‐amylases to the ingredients distinctly increased the specific volume of the bread and improved the softness of breadcrumb as compared with the addition of Novamyl (NM), an exo‐type α‐amylase. M77 was the most effective in retarding the staleness of breadcrumb. The softness of breadcrumb during storage, however, was not correlated with the thermostability. All mutant α‐amylases weakened the mixing property of the dough, whereas they strengthened the property of fermented dough. Especially, M77 and NM had different effects on the dough properties, but their bread qualities were similar to each other. The strong tolerance of M77 dough to the long baking process might be due to the production of hydrolyzed starches, oligosaccharides in the range of maltopentaose to maltohexaose, as compared with NM. Therefore, in the light of present findings, these mutant α‐amylases are possible substitutes for NM as bread improvers.     

Influence of extreme K:Na ratios and high substrate salinity on plant metabolism of crops differing in salt tolerance

K⁺/Na⁺ and Cl^‐ effects on activity of amylases as well as on their isoenzyme pattern in leaves of bushbeans and sugarbeets at the beginning of salinity stress were investigated, in plants grown in water culture under controlled environmental conditions. Alpha‐ and beta‐amylase activity in beans increased, particularly due to K⁺ and Cl^‐ supplied. In sugarbeets amylase activity remained unchanged as a result of K/Na treatment in combination with Cl and decreased using SO₄ ^2‐ as counterion. A direct correlation of amylase activity to the starch content of both species was not detctable. Particularly α‐but also ß‐amylase was most strongly inhibited by KCl “in vitro”. Independent on their origin, amylases from bushbeans and sugarbeets did not show any differences in ionic inhibition “in vitro”. The isoenzyme pattern of the species was different, but no clear ionic effect was detectable. Amylolytic activity is evidently not a causative factor for restricted starch mobilization in leaves under an early salinity stress. It is suggested that amylases are indirectly involved in starch formation via degradation due to a lack of a carbohydrate sink under salinity stress. Differences in salt tolerance of the investigated crops are obviously not related to different “in vitro” properties of amylases.     

Response to the Views and Opinions of Maningat,Seib, and Bassi Regarding McCleary et al (2013)

Phosphate cross‐linked starch, referred to as resistant starch 4 (RS₄) is hydrolyzed much more rapidly under physiological conditions (i.e., pH 6, 37°C, pancreatic α‐amylase) than under the conditions used in AOAC dietary fiber method 985.29/AACC International Approved Method 32‐45.01 (the Prosky method), in which samples are incubated with thermostable bacterial α‐amylase at 95–100°C. For this reason, the author concludes that the Prosky method overestimates the dietary fiber content of these materials. A more accurate estimate of dietary fiber content is obtained with AOAC Method 2009.01/AACCI Approved Method 32‐45.01.     

Evaluation of the Displacement Value as a Method to Detect Reduced Corn Wet-Milling Quality

A procedure based on the resistance and capacitance (RC) properties of corn to calculate a displacement value (DV) was evaluated for detection of corn that had reduced wet-milling quality. In 1991 and 1992, three hybrids were dried at air temperatures between ambient and 115°C in batch dryers. Additional samples, obtained from commercial elevators in 1992, had been dried with air temperatures ranging from 52 to 136°C. A baseline reference relationship was developed between log₁₀-resistance and capacitance with data from ambient-dried samples. A DV was defined as the horizontal distance along the capacitance axis from a sample RC data point to the baseline reference. RC properties of samples dried at air temperatures >50°C were compared to the baseline and the DV determined. Selected drying treatments were wet-milled by a laboratory-scale procedure to verify milling quality and correlation with DV. The effects attributed to hybrid and harvest moisture content on the RC properties of ambient-dried samples were small, allowing the baseline reference to be applied to a wide range of corn samples. In 1992, the baseline shifted upward from the 1991 baseline by 0.5 units on the log₁₀-resistance axis. DV increased significantly at drying air temperatures >50°C for batchdried samples. While DV correlated with drying temperature in batchdried samples (r = 0.66), it did not correlate with starch yield or recovery of commercial samples (r ≤0.10). Although the specific causes could not be determined, the shift in the baseline indicates the method would be difficult to implement on a practical scale. Although not indicated by DV, starch recovery decreased significantly for samples batch-dried at air temperatures ≥70°C. All samples dried at 115–136° had significantly lower starch recoveries.     

Release of Bound Lipids in Cereal Starches Upon Hydrolysis by Glucoamylase

The raw starch granules from corn, rice, and wheat were hydrolyzed by practically pure glucoamylase (Rhizopus niveus). The bound lipids remaining in the residual starches were investigated, of which the major components of the lipids, free fatty acids (FFA) in corn starch, FFA and phospholipids (PL) in rice starch, and PL in wheat starch were determined. In each case, the bound FFA and PL were decreased to some extent during the initial stage of hydrolysis. During the later stages, the FFA continued to gradually decrease, while the level of PL stabilized. It was interesting that some of the bound lipids were released from the granules upon glucoamylase hydrolysis, differing from the model amylose-lipid complexes. Furthermore, the structures of the residual starches were investigated. The blue value and λ_max of the starches were increased by partial hydrolysis of the starch granules using practically pure glucoamylase. Two gel-permeation chromatography analyses revealed that the relative amount of amylose fraction was increased by glucoamylase hydrolysis, and also that the increments were reduced by the defatting of bound lipids. The results suggest that the increase in amylose fraction is attributable to the existence of bound lipids in the granules.     

Influence of Annealing on Gel Properties of Mung Bean Starch

Mung bean starch gels (8% solids) were prepared after annealing at 45–60°C for 1–24 hr, and the relationship between the physical properties of gels and the swelling power (SP) and solubility of starch was investigated. The SP and solubility decreased with increasing annealing temperature and time, mostly in the first 6 hr. The solubles were mainly composed of amylose. Gel hardness at a 5 mm depth of annealed starch was larger than that of native starch, and gel hardness increased as SP decreased (r = ‐0.94). Upon continued compression, the yield force of gel showed a different function. Above SP of ≈12.5, the yield force of annealed starch gels decreased, but at <12.5 the yield force increased with increasing SP. Both granular rigidity and extent of packing appeared to determine the yield force. Although annealing increased the gel hardness, α‐amylase digestibility of gel was not affected. Pasting analysis in the Rapid Visco Analyser (RVA) revealed that annealing increased pasting temperature. A pasting peak was found only in 45 and 50°C annealed starches. Overall paste viscosities of the starches annealed at >55°C were lower than that of the control starch. Final viscosities in RVA were correlated with the yield force of gel (r = 0.99).     

Observations on the α‐Amylolysis Pattern of Some Waxy Maize Starches from Inbred Line Ia453

Maize starches of the endosperm mutants waxy (wx), dull:waxy (duwx), and amylose‐extender:dull:waxy (aeduwx) from inbred line Ia453 lack amylose. However, in addition to high molecular weight (HMW) amylopectin, the duwx and aeduwx starches contained 40 and 80%, respectively, intermediate branched material of low molecular weight (LMW). As gelatinized, the amylopectin of the wx starch was easily hydrolyzed into small dextrins by the α‐amylase of B. amyloliquefaciens, but components of duwx and aeduwx possessed partial resistance to amylolytic attack. Residual material of intermediate size obtained by a 4‐hr α‐amylolysis could not be separated from LMW dextrins by fractional precipitation in methanol. It is suggested that this material possessed a more regularly branched structure, in which the d ‐glucosyl chain segments were too short to allow α‐amylase action. The granular starches of duwx and aeduwx genotypes were initially considerably more resistant than the wx sample to α‐amylase attack. This was possibly due to an altered structure in the amylopectin component or the high content of intermediate material in the former granules.     

Staling of Bread: Role of Amylose and Amylopectin and Influence of Starch‐Degrading Enzymes

The present investigation aims at understanding the mechanism of bread firming during staling. Changes in the starch fraction due to the addition of amylases and their influence on the texture of bread crumb were studied during aging and after rebaking of stale bread. Pan bread was prepared by a conventional baking procedure. The influence of three different starch‐degrading enzymes, a conventional α‐amylase, a maltogenic α‐amylase, and a β‐amylase were investigated. The mechanical properties of bread were followed by uniaxial compression measurements. The microstructure was investigated by light microscopy, and starch transformations were assessed by differential scanning calorimetry (DSC) and wide‐angle X‐ray powder diffraction. Firming of bread crumb and crystallization of starch are not necessarily in agreement in systems with added amylases. Reorganization of both starch fractions, amylopectin and amylose, and the increase of starch network rigidity due to increase of polymer order are important during aging. Starch‐degrading enzymes act by decreasing the structural strength of the starch phase; for instance, by preventing the recrystallization of amylopectin or by reducing the connectivity between crystalline starch phases. On the other hand, starch‐degrading enzymes may also promote the formation of a partly crystalline amylose network and, by this, contribute to a kinetic stabilization of the starch network. Based on the results, a model for bread staling is proposed, taking into account the biphasic nature of starch and the changes in both the amylose and amylopectin fraction.     

Effect of Sorghum Decortication and Use of Protease Before Liquefaction with Thermoresistant α‐Amylase on Efficiency of Bioethanol Production

The aim was to study the dual effect of sorghum decortication and protease treatment before liquefaction with α‐amylase on the performance of subsequent steps of saccharification and fermentation. A bifactorial experiment with a level of confidence of P < 0.05 was designed to study differences among grains (maize, whole, and decorticated sorghum) and the effectiveness of the protease before liquefaction. Sorghum was decorticated to remove most of the pericarp and part of the germ and increase starch concentration of the feedstock. The decorticated sorghum had significantly higher starch hydrolysis during liquefaction compared with the whole kernel. These hydrolyzates contained ≈50% more reducing sugars than the untreated counterparts. At the end of saccharification, the final glucose concentration in hydrolyzates treated without protease was the highest for maize (180 mg/mL), followed by decorticated sorghum (165 mg/mL), and whole sorghum (145 mg/mL). Decortication and protease treatment had a significant effect on fermentation times. In decorticated sorghum mash treated with and without protease, fermentation times were 22 and 60 hr, respectively. The decorticated sorghum treated with protease yielded similar amounts of ethanol compared with maize and 44% more ethanol compared with the untreated whole sorghum. Both sorghum decortication and protease treatments before hydrolysis with α‐amylase are recommended to increase ethanol yields, lower yields of distilled grains, and save liquefaction, saccharification, and fermentation times.     

Effects of Additives and Storage Temperature on Staling Properties of Bagels

The effects of xanthan gum, Novamyl (a type II α‐amylase), Instant Tender‐Jel C starch (a modified starch), and GMS‐90‐SSK (a hydrated monoglyceride) on the staling properties of bagels stored at 4 and 22°C from 0–7 days were studied. Texture analysis and moisture determination were conducted on the bagels before lyophilization. Analysis of percent soluble starch, crumb pasting (Rapid Visco Analyser) and degree of amylopectin recrystallization (differential scanning calorimeter) were conducted on lyophilized bagel crumb. Novamyl‐treated bagels appeared to be the most resistant to staling over time at both storage temperatures in relation to the enthalpy of gelatinization (ΔH). Bagels containing xanthan gum, Instant Tender‐Jel C starch, and GMS‐90‐SSK showed some improvements over the control bagels, although the effects of the additives on the characteristics of the bagels varied. Bagels made with xanthan gum or monoglyceride retained slightly higher crumb moisture percentages over most days of storage. The monoglyceride‐treated bagels had higher enthalpy values, lower percentages of soluble starch, and a higher pasting profile but had the softest texture. The apparent onset of increased staling of the monoglyceride‐ treated bagels was attributed to complexes formed with the starch fractions.     

Cross‐Linked Resistant Starch: Preparation and Properties

Resistant starches (RS) were prepared by phosphorylation of wheat, waxy wheat, corn, waxy corn, high‐amylose corn, oat, rice, tapioca, mung bean, banana, and potato starches in aqueous slurry (≈33% starch solids, w/w) with 1–19% (starch basis) of a 99:1 (w/w) mixture of sodium trimetaphosphate (STMP) and sodium tripolyphosphate (STPP) at pH 10.5–12.3 and 25–70°C for 0.5–24 hr with sodium sulfate or sodium chloride at 0–20% (starch basis). The RS₄ products contain ≤100% dietary fiber when assayed with the total dietary fiber method of the Association of Official Analytical Chemists (AOAC). In vitro digestion of four RS₄ wheat starches showed they contained 13–22% slowly digestible starch (SDS) and 36–66% RS. However after gelatinization, RS levels fell by 7–25% of ungelatinized levels, while SDS levels remained nearly the same. The cross‐linked RS₄ starches were distinguished from native starches by elevated phosphorus levels, low swelling powers (≈3g/g) at 95°C, insolubilities (<1%) in 1M potassium hydroxide or 95% dimethyl sulfoxide, and increased temperatures and decreased enthalpies of gelatinization measured by differential scanning calorimetry.     

Wet‐Milling and Dry‐Milling Properties of Dent Corn with Addition of Amylase Corn

A transgenic corn (amylase corn) has been developed that produces an endogenous α‐amylase that is activated in the presence of water and elevated temperature (>70°C). Wet‐ and dry‐milling characteristics of amylase corn were evaluated using laboratory wet‐ and dry‐milling procedures. Different amounts of amylase corn (0.1–10%) were added to dent corn (with the same genetic background as the amylase corn) as treatments. Samples were evaluated for wet‐ and dry‐milling fraction yields using 1‐kg laboratory procedures. Milling yields for all amylase corn treatments were compared with the control treatment (0% amylase corn or 100% dent corn). No significant differences were observed in wet‐ and dry‐milling yields between the control and the 0.1, 1, and 10% amylase corn treatments. Most of the amylase activity (77%) in wet‐milling fractions was detected in the protein fraction. In dry‐milling, amylase activity (68.8%) was detected in endosperm fractions (fines, small grits, and large grits).     

Effect of Complexation Temperature on Thermal Properties and Enzymatic Hydrolysis of Starch–Oleic Acid Complexes

The effect of complexation temperature (30, 60, and 90°C) on the gelatinization properties, glass transition, enzymatic hydrolysis, and crystalline structure of high‐amylose corn starch–oleic acid (HACS‐OA) complexes created by a dimethyl sulfoxide (DMSO)‐based complexation method and of normal corn starch–oleic acid (NCS‐OA) complexes created by an alkaline‐based complexation method were investigated by using differential scanning calorimetry, thermogravimetric analysis, and X‐ray diffractometry. The results indicated that the highest complex indices were found in the complexes created at 30 and 60°C with the DMSO‐based complexation method and alkaline‐based complexation method, respectively. The X‐ray diffraction patterns of both HACS‐OA complexes and NCS‐OA complexes created at different complexation temperatures were the V‐type pattern. For the complexes created by the two methods, both the melting temperature and the glass transition temperature increased obviously with increasing complexation temperature. Complexation temperature also influenced the enzymatic hydrolysis rate of starch‐OA complexes.