Effect of pH and ethanol content of solvent on rheology of zein solutions

Zein, a corn protein, is a mixture of the polypeptides α-, γ-, β-, and δ-zein. α-Zein and γ-zein comprise 70–85% and 10–20% of total zein mass, respectively. Both peptides have similar amino acid composition, except γ-zein is rich in cysteine. The presence of cysteine has been associated with gelation of zein solutions. A common solvent for zein is aqueous ethanol. Preliminary results suggested that pH and ethanol content affect the rheology of zein solutions. Our objective was to investigate the effect of ethanol content (65–90%) and pH of the solvent (2, 6, and 12) on rheological properties of zein solutions (20% w/w) containing γ-zein. Steady shear tests and oscillatory time sweeps were performed to determine flow behavior and gelation time of zein solutions. Results indicated that α-zein solutions were nearly Newtonian while those containing γ-zein showed shear thinning behavior. At high pH, γ-zein increased the consistency index (K) and shortened gelation time. Results were attributed to the cysteine in γ-zein. High pH promoted formation of disulfide bonds leading to higher K values and shorter gelation times. Results of this work are expected to be useful in the design of zein extraction processes and the development of new zein applications.     

Effect of hydrostatic pressure and temperature on the chemical and functional properties of wheat gluten II. Studies on the influence of additives

Cysteine, N-ethylmaleinimide, radical scavengers, various salts or urea were added to wheat gluten. After treatment at increasing pressure (0.1–800 MPa) and temperature (30–80 °C) the resulting material was analysed by micro-extension tests and an extraction/HPLC method to measure protein solubility. Furthermore, cysteine was added to isolated gliadin and glutenin prior to high-pressure treatment and protein solubility was determined. The resistance to extension of gluten strongly increased and the solubility of gliadin in aqueous ethanol decreased with increasing pressure and temperature. As compared to experiments without additive the observed effects were much stronger. Isolated gliadin turned largely insoluble in aqueous ethanol when cysteine was added prior to high-pressure treatment. The S-rich α- and γ-gliadins were much more strongly affected than the S-poor ω-gliadins pointing to a disulphide related mechanism. Monomeric gliadin components were completely recovered after reduction of the aggregates with dithioerythritol. In contrast, samples without free thiol groups such as isolated gliadins or with SH groups, which had been blocked by N-ethylmaleinimide, were hardly affected by high-pressure treatment. The addition of radical scavengers to gluten showed no effect in comparison to the control experiment, indicating that a radical mechanism of the high-pressure effect can be excluded. The observed effects can be explained by thiol-/disulphide interchange reactions, which require the presence of free thiol groups in the sample. The addition of salts and urea showed that unfolding of the protein due to weakening of interprotein hydrogen bonds is strongest for ions with a high radius (e.g. thiocyanate). This leads to weakening of gluten at ambient pressure but it facilitates high pressure induced reactions, e.g. of disulphide bonds.     

Aggregate formation of zein and its structural inversion in aqueous ethanol

Zein is a prolamine of maize. Conventionally, 70–90% aqueous ethanol is used to dissolve zein. When the hydrodynamic radii of zein molecules in aqueous ethanol were monitored with a dynamic light scattering instrument, it was found that zein aggregates in the solvent and the degree of aggregation depends on the composition of the solvent mixture. As the ethanol content of solvent increased from 70% to 90%, the aggregation number of zein molecules decreased from 10,000 until it reached a minimum. The aggregation number then increased abruptly to greater than 10,000 as the ethanol content of the solvent mixture increased to 92%. Since zein has amphiphilic characteristics, this behavior was interpreted as the formation of a micelle-like structure in its solution whereby ca. 90% ethanol that showed minimum aggregation number is regarded as a structural inversion point. This point of view was supported by a simple experiment that showed selective interaction of zein molecules with hydrophilic or hydrophobic particles.     

Gliadin and zein show similar and improved rheological behavior when mixed with high molecular weight glutenin

Small amplitude and lubricated squeezing flow tests were performed to investigate the effect of high molecular weight glutenin (HMWG) on the rheological properties of gliadin and zein dough composites. It was hypothesized that addition of small amounts of HMWG to zein cause changes in its viscoelastic properties in the same way as its addition to gliadin. Starch (87%, w/w) and protein/protein composites (13%, w/w), and water were mixed into dough. The water content of prepared dough was in the 41.7–45.3% range. Composites were gliadin-HMWG and zein-HMWG. Phase angle and complex modulus (G*) were obtained from frequency sweep tests at 0.5% strain amplitude over a 0.01–100 rad/s frequencies. Lubricated squeezing flow test were carried out at a range of strain rates to determine the sample extensional viscosities.     

Ethanolic extraction of zein from maize

Zein can be extracted from maize (Zea mays) using ethanol solutions, and recovered from the extract by diluting with water. Dilution to 40% ethanol will precipitate a mixture of zein and maize lipid with ∼15% lipid. For possible zein applications, such as film production, purer zein is required. Most of the lipid can be removed from the precipitate by selective re-extraction with hexane or ethanol, but this is expensive. A precipitate containing 90% or more of the protein in the extract, with less than 10% lipid, can be obtained by diluting the extract in stages. An initial dilution will precipitate a solid with 75% or more lipid, this precipitate can be centrifuged from the extract and the centrifugate diluted to precipitate a solid of 70% or more zein. The mass and composition of solid centrifuged from a maize extract that was diluted in several steps to characterize the composition of precipitates so formed. The lipid content of a zein product precipitated from an extract diluted from 55% ethanol to 50% is significantly lower than the content of precipitate of an extract diluted in a single step to 40% ethanol.     

Nano-structure and properties of maize zein studied by atomic force microscopy

Atomic force microscopy was used to investigate the nanostructure of zein from maize (Zea mays). In aqueous ethanol solution zein exists as small globules with diameters between 150 and 550 nm. The mechanism of zein film formation was also explored. The characteristic film structure consists of a meshwork composed of doughnut structures formed from asymmetric rods joined to each other.     

Optimisation of a solvent for the complete extraction of prolamins from heated foods

Wheat bread was lyophilised, ground, extracted and centrifuged. The supernatants were analysed for gluten content by RP-HPLC and a commercial sandwich ELISA. Prolamin extraction solvents contained tris(2-carboxyethyl)phosphine (TCEP; 1, 2, 5, 10, 20, 50 mmol/L), guanidine hydrochloride (GUA; 0 or 2 mol/L) and a buffered salt solution. A commercial cocktail solution (250 mmol/L mercaptoethanol (ME), 2 mol/L GUA, buffered salt solution) as well as 60% (v/v) ethanol were used as control solvents. Wheat flour was the control for the extractability of the native prolamins. 60% ethanol only extracted 37% of the prolamins from wheat bread (cocktail = 100%). When ME was replaced by TCEP the protein yield increased from 35% at the lowest TCEP-level to 95% when 20–50 mmol/L TCEP were used. The use of GUA was essential to extract prolamins quantitatively. Comparative protein analysis using RP-HPLC and ELISA showed that both methods provided comparable prolamin (gliadin) concentrations of the wheat flour (40.3–45.7 mg/g), when 60% ethanol was used as extraction solvent. The extraction yields from bread were considerably lower (16.7–24.7 mg/g). Cocktail and TCEP extracted almost the same amount of protein from flour and bread with TCEP showing slightly lower yields. Total extractable protein (gliadin + glutenin) as determined by RP-HPLC was 70.5–75.3 mg/g, and total gliadin as determined by ELISA was 42.7–44.2 mg/g. Thus, the study has shown that TCEP in combination with GUA extracts proteins from heated, gluten-containing foods as effective as the commercial cocktail solution.     

Towards a new gliadin reference material–isolation and characterisation

Twenty-eight wheat cultivars representative of the three main European wheat producing countries, France, UK and Germany, were selected as a source for the preparation of a reference gliadin. One kilogram of kernels from each cultivar were mixed and milled. The resulting white flour was defatted and vacuum dried. Albumins and globulins were eliminated by extraction using 0.4 M NaCl solution and gliadins were extracted with 60% ethanol. The gliadin extracts were concentrated, desalted by ultrafiltration, freeze-dried, and homogenised. After tests had shown good solubility and homogeneity, aliquots of the reference gliadin were sent to 16 different laboratories for further investigations: The material was analysed by various methods including RP-HPLC, SE-HPLC, RP-HPLC-ESI-MS, MALDI-TOF, capillary electrophoresis, acid-PAGE, 2D-PAGE, SDS-PAGE and immunoblotting and ELISA-tests with different monoclonal and polyclonal antibodies. The results showed that the gliadin composition of the source flour and the reference gliadin matched perfectly, demonstrating that no major gliadin components had been lost during the isolation procedure. The reference gliadin showed good immunochemical sensitivity with different gliadin antibodies in enzyme immunoassays. Because of its high protein and gliadin content, good solubility, homogeneity, stability and representative character, the product is regarded as a suitable universal reference material.     

Effect of different fractions of zein on the mechanical and phase properties of zein films at nano-scale

The phase behavior of zein films has been investigated at nano-scale using atomic force microscopy (AFM) and compared to the phase behavior of the bulk using a thermal characterization technique. The local surface properties of the films were evaluated as a function of water activity using AFM. The glass transition temperature (T_g) of zein films decreased with increasing water activity. Adhesion forces measured by the AFM force curves increased with increasing water activity. Topography of zein and zein fractions were evaluated both qualitatively and quantitatively by the use of AFM and dedicated software to calculate the surface roughness. It has been found that processing technologies (solvent casting, drop deposition and spin casting) has influence on the surface structures of films. The films which were formed by the alpha zein rich fraction were found to have highest roughness values. Sectional surface profiles revealed that α-zein films have mean roughness (R_a) of 1.808 nm and root mean square roughness (RMS) of 2.239 nm while β-zein films have mean roughness (R_a) of 1.745 nm and root mean square roughness (RMS) of 3.623 nm. The discussions conducted on the differences/similarities in the observations were based on the hydrophobic/hydrophilic properties and interactions of these zein fractions.     

Effects of ultrasound-assisted freezing on the water migration of dough and the structural characteristics of gluten components

The effects of ultrasound-assisted freezing on the freezing time and water migration of dough, and the structural characteristics of gluten components were investigated. The effects of ultrasound-assisted freezing in the whole immersion freezing process (UWF) on the freezing time were better than those of ultrasound-assisted freezing in the maximum ice crystal generation zone. The shortest freezing time was obtained at 80 W/L, and was 577 s shorter than that with traditional immersion freezing. The UWF treatment at 80 W/L significantly (p < 0.05) affected the absorption enthalpy, freezable water content and water migration of frozen dough. In UWF compared with traditional immersion freezing, the SH content of gluten, glutenin and gliadin was significantly (p < 0.05) higher, by 12.06%, 27.55% and 21.65%, respectively. The surface hydrophobicity of gluten, glutenin and gliadin in UWF treated samples significantly (p < 0.05) decreased, by 19.67%, 13.21% and 9.17%, respectively. The secondary structure of gluten components was also significantly changed by UWF. The network of gluten, the chain structure of glutenin and the gliadin particles were all changed by UWF treatment. These findings indicated that UWF is an effective method to improve the moisture distribution in dough and reduce the damage to protein molecular structure caused by freezing.     

Effect of zein extrusion and starch type on the rheological behavior of gluten-free dough

Previous research has shown that zein, above its glass transition temperature, may adopt molecular structures that are able to form doughs with viscoelastic properties comparable to those of wheat gluten. It is hypothesized that extrusion can promote molecular changes in zein and favor interactions with starch that enhance dough viscoelasticity. Thus, the effects of extruding zein at 90–160 °C on the rheological properties of doughs prepared with potato, rice, and maize starches were determined.Formulations were optimized to provide similar mixing profiles to that of a standard wheat dough. For all zein samples, creep-recovery tests demonstrated that doughs prepared with maize and potato starches were less elastic when compared to doughs prepared with rice starch. Zein doughs produced using rice starch were comparable to wheat-dough. Extensional tests showed that zein extruded at 160 °C provided a larger increase in strain-hardening behavior, which is important for bread production. These samples also exhibited larger extensional stresses. Gel electrophoresis of zein extruded at 160 °C revealed an increase in protein aggregates and the presence of smaller peptides when compared to samples subjected at lower extrusion temperatures.Scanning electron micrographs of doughs containing zein showed starch granules embedded within an amorphous material and fibrous structures, which is attributed to elongated zein.     

Characterization of zein modified with a mild cross-linking agent

Zein, a predominant corn protein, is an alcohol-soluble protein extracted from corn and is an excellent film former. The characteristic brittleness of zein diminishes its usefulness as a film. It is well known that zein has a propensity for forming aggregates in solution. When zein molecules were cross-linked with 1-[3-dimethylaminopropyl]-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), it was found that the film-forming property was improved and the aggregation phenomenon in solution was suppressed. At the air/water interface, native zein forms brittle film with rough surface, whereas cross-linked zein forms rigid film with very smooth and even surface. Tensile strengths of the films were shown to be greatly increased by cross-linking. Objectives of this study are to determine the cross-linking mechanism of zein, the optimum reaction conditions, characteristics of the reaction product, and mechanical properties of cross-linked zein film. Through viscosity and dynamic light scattering, the cross-linking reaction was monitored. Optimum amount of EDC and NHS was determined to be 30 mg each per gram of zein. The cross-linking of zein with EDC and NHS seemed to be self-terminating because the cross-linking reaction did not proceed to precipitation.     

Two fraction extraction of α-zein from DDGS and its characterization

Zein was recovered from corn distiller's dried grains with solubles (DDGS) by a modified method using 70% (w/w) aqueous 2-propanol (70-IPA) or 70% (v/v) aqueous ethanol (70-EtOH) solvents, and a commercial method using 88% (w/w) aqueous 2-propanol (88-IPA). Yield, purity, and film properties of the isolated zein were determined. The modified procedure extracted two fractions of zeins: a mostly α-zein fraction, and a mostly γ-zein fraction. The modified method increased α-zein yield from 4% to 14%. Enzyme cellulase pretreatment did not improve zein yield, but grinding did. The α-zein fraction showed electrophoretic bands at 40, 22, 19, and 10 kDa, corresponding to α-zein dimer, α₁-zein, α₂-zein, and δ-zein, respectively. The α-zein of DDGS retained its film forming capability. The α-zein film of unmodified DDGS was cloudy and rough, unlike the clear and smooth films of α-zeins isolated from corn gluten meal and enzyme-treated DDGS.     

Conversion of starch from dry common beans (Phaseolus vulgaris L.) to ethanol

Dry common beans (Phaseolus vulgaris L.) were evaluated for potential conversion of starch to ethanol. Eight varieties of beans with average starch content of 46% (db) were assayed in a laboratory-scaled process based upon the commercial corn dry grind fermentation process. Ethanol yield was 0.43-0.51 g ethanol/g glucose (0.19-0.23 g ethanol/g beans). The average ethanol yield for the eight bean types was 92% of maximum theoretical yield, demonstrating that starch from beans could be efficiently converted to ethanol. Ethanol concentration obtained from 20% (w/w) solids loading was 3.5-4.4% (w/v). The residual fermentation solids contained, on a dry basis, 37.1-43.6% crude protein, 10.8-15.1% acid detergent fiber and 19.1-31.3% neutral detergent fiber.     

Effects of protein oxidation on gelatinization characteristics during rice storage

Gliadin and glutelin are major rice storage proteins. In this study, we evaluated changes in these proteins to determine the influence of pasting properties on rice storage. Notably, the physical and chemical properties of these proteins changed steadily. We analyzed protein oxidation indexes and determined correlations between the protein oxidation index and gelatinization property index. The results showed that significant oxidation of gliadin and glutelin occurred during the process of rice storage (P < 0.05). Protein oxidation had a significant impact on pasting properties. For glutelin, changes in the structure and function of the protein during rice storage had a significant impact on the five rice pasting property indexes, including peak viscosity (PV), holding strength (HS), breakdown, final viscosity (FV), and setback (SB; P < 0.05). In particular, the levels of carbonyl compounds and active sulfur and the surface hydrophobicity of glutelin were significantly correlated with rice pasting property indexes (P < 0.05). However, gliadin only significantly affected three indexes, i.e., PV, HS, and FV (P < 0.05). Thus, these findings suggested that the carbonyl compounds, active sulfur, and surface hydrophobicity of glutelin could be used as sensitive indexes for changes in rice quality evaluation.     

Ethanol production from supercritical-fluid-extrusion cooked sorghum

Sorghum (Sorghum bicolor (L.) Moench) is a starch-rich grain similar to maize (Zea mays L.), but sorghum has been underutilized for biobased products and bioenergy. This study was designed to investigate the effects of supercritical-fluid-extrusion (SCFX) of sorghum on ethanol production. Morphology, chemical composition, and thermal properties of extruded sorghum were characterized. Analysis of extruded sorghum showed increased measurable starch content, free sugar content, and high levels of gelatinized starch. SCFX cooked and non-extruded sorghum were further liquefied, saccharified, and fermented to ethanol by using Saccharomyces cervisiae. The ethanol yield increased as sorghum concentration increased from 20 to 40% for both extruded and non-extruded sorghum. Ethanol yields from SCFX cooked sorghum were significantly greater than that from non-extruded sorghum (>5%).     

The dynamic rheological properties of glutens and gluten sub-fractions from wheats of good and poor bread making quality

The dynamic rheological properties of glutens and gluten fractions (gliadin and glutenin) of two U.K.-grown wheat cultivars, Hereward and Riband, having good and poor bread quality, respectively, were studied. Gluten and glutenin doughs from cv. Hereward had higher G' and lower tan δ values than those from cv. Riband at all frequencies studied. A more pronounced difference in G' and tan δ was observed between the glutenin doughs of the two wheats than between their respective gluten doughs. The rheological properties, i.e. G' and tan δ values, of gliadin doughs were similar for both wheats. Varying the gliadin/glutenin ratio by adding the isolated gliadin or glutenin sub-fractions to the parent glutens showed that the G' values decreased and the tan δ values increased as the gliadin/glutenin ratio was increased for both cultivars, indicating a considerable decrease in elasticity as the gliadin/glutenin ratio increased. The decrease in G' may be attributed to a plasticising effect of gliadin and ‘interference’ of gliadin with glutenin-glutenin interactions. The reduction in G' was much more pronounced when the gliadin/glutenin ratio was increased between 0.15 and 1.0 than between 1.0 and above. Gluten from cv. Hereward had higher G' and lower tan δ values than cv. Riband gluten at all gliadin/glutenin ratios, indicating that cv. Hereward gluten had greater elastic character than cv. Riband gluten. Although significant effects of other non-protein hydrocolloid components cannot be discounted, these observations are consistent with the view that the viscoelasticity of the glutenin sub-fraction of gluten and differences in the ratio of gliadin to glutenin are the main factors governing inter-cultivar differences in the viscoelasticity of wheat gluten.     

Enhanced molecular flexibility of α-zein in different polar solvents

α-Zein is the most abundant prolamin of zein; a specified molecular model of its structure has been proposed. α-Zein was modified by alkali and heat treatment in 70% ethanol aqueous solutions and ultrapure water to characterize the improvement in its amphiphilic property. The globular-structured aggregates formed by native α-zein were broken up after alkali and heat treatment. The size of the resulting particles was significantly reduced. The improved emulsifying stability and emulsifying activity of the modified α-zein were observed. The best emulsifying stability and emulsifying activity were observed for α-zein modified by alkali and heat treatment in ultrapure water. The enhanced amphiphilic property of α-zein resulted from the structure inversion ability achieved during treatment, which was confirmed by the recovery and unfolding of the α-helix secondary structure as the solvent polarity changed. The deamidation reaction improved the α-zein molecular flexibility, which was the fundamental reason for its structural inversion ability. Finally, a solvent-responsive pathway model was developed for alkali-heat modified α-zein.     

Chemical Characterization and Release of Polyphenols from Pecan Nut Shell [<Emphasis Type="Italic">Carya illinoinensis</Emphasis> (Wangenh) C. Koch] in Zein Microparticles for Bioactive Applications

The pecan nut [Carya illinoinensis (Wangenh) C. Koch] is a natural source of polyphenols with antioxidant properties. In this study, the encapsulation of aqueous and hydroalcoholic extracts of pecan nut shell were evaluated for the release of bioactive compounds and antioxidant potential in order to explore food applications using zein as encapsulating agent. The extracts showed high contents of total phenolics, condensed tannins and high antioxidant activity. Concentrations of proanthocyanidins were 9-fold higher in hydroalcoholic extracts. The LC-DAD analysis showed that catechins were the major phenolic compounds in samples, with epigallocatechin levels up to 138.62 mg mL^?1. Zein microparticles loaded with aqueous extract released 2.3 times more phenolic compounds than the hydroalcoholic extracts and the DSC thermograms showed that extracts of pecan nut shell remained thermally stable up to 240 °C. The zein microcapsules obtained in this study were efficiently encapsulated and represent an interesting additive due its high antioxidant capacity, physicochemical characteristics and morphology. The use of zein microparticles combined with natural extracts constitute a step forward in the improvement of current technology for delivering phenolic compounds with applications in functional foods and nutraceuticals.     

Slow digestion property of microencapsulated normal corn starch

Starch is the main glycemic dietary carbohydrate, and its nutritional quality is associated with the amount of slowly digestible starch (SDS) that is beneficial to glycemic control. In the current study, a microencapsulation of normal corn starch by zein protein and its slow digestion property were investigated. A significant increase of SDS and RS was shown for starch capsules (weight ratio of zein to starch: 1:6) containing plasticizers of glycerol and oleic acid after high temperature (≥70 °C) treatment. Further studies showed a substantially decreased viscosity and the formation of an amylose–lipid complex after starch gelatinization. Thus, the hydrophobic physical barrier of the zein matrix and the amylose–lipid complex might together limit the water accessibility and starch swelling leading to a dense packing of starch materials with a high amount of SDS. The acceptable sensory property makes it an ideal ingredient for specialty food preparation and glycemic control.