Extraction and Film Properties of Kafirin from Coarse Sorghum and Sorghum DDGS by Percolation

The high cost of kafirin and zein restricts their use for bioplastic and food applications. Effective, simple, and rapid kafirin/zein isolation processes are required. Here a percolation‐type aqueous ethanol solvent extraction process from coarse meals (grits) and coarse sorghum distillers dried grains and solubles (DDGS) for kafirin and zein isolation employing a low ratio of extractant to meal (2.5:1) was investigated, which is potentially applicable in the grain bioethanol industry. Postextraction filtration times were more than twice as fast using coarse meals compared with fine flours. Washing the meals prior to extraction to remove starch improved protein preparation purity to 73–85% compared with 68–72% for unwashed meals. Hence, no subsequent filtration or centrifugation step is required to clean up the kafirin/zein solution prior to solvent evaporation. With a single extraction step, kafirin/zein yields were 48% (protein basis) for DDGS and 53–70% for washed sorghum/maize meals. Cast films were used as a model bioplastic system to evaluate extracted kafirin/zein functional properties. DDGS kafirin films had rough surfaces but had the lowest water uptake and in vitro digestibility, owing to heat‐induced disulfide crosslinking during DDGS processing. Extraction by percolation using coarse meal/DDGS has potential to improve kafirin/zein viability.     

Physical,Mechanical, and Barrier Properties of Kafirin Films from Red and White Sorghum Milling Fractions

Fractions from the sorghum dry milling industry, including bran, are a potential source of kafirin. Free‐standing plasticized cast films were prepared from defatted kafirin preparations from red and white sorghum flour and bran fractions, and from commercial zein. All the kafirin preparations were able to form films. However, there were differences in film thickness, clarity, flexibility, surface texture, odor, and color between the different kafirin films. Bran kafirin films were highly colored, less flexible with a less smooth surface texture compared with films from flour, probably due to higher levels of contaminants in the bran kafirins. The strong color of the bran films could limit their use in certain coating applications. The kafirin films had much higher tensile strength and lower extensibility than zein film, probably because of the presence of β‐ and γ‐kafirins in the kafirin, giving high levels of disulfide cross‐linking in the kafirin films. The kafirin films had poorer water barrier properties than zein film, possibly due to greater thickness or to poorer flexibility, which may have caused microcracks.     

Improvement of Sorghum-Wheat Composite Dough Rheological Properties and Breadmaking Quality Through Zein Addition

Addition of sorghum flour to wheat flour produces marked negative effects on rheological properties of dough and loaf volume. Although there are notable differences in the chemical composition of sorghum proteins (kafirins) compared with wheat gluten that might imply poor functionality in breadmaking systems, a larger constraint may be the unavailability of kafirins due to encapsulation in protein bodies. In this study, zein, the analogous maize prolamin to kafirin, was used to determine the potential effects of protein-body-free prolamins on dough rheology and baking quality of wheat-sorghum composite flour. Mixograms run at 35°C (above the glass transition temperature of zein) were significantly (P < 0.01) improved with addition of zein. Mixogram peak heights increased while mixing time decreased uniformly with addition of zein. Dough extensibility studies showed an increase in maximum tensile stress, while baking studies showed an increase in loaf volume with increasing amounts of added zein. These data are supported by a previous study showing that, in a model system, zein mixed with starch can form viscoelastic networks, and suggest that kafirin, if made available, could contribute to dough formation.     

Alteration of kafirin and kafirin film structure by heating with microwave energy and tannin complexation

Heating with microwave energy and tannin complexation of kafirin both increase the tensile strength of cast kafirin bioplastic films. The effects of these treatments on the molecular structure of kafirin and of kafirin in the film were investigated. SDS-PAGE of heated wet kafirin showed an increase in kafirin oligomers. Disulfide groups increased in heated kafirin and in films made from the heated kafirin. Fourier transform infrared (FTIR) spectroscopy of heated kafirin and films made from the heated kafirin indicated an increase in beta-sheet conformation. In contrast, kafirin complexation with tannic acid (TA) and sorghum condensed tannin (SCT) resulted in a slight decrease in beta-sheet conformation in the kafirin and a larger decrease in the kafirin in the films. Raman spectroscopy showed that, with TA, there was a shift in peak from 1710 to 1728 cm(-1) for kafirin-tannic acid complexes, indicating kafirin and tannic acid interaction. The protein conformational changes presumably facilitated cross-linking between kafirin molecules and/or between kafirin and the tannins. Thus, although both heating with microwave energy and tannin complexation cause cross-linking of kafirin to increase film tensile strength, their effects on kafirin structure appear to be different.     

Analysis of zein by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) was used to analyze the protein composition of corn prolamine (zein). Mass spectra were obtained from commercial zein and zein extracted with aqueous 2-propanol and aqueous ethanol from consumer corn meal. For the commercial zein, three major zein fractions with m/z 26.8k, 24.1k, and 23.4k were clearly seen with two minor fractions (m/z 14.5k and 20.4k) also present. As compared with the results from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these three fractions were identified as alpha-zeins (24.1k and 23.4k combined as Z19; 26.8k as Z22). When extracted with 55% aqueous 2-propanol, three alpha-zein fractions with m/z 26.8k, 24.1k, and 23.4k were predominant. When extracted with ethanol, extraction temperature had an effect on the final products. When extracted with 75% aqueous ethanol at room temperature, alpha-zein and some 17-18k species were observed, whereas at 60 degrees C, a small amount of delta-zein was also present. Comparison of the MALDI/MS results with SDS-PAGE and gene sequence analysis shows that the MALDI/MS method is superior to SDS-PAGE in having higher resolution and mass accuracy.     

Encapsulation of essential oils in zein nanospherical particles

Three essential oils, oregano, red thyme, and cassia (100% pure oil), were encapsulated by phase separation into zein nanospheres. Topographical images indicated that the powders were made up of irregularly shaped particles ( approximately 50 mum) containing close-packed nanospheres. Approximately 31% of the oregano encapsulated particles had mean diameters greater than 100 nm compared to 19% for the zein alone particles. In vitro digestion of zein particles with pepsin at a concentration ratio of 10:1 was complete after 52 h in phosphate-citrate buffer, pH 3.5, at 37 degrees C by spectroscopic analysis. Nonenzymatic, aqueous in vitro release of essential oils from encapsulated zein particles was carried out in phosphate buffered saline at pH 7.4 and 37 degrees C. Release occurred at varying rates over 20 h probably from different locations within the closely packed nanospheres of different sizes. Gel electrophoresis SDS-PAGE of zein incubated with freeze-dried swine manure solids at 37 degrees C indicated that preformed microbial enzymes capable of digesting zein within minutes were present in the manure. Except for differences in size of nanospheres, no structural differences were resolved by several microscopic methods, suggesting that the oil and proteins phases were blended during phase separation.     

Reducing the brittleness of zein films through chemical modification

Zein protein is a major coproduct of biofuel from corn. To reduce the brittleness of zein films, a new type of zein-based biomaterial, was synthesized by chemical modification of zein with lauryl chloride through an acylation reaction. The final products were confirmed by (1)H NMR, FT-IR analysis, and SDS-PAGE. Thermal analysis detected no microphase separation in the synthesized polymer matrix. As the content of lauryl moiety increased, the glass transition temperatures of modified zein decreased by as large as 25.8 °C due to the plasticization effect of the lauryl moiety. In addition, mechanical and surface properties of cast films from acylated zein were also investigated. The elongation at break of modified zein sheet was increased by about 7-fold at the high modification level with some loss of mechanical strength. The surfaces of modified zein films were as uniform as unmodified zein film but more hydrophobic, further suggesting that no microphase separation happened during the film formation process. This work indicated the potential of these new biomaterials in the development of biodegradable food packaging materials and delivery systems.     

Chemistry and physical properties of melt-processed and solution-cross-linked corn zein

Corn zein was cross-linked with glutaraldehyde (GDA) using glacial acetic acid (HAc) as catalyst. The objectives are to evaluate the swelling characteristics of GDA cross-linked zein gels in water, ethanol, and their combinations. Similar formulations, upon solvent evaporation, form films. The mechanical properties of the films are compared to compression molded tensile bars from GDA melt-processed zein as a second objective. Chemistry of the cross-linking reaction was based on the aldehyde binding characteristics defined by use of fluorescence spectroscopy; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to demonstrate the cross-linking reaction; FTIR to observe absorption differences of the cross-linked product; differential scanning calorimetry, dynamic mechanical analysis and thermogravimetric analysis to assess thermal properties; and the use of Instron Universal Testing Machine to evaluate mechanical properties. A reaction mechanism for acid catalyzed GDA cross-linking of zein is proposed. Thermal and mechanical properties of tensile bars cut from either film or formed by compression molding were similar, where both showed increased tensile strengths, ductility and stiffness when compared with unmodified controls. Samples that were reacted with 8% GDA by weight based on weight of zein from either process retained their integrity when tensile bars from each were subjected to boiling water for 10 min or soaking in either water or HAc for 24 h. The melt-processed, cross-linked zein is a more environmentally friendly method that would eliminate the need for HAc recovery.     

Variation among maize (Zea mays L.) accessions of Bendel State,Nigeria

Summary Variability in maize zein protein band mobilities in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was characterized to classify 27 maize accessions (OTUs) collected from Bendel State, Nigeria. The classification of the OTUs was achieved using two numerical procedures: average linkage cluster analysis and principal component analysis (PCA). Five clusters were delineated by the cluster analysis whereas the PCA complemented the cluster analysis by separating the OTUs with yellow kernels into one group and OTUs with early maturity into another. OTUs from the same geographical contiguity commonly grouped together. However some regional overlappings of the OTUs occurred. Results of the PCA revealed that zein bands that stained less intensely more strongly separated the OTUs into various clusters than did those that stained more intensely.     

Sorghum Bran as a Potential Source of Kafirin

Sorghum bran, a coproduct of sorghum dry milling, could be a source of protein for industrial applications. Condensed tannin‐free red and white sorghum samples were decorticated by abrasion until ≈10 or 25% grain by weight was removed. Kafirin was then extracted from the milling fractions using an aqueous ethanol based solvent system. The brans were darker and considerably higher in protein and fat compared with the whole grain flours and decorticated grain flours, with the 25% bran having higher protein than the 10% bran. This is due to increased contamination of the bran with protein‐dense, corneous endosperm. The protein extracted from all the milling fractions, including the brans, was pure kafirin. However, the yield of kafirin from the brans (15.9–26.7% of total protein present) was somewhat lower than that from whole grain and decorticated grain flours (45.0–57.9% of total protein present), due to the fact that kafirin is located solely in the endosperm. Also, the kafirin from bran was more contaminated with fat, polyphenols, and other substances, and more highly colored, particularly the kafirin from red sorghum. Thus, sorghum bran could be used as a source of kafirin but further purification steps may be necessary.     

Plasticization of a protein-based film by glycerol: a spectroscopic, mechanical, and thermal study

Kafirin, the seed storage protein of the cereal sorghum, is highly homologous with the maize storage protein zein. The effects of plasticisation of a kafirin film by glycerol in the absence of water were examined by a combination of spectroscopic (NMR and infrared), rheological, and calorimetric methods. The results suggest that at low glycerol levels the glycerol is absorbed onto and possibly into the protein. Increasing the level of glycerol increases the motion of the protein and changes the protein conformation. There are corresponding changes of the mechanical properties of protein films. At 40% (w/w) of glycerol, two glass transition temperatures were observed, one of which corresponded to the glass transition temperature of pure glycerol. This result indicates that at this level of plasticizer there are sufficient glycerol/glycerol interactions occurring to allow a separate glass formation process for glycerol.     

Protein-lipid interactions in zein films investigated by surface plasmon resonance

Experiments on the adsorption of alpha-zein (characterized by SDS-PAGE) from aqueous ethanol and 2-propanol solutions onto hydrophilic and hydrophobic surfaces are reported. Zein adsorption onto self-assembled monolayers (SAMs) was detected by surface plasmon resonance (SPR). Gold substrates were prepared by thermal evaporation on glass slides. Gold-coated surfaces were modified by depositing SAMs of either a long-chain carboxylic acid terminated thiol [COOH(CH2)(10)SH] or a methyl-terminated alkanethiol [CH3(CH2)(7)SH]. Experimental measurements indicated that zein interacted with both hydrophilic and hydrophobic surfaces. Zein concentration affected the thickness of bound zein layers. The estimated thickness of the zein monolayer deposited on hydrophilic surfaces was 4.7 nm. Zein monolayer thickness on hydrophobic surfaces was estimated at 4.6 nm. The topography of zein layers was examined by atomic force microscopy (AFM) after solvent was evaporated. Surface features of zein deposits depended on the adsorbing surface. On hydrophilic surfaces, roughness values were high and distinct ring-shaped structures were observed. On hydrophobic surfaces, zein formed a uniform and featureless coverage.     

Recovery and characterization of α-zein from corn fermentation coproducts

Zeins were isolated from corn ethanol coproduct distiller's dried grains (DDG) and fractionated into α- and β γ-rich fractions. The effects of the ethanol production process, such as fermentation type, protease addition, and DDG drying temperature on zein recovery, were evaluated. Yield, purity, and molecular properties of recovered zein fractions were determined and compared with zein isolated from corn gluten meal (CGM). Around 29-34% of the total zein was recovered from DDG, whereas 83% of total zein was recovered from CGM. Process variations of cooked and raw starch hydrolysis and fermentation did not affect the recovery, purity, and molecular profile of the isolated zeins; however, zein isolated from DDG of raw starch fermentation showed superior solubility and film forming characteristics to those from conventional 2-stage cooked fermentation DDG. Protease addition during fermentation also did not affect the zein yield or molecular profile. The high drying temperature of DDG decreased the purity of isolated zein. SDS-PAGE indicated that all the isolated α-zein fractions contained α-zein of high purity (92%) and trace amounts of β and γ-zeins cross-contamination. Circular dichroism (CD) spectra confirmed notable changes in the secondary structure of α-zeins of DDG produced from cooked and raw starch fermentation; however, all the α-zeins isolated from DDG and CGM showed a remarkably high order of α-helix structure. Compared to the α-zein of CGM, the α-zein of DDG showed lower recovery and purity but retained its solubility, structure, and film forming characteristics, indicating the potential of producing functional zein from a low-value coproduct for uses as industrial biobased product.     

Transgenic sorghum with altered kafirin synthesis: kafirin solubility, polymerization, and protein digestion

Transgenic sorghum (TG) lines with altered kafirin synthesis, particularly suppression of γ-kafirin synthesis, and improved protein quality have been developed. The proportion of kafirin extracted with 60% tert-butyl alcohol alone was greatly increased in the TG lines. However, the total amount of kafirin remained unchanged. Further, in the TG lines, the kafirin was much less polymerized by disulfide bonding. There was also evidence of compensatory synthesis of other kafirin proteins. Cooked protein digestibility was increased in the TG form, even after removal of interfering starch. The TG protein bodies were intermediate in appearance between the normal type and the invaginated high digestibility mutants. Hence, the increased protein digestibility of these TG lines is probably related to their lower levels of disulfide-bonded kafirin polymerization, allowing better access of proteases. This work appears to confirm that disulfide bond formation in kafirin is responsible for the reduced protein digestibility of cooked sorghum.     

Effect of Microwave Heating of Kafirin on the Functional Properties of Kafirin Films

To improve the functional properties of cast kafirin films, dry kafirin, extracted with an aqueous ethanol‐based solvent at 70°C, was microwave‐heated. No effect on film tensile properties was found. Two strategies were employed to improve the effect of microwaving: extraction of kafirin using an aqueous tert‐butanol‐based solvent at ambient temperature to minimize temperature‐induced denaturation and wetting the kafirin to increase its dielectric properties. Microwave heating this kafirin to 90 or 96°C and holding for 1–2 min more than doubled maximum tensile strength and Young's modulus, and decreased strain by about one‐third compared with films made from nonmicrowaved kafirin. Film water vapor permeability was reduced by at least one‐third. Digestibility of microwaved kafirin and films was also substantially decreased, and film biodegradability was slowed slightly. Microwave heating gave a film microstructure with fewer and smaller size pores. SDS‐PAGE showed microwave‐induced intermolecular cross‐linking of the kafirin monomers, which was possibly responsible for the modification of film properties. Microwave heating of kafirin can be used to modify kafirin film properties, but the kafirin must be microwaved wet and be as close as possible to its native state.     

Improvement in water stability and other related functional properties of thin cast kafirin protein films

Improvement in the water stability and other related functional properties of thin (<50 μm) kafirin protein films was investigated. Thin conventional kafirin films and kafirin microparticle films were prepared by casting in acetic acid solution. Thin kafirin films cast from microparticles were more stable in water than conventional cast kafirin films. Treatment of kafirin microparticles with heat and transglutaminase resulted in slightly thicker films with reduced tensile strength. In contrast, glutaraldehyde treatment resulted in up to a 43% increase in film tensile strength. The films prepared from microparticles treated with glutaraldehyde were quite stable in ambient temperature water, despite the loss of plasticizer. This was probably due to the formation of covalent cross-linking between free amino groups of the kafirin polypeptides and carbonyl groups of the aldehyde. Thus, such thin glutaraldehyde-treated kafirin microparticle films appear to have good potential for use as biomaterials in aqueous applications.     

Effect of γ-zein on the rheological behavior of concentrated zein solutions

Zein, the prolamin of corn, is attractive to the food and pharmaceutical industries because of its ability to form edible films. It has also been investigated for its application in encapsulation, as a drug delivery base, and in tissue scaffolding. Zein is actually a mixture of proteins, which can be separated by SDS-PAGE into α-, β-, γ-, and δ-zein. The two major fractions are α-zein, which accounts for 70-85% of the total zein, and γ-zein (10-20%). γ-Zein has a high cysteine content relative to α-zein and is believed to affect zein rheological properties. The aim of this study was to investigate the effect of γ-zein on the often observed phenomena of zein gelation. Gelation affects the structural stability of zein solutions, which affects process design for zein extraction operations and development of applications. The rheological parameters, storage modulus (G') and loss modulus (G″), were measured for zein solutions (27% w/w solids in 70% ethanol). β-Mercaptoethanol (BME) was added to the solvent to investigate the effect of sulfhydryl groups on zein rheology. Modulus data showed that zein samples containing γ-zein had measurable gelation times under experimental conditions, contrary to samples with no γ-zein, where gelation was not detected. Addition of BME decreased the gelation time of samples containing γ-zein. This was attributed to protein unfolding. SEM images of zein microstructure revealed the formation of microspheres for samples with relatively high content of α-zein, whereas γ-zein promoted the formation of networks. Results of this work may be useful to improve understanding of the rheological behavior of zein.     

Quantitative Analysis of Corn Zeins by Capillary Electrophoresis

Kjeldahl analysis is commonly used to measure zein proteins in corn maize (N × 5.7) with no attempt to eliminate contribution from other nitrogen sources. In this study, dry milled corn was extracted with 70% ethanol or 0.1N NaOH and the zein content of the extract measured using capillary electrophoresis in the presence of sodium dodecyl sulfate. The amount of zein protein in alcohol extracts, using this method, was in good agreement with that determined by Kjeldahl nitrogen analysis. However, less than half of the Kjeldahl nitrogen in the alkaline extracts could be attributed to corn zein. Reproducibility expressed as relative standard deviation for migration time and peak area was 0.10 and 1.05, respectively. The technique permits rapid analysis of a large number of samples without interference from other compounds present in the extracts.     

丁香酚对高粱醇溶蛋白可食性膜结构及性能的影响

为开发天然的可降解、可食性包装材料,以高粱醇溶蛋白为原料,采用溶液共混的方法制备可食性丁香酚/高粱醇溶蛋白复合膜,分析不同浓度丁香酚对可食性高粱醇溶蛋白膜物理性能及微观结构的影响并探讨其变化机理。结果表明,添加4%丁香酚可优化蛋白膜的机械性能,提升膜的拉伸强度(TS)和断裂伸长率(EAB);添加丁香酚不影响蛋白膜的水蒸气透过系数(WVP),但略微提高了蛋白膜的溶解度;添加4%丁香酚可增加蛋白膜对紫外光和可见光的吸光度值,即增强膜的光阻隔性能。DSC测量显示,添加丁香酚后降低了高粱醇溶蛋白的玻璃态转变温度(Tg),表明丁香酚提高了丁香酚/高粱醇溶蛋白复合膜的延展性;FTIR分析结果表明,添加丁香酚后使得高粱醇溶蛋白二级结构中的α-螺旋、无规则卷曲转变为β-折叠、β-转角,表明丁香酚有助于提高丁香酚/高粱醇溶蛋白复合膜的机械性能;SEM结果显示,4%丁香酚与高粱醇溶蛋白的相容性良好,制备的复合膜截面光滑紧致。本研究结果为可降解、可食性膜新材料的研究及应用推广提供了理论参考。     

Effect of preparation conditions on protein secondary structure and biofilm formation of kafirin

Various extraction and drying conditions for the isolation of kafirin from dry-milled, whole grain sorghum have been investigated, with a view to optimizing extraction of the protein for commercial food coatings and packaging films. The addition of sodium hydroxide to an aqueous ethanol extractant increased the yield and solubility of kafirin. Subsequent heat drying at 40 degrees C was shown to cause the kafirin to aggregate as indicated by an increase in intermolecular beta-sheets. Extraction of the flour using ethanol (70%, w/w) with 0.5% (w/w) sodium metabisulfite and 0.35% (w/w) sodium hydroxide at 70 degrees C followed by freeze-drying of the protein was found to produce a yield of 54% kafirin with good film-forming properties. The kafirin films were assessed for their sensory properties, tensile strength, strain, and water vapor permeability. Fourier transform infrared spectroscopy was used to study the secondary structure of the extracted kafirins. The best films were made with kafirin containing a large proportion of nativelike alpha-helical structures with little intermolecular beta-sheet content as indicated by the Fourier transform infrared reflectance peak intensity ratios associated with these secondary structures. The principal factor affecting the secondary structure of the protein appeared to be the temperature at which the protein was dried. Heat drying resulted in a greater proportion of intermolecular beta-sheets. Any industrial-scale extraction must therefore minimize protein aggregation and maximize native alpha-helical structures to achieve optimal film quality.