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.     

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.     

Improved Isolation of Zein from Corn Gluten Meal Using Acetic Acid and Isolate Characterization as Solvent

An improved means of isolating zein is needed to develop new uses for corn zein. We have measured the yield of zein and evaluated the ability of acetic acid to remove zein from corn gluten meal, distillers dried grains, and ground corn using acetic acid as solvent. Acetic acid removed zein more quickly, at lower temperatures, and in higher yields when compared with alcoholic solvents. After 60 min at 25°C, ≈50% of the zein in corn gluten meal was removed. A step change in yield from 43 to 50% occurs as the extraction temperature is increased from 40 to 55°C after mixing for 30 min at 25% solids. The protein composition of the zein removed from corn gluten meal using acetic acid is very similar to that of commercial zein by SDS‐PAGE. The zein obtained from corn gluten meal using acetic acid had higher amounts of fatty acids and esters according to IR analysis, leading to slightly lower protein content. Films made from zein extracted from corn gluten meal using acetic acid had lower tensile strength (≈60% lower) than films produced from commercial zein. Fibers with very small diameter (0.4–1.6 μm) can be produced by electrospinning using the AcOH solution obtained after corn gluten meal extraction.     

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.     

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.     

Formulation and Characterization of Drug‐Loaded Microparticles Using Distillers Dried Grain Kafirin

Kafirin, a protein extracted from sorghum grain, has been formulated into microparticles and proposed for use as a delivery system owing to the resistance of kafirin to upper gastrointestinal digestion. However, extracting kafirin from sorghum distillers dried grains with solubles (DDGS) may be more efficient, because the carbohydrate component has been removed by fermentation. This study investigated the properties and use of kafirin extracted from DDGS to formulate microparticles. Prednisolone, an anti‐inflammatory drug that could benefit from a delayed and targeted delivery system to the colon, was loaded into DDGS kafirin microparticles by phase separation with sodium chloride. Scanning electron micrographs revealed that the empty and prednisolone‐loaded microparticles were round in shape and varied in size. Surface binding studies indicated prednisolone was loaded within the microparticles rather than being solely bound on the surface. These findings demonstrate DDGS kafirin can be formulated into microparticles and loaded with medication. Future studies could investigate the potential applications of DDGS kafirin microparticles as an orally administered targeted drug‐delivery system.     

An acidic method of zein extraction from DDGS

Zein with a higher intrinsic viscosity and phosphorus content, similar protein content, lower yellowness, and at potentially much lower cost than commercially available zein was obtained from distillers dried grains with solubles (DDGS). A novel extraction method using acidic conditions in the presence of a reducing agent has been used to obtain about 10% aqueous ethanol soluble zein from DDGS. The optimum pH, time, temperature, and amount of reducing agent that can produce zein with high quality and yield have been developed. In addition to the zein, about 17% oil based on the dry weight of DDGS has also been obtained during zein extraction. The zein obtained from this research is expected to be suitable for use as fibers, films, and binders and in paints.     

REVIEW: Zein Extraction from Corn,Corn Products,and Coproducts and Modifications for Various Applications: A Review

Corn can be fractioned to produce starch, fiber, oil, and protein in relatively pure forms. The corn kernel contains 9–12% protein, but half of this is an industrially useful protein called zein. Dry milled corn (DMC), corn gluten meal (CGM), and distiller's dried grains with solubles (DDGS) are all coproducts from corn that contain zein and are used for zein extraction. Because it is insoluble in water, zein has found uses in many products such as coatings, plastics, textiles, and adhesives. Newer applications are taking advantage of zein's biological properties for supporting growing cells, delivering drugs, producing degradable sutures, and producing biodegradable plastics. This review covers zein characteristics and nomenclature, past and current practices in processing and extraction of zein from corn products and coproducts, and the modifications of zein for various applications.     

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.     

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.     

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。     

A Rapid Protein Digestibility Assay for Identifying Highly Digestible Sorghum Lines

Protein digestibility in sorghum (Sorghum bicolor (L.) Moench) lines was determined using two standard procedures (pepsin digestibility and pH‐stat) and compared with a newly developed, rapid electrophoresis‐based screening assay. The new assay was based on the rate of α‐kafirin disappearance after pepsin digestion. α‐Kafirin, the major sorghum storage protein, makes up ≈60–70% of the total protein in the grain. In the new assay, samples were first digested with pepsin for 1 hr, and undigested proteins were then analyzed by SDS‐PAGE. The intensitizes of the undigested α‐kafirin bands were measured. Higher band intensity indicated lower protein digestibility. The new assay was significantly correlated with the standard pepsin digestibility assay (r = −0.96, n = 16) after which it was patterned. The same was true of the pH‐stat procedure (r = −0.85, n = 16). This implies that the new assay is comparable to existing procedures and can be used for screening sorghum lines for protein digestibility. Two groups consisting of high‐protein digestibility and wild‐type sorghum lines were identified when the new assay was tested on 48 sorghum lines derived from crosses of wild‐type and mutant high protein digestibility lines, indicating that the new assay was efficient in differentiating between the two groups. Advantages of the new assay over the standard procedures include considerable reduction in analysis time and sample size required for the analysis. For example, analysis time was reduced by 20% and sample size by 10% when the new assay was used as compared with the pH‐stat procedure. We estimate that ≈60 sorghum lines can be screened in a day by a single operator using the new assay.     

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.     

Development of New Method for Extraction of α‐Zein from Corn Gluten Meal Using Different Solvents

A modified procedure for the extraction of α‐zein from corn gluten meal was developed and compared against a commercial extraction method. The modification involved raising the concentration of alcohol in solvent and removing the precipitate by centrifugation. Five organic solvent mixtures were compared using the modified extraction procedure developed along with the reductant sodium bisulfite and NaOH. The modified procedure precipitated most of the non‐α‐zein protein solids by increasing the concentration of alcohol. The supernatant had α‐zein‐rich fraction, resulting in higher yield of α‐zein than the commercial method when cold precipitated. The commercial extraction procedure had a zein yield of 23% and protein purity of 28% using 88% 2‐propanol solvent. The three best solvents, 70% 2‐propanol, 55% 2‐propanol, and 70% ethanol, yielded ≈35% of zein at protein purity of 44% using the modified extraction procedure. Zeins extracted using the novel method were lighter in color than the commercial method. Densitometry scans of SDS‐PAGE of α‐zein‐rich solids showed relatively large quantities of α‐zein with apparent molecular weights of 19,000 and 22,000 Da. The α‐zein‐rich solids also had small amounts of δ‐zein (10,000 Da) because it shares similar solubility properties to α‐zein. A solvent mixture with 70% 2‐propanol, 22.5% glycerol, and 7.5% water extracted significantly less zein (≈33%) compared to all other solvents and had α‐zein bands that differed in appearance and contained little to no δ‐zein.     

Solvent Extraction of Zein from Dry‐Milled Corn

Batch extraction of zein from dry‐milled whole corn with ethanol was optimum with 70% ethanol in water, an extraction time of 30–40 min, and temperature of 50°C. High yields (60% of the zein in corn) and high zein contents in the extracted solids (50%) were obtained at a solvent‐to‐solids ratio of 8 mL of 70% ethanol/g of corn. However, zein concentration in the extract was higher at lower ratios. Multiple extraction of the same corn with fresh ethanol resulted in a yield of 85% after four extractions, whereas multiple extractions of fresh corn with the same ethanol resulted in high (15 g/L) zein concentration in the extract. Optimum conditions for batch extraction of zein were 45°C, with 68% ethanol at a solvent‐to‐solids ratio of 7.8 mL/g for an extraction time of 55 min. Column extractions were also best at 50°C and 70% ethanol; a solvent ratio of 1 mL/g resulted in high zein concentrations in the extract (17 g/L) but yields were low (20%).     

Impacts of Kafirin Allelic Diversity,Starch Content,and Protein Digestibility on Ethanol Conversion Efficiency in Grain Sorghum

Seed protein and starch composition determine the efficiency of the fermentation process in the production of grain‐based ethanol. Sorghum, a highly water‐ and nutrient‐efficient plant, provides an alternative to fuel crops with greater irrigation and fertilizer requirements, such as maize. However, sorghum grain is generally less digestible because of extensive disulfide cross‐linking among sulfur‐rich storage proteins in the protein– starch matrix. Thus, the fine structure and composition of the seed endosperm directly impact grain end use, including fermentation performance. To test the hypothesis that kafirin (prolamin) seed storage proteins specifically influence the efficiency of ethanol production from sorghum, 10 diverse genetic lines with allelic variation in the β‐, γ‐, and (δ‐kafirins, including three β‐kafirin null mutants, were tested for ethanol yield and fermentation efficiency. Our selected lines showed wide variation in grain biochemical features, including total protein (9.96–16.47%), starch (65.52–74.29%), and free amino nitrogen (FAN) (32.84–73.51 mg/L). Total ethanol yield (ranging from 384 to 426 L/metric ton), was positively correlated to starch content (R² = 0.74), and there was a slight positive correlation between protein digestibility and ethanol yield (R² = 0.52). Increases in FAN content enhanced fermentation efficiency (R² = 0.65). The highest ethanol producer was elite staygreen breeding line B923296, and the line with the highest fermentation efficiency at the 72 h time point was inbred BT×623. A large‐seeded genotype, KS115, carrying a novel γ‐kafirin allele, was rich in FAN and exhibited excellent short‐term fermentation efficiency at 85.68% at the 20 h time point. However, the overall ethanol yield from this line was comparatively low at 384 L/metric ton, because of insufficient starch, low digestibility, and high crude protein. Multivariate analysis indicated an association between the β‐kafirin allele and variation in grain digestibility (P = 0.042) and FAN (P = 0.036), with subsequent effects on ethanol yield. Reversed‐phase HPLC profiling of the alcohol‐soluble kafirin protein fraction revealed diversity in protein content and composition across the lines, with similarities in peak distribution profiles among β‐kafirin null mutants compared with normal lines.     

Extraction and solubility characteristics of zein proteins from dry-milled corn

Zein isolation by aqueous ethanol extraction from dry-milled corn produces a mixture of zeins, covalently linked polymers (dimers, tetramers, etc.) and higher-molecular-weight aggregates, some of which were not soluble in aqueous alcohol. The insoluble particles were identified as protein aggregates which form when the extraction solution is heated, particularly under alkaline conditions. The insoluble protein aggregates were not present in zein isolated by the same method from corn gluten meal. Zeins extracted from corn gluten meal and dry-milled corn were fractionated (by differential solubility) to identify differences in their polypeptide compositions. Using polyacrylamide gel electrophoresis, beta- and gamma-zeins were detected in dry-milled corn, but only trace amounts of beta-zein were found in corn gluten meal. Treatment of dry-milled corn with 0.55% lactic acid and 0.2% sulfur dioxide at 50 degrees C for 6 h before ethanol extraction resulted in a 50% increase in zein isolate yield with high solubility (98%). This pre-extraction treatment cleaved disulfide linkages of the beta- and gamma-zeins and significantly reduced insoluble aggregates in zein isolates.     

Phenolic Content,Antioxidant Activity,and Consumer Acceptability of Sorghum Cookies

Cookies were produced from different sorghum flours to determine their potential as vectors of antioxidants. Different sorghum cultivars and their flour extraction rates were evaluated for their effects on phenolic content and antioxidant activity of the cookies. Consumer acceptance of the sorghum cookies was compared with that of wheat flour cookies. For each sorghum cultivar, cookies of 100% extraction rate flours had two to three times more total phenolics compared with those of 70% extraction rate flours, while antioxidant activity was 22–90% higher. Cookies of the condensed tannin sorghum had two to five times more phenolics compared with those of condensed tannin‐free sorghum. Antioxidant activity was 145–227 μMol Trolox equivalents (TE)/g in cookies of condensed tannin sorghum compared with 10–102 μMol TE/g in those of condensed tannin‐free sorghum. The sorghum flours had slightly higher phenolic content and antioxidant activity values than their corresponding cookies. Cookies of the red tannin‐free sorghum flours (PAN 8564/8446) were equally liked as wheat flour cookies, except for texture. However, cookies of condensed tannin sorghum were least accepted compared with wheat flour cookies despite their high antioxidant activity.