Antifungal edible coatings for fruits based on zein and chitosan nanowhiskers.

Fresh produce have a more limited shelf life than processed ones. Their sensory attributes such as appearance and surface texture are important features in consumer perception and liking. The decomposition of fresh produce, which is caused by enzymes, chemical reactions, and microbial infections, often caused by Colletotrichum species, is inevitable. However, it can be slowed down. Several materials have been developed for this purpose, with an emphasis on active coatings using nanomaterials. In this study, the protective effects of a zein coating containing chitosan nanowhiskers (CSW) for the maintenance of fruit quality were investigated using guava (Psidium guajava L.) as a model fruit. CSW were previously characterized, and their antifungal effects against distinct Colletotrichum species (Colletotrichum asianum, Colletotrichum tropicale, Colletotrichum gloeosporioides, and Colletotrichum brevisporum) were proven. Coatings were characterized by thermogravimetric analysis, optical profilometry, and mechanical properties. Total soluble solids, pH, mass loss, and visual inspection of uncoated and coated guava fruits were also verified during 9 days. Results show that CSW length and aspect ratio decreased for longer extraction times. A similar behavior was found for x-ray diffraction in which peak intensity decreases under the same conditions. CSW degradation (ca. 250-400°C) also depends on extraction time in which more crystalline whiskers are the most thermally stable ones. The addition of CSW did not significantly (p < 0.05) modify the homogeneity and continuity of coating but prevented microbial growth assuring fruit quality during storage. In summary, coatings protected guava fruits from post-harvest spoilage while preserving quality and extending shelf life. PRACTICAL APPLICATION: Fresh foods such as fruits and vegetables have a more limited shelf life than processed ones.

Endosperm-specific accumulation of human α-lactalbumin increases seed lysine content in maize.

KEY MESSAGE: This study demonstrated high expression and accumulation of human α-lactalbumin in transgenic maize, and significant improvement of lysine content in maize endosperm. As a high-yield crop, lack of lysine in endosperm storage protein is a major defect of maize (Zea mays L.). Specifically expression of foreign proteins is a potential way to improve lysine content in maize endosperm. Human α-lactalbumin is such a protein with high lysine content and high nutritional value. In this study, the codon-optimized human lactalbumin alpha (LALBA) gene was driven by maize endosperm-specific 27 kD γ-zein promoter, and transformed into maize. Five independent transgenic lines were obtained, and LALBA was highly expressed in endosperm in all these lines. Protein assay indicated that human α-lactalbumin was highly accumulated in maize endosperm. Immuno-localization assay indicated that human α-lactalbumin was mainly deposited into the protein body (PB). Protein interaction assay showed that human α-lactalbumin interacted with 16 kD γ-zein, which might lead to its deposition to the PBs. Amino acid analysis of two independent transgenic lines showed significant increase of lysine contents in transgenic endosperm, with 47.26% and 45.15% increase to their non-transgenic seeds, respectively. We obtained transgenic maize with endosperm-specific accumulation of human α-lactalbumin at high level and increased the lysine content in maize endosperm. This study demonstrated an effective way to improve the nutritional value of maize seeds.

Factors Influencing Zein-Whole Sorghum Flour Dough Formation and Bread Quality.

Zein is known to able to form viscoelastic dough with wheat-like properties under certain conditions. Several studies have been conducted to explain the mechanism behind this ability and to improve the functionality and end-use quality of zein-based dough systems. However, most of this research has been conducted using zein in combination with isolated starches or high-starch flours. To investigate the production of additional zein-whole sorghum flour breads, experiments were conducted to determine factors impacting zein-whole sorghum flour dough and bread quality. Optimizing water levels, using defatted zein and/or sorghum flour, and increasing zein content in dough formulas were investigated as initial formulation steps. Of these factors, increasing zein content from 20% to 30% (flour weight basis) had the greatest impact, resulting in stronger zein-based dough and improved bread quality. Additives and zein treatments shown to impact zein functionality were then investigated for their effect of zein-whole sorghum flour breads. Mixing zein and whole sorghum flour with 0.5% hydrogen peroxide, 5% ethanol, or 3% hydroxypropyl methylcellulose resulted in improved dough strength and bread quality. Breads made from whole white sorghum flour had improved quality compared to zein-based breads made with black or high-tannin whole sorghum flour. PRACTICAL APPLICATION: Zein is known to be able to form wheat-like dough when mixed under the right conditions. Most of the research on zein-based dough and food products has used high-starch flours. This project investigated optimizing the production of zein-whole sorghum flour dough and bread as an alternative. Increasing the zein content in the formula and using additives including ethanol and HPMC produced breads from zein-whole sorghum flour that were like those made with zein and pure starch.

Mass spectrometric characterization of the zein protein composition in maize flour extracts upon protein separation by SDS-PAGE and 2D gel electrophoresis.

Highly homogenous α zein protein was isolated from maize kernels in an environment-friendly process using 95% ethanol as solvent. Due to the polyploidy and genetic polymorphism of the plant source, the application of high resolution separation methods in conjunction with precise analytical methods, such as MALDI-TOF-MS, is required to accurately estimate homogeneity of products that contain natural zein protein. The α zein protein product revealed two main bands in SDS-PAGE analysis, one at 25 kDa and other at 20 kDa apparent molecular mass. Yet, high resolution 2DE revealed approximately five protein spot groups in each row, the first at ca. 25 kDa and the second at ca. 20 kDa. Peptide mass fingerprinting data of the proteins in the two dominant SDS-PAGE bands matched to 30 amino acid sequence entries out of 102 non-redundant data base entries. MALDI-TOF-MS peptide mapping of the proteins from all spots indicated the presence of only α zein proteins. The most prominent ion signals in the MALDI mass spectra of the protein mixture of the 25 kDa SDS gel band after in-gel digestion were found at m/z 1272.6 and m/z 2009.1, and the most prominent ion signals of the protein mixture of the 20 kDa band after in-gel digestion were recorded at m/z 1083.5 and m/z 1691.8. These ion signals have been found typical for α zein proteins and may serve as marker ion signals which upon chymotryptic digestion reliably indicate the presence of α zein protein in two hybrid corn products.

From 'green' technologies to 'red' antioxidant compounds extraction of purple corn: a combined ultrasound-ultrafiltration-purification approach.

BACKGROUND: A pilot scale process consisting of ultrasound-assisted extraction, ammonium sulfate precipitation, cross-flow ultrafiltration and AB-8 macroporous resins purification aiming to recover anthocyanins and zein from purple corn (PC) was optimized and scaled-up. The effects of five independent variables (ethanol concentration, liquid to solid ratio, ultrasound temperature, time and power) were discussed and the most influential factors were optimized. RESULTS: The highest total anthocyanin (0.45 ± 0.01 g kg-1 ) and zein (17.14 ± 1.73 g kg-1 ) contents from purple corn were obtained using an ultrasound power of 105 W, an extraction time of 90 min, an ethanol concentration of 74% and a liquid to solid ratio of 26:1, at 70 °C, and this was consistent with the predicted values (0.46 and 17.36 g kg-1 , for anthocyanin and zein, respectively). Subsequently, ammonium sulfate precipitation was used to isolate anthocyanins and zein. After cross-flow ultrafiltration, zein (6.30 g) was obtained with 80% purity. Anthocyanins were purified by AB-8 macroporous resins, resulting in 1.60 g of anthocyanins. High-performance liquid chromatography-electrospray ionization-mass spectrometry analysis revealed eight different anthocyanins in purple corn extracts. CONCLUSION: From the results obtained in the present study, it can be concluded that the proposed extraction-separation-filtration-purification method applied under the optimal conditions could be scaled-up to recover anthocyanins and zein simultaneously. Moreover, under the selected conditions, no significant degradation of anthocyanins was observed. © 2018 Society of Chemical Industry.

Zein Nanoparticles Uptake and Translocation in Hydroponically Grown Sugar Cane Plants.

The main objective of this study was to investigate the uptake and translocation of positively charged zein nanoparticles (ZNPs) in hydroponically grown sugar cane plants. Fluorescent ZNPs (spherical and measuring an average diameter 135 ± 3 nm) were synthesized by emulsion-diffusion method from FITC-tagged zein. Fluorescent measurement following digestion of plant tissue indicated that sugar cane roots had a significant adhesion of ZNPs, 342.5 ± 24.2 µg NPs/mg of dry matter, while sugar cane leaves contained a very limited amount, 12.9 ± 1.2 µg NPs/mg dry matter for high dose(1.75 mg/ml) after 12 h. Confocal microscopy studies confirmed presence of fluorescent ZNPs in the epidermis and endodermis of the root system. Given their ability to adhere to roots for extended periods of time, ZNPs are proposed as effective delivery systems for agrochemicals to sugar cane plants, but more studies are needed to identify effect of nanoparticle exposure to health of the plant.

A practical method for extending the biuret assay to protein determination of corn-based products.

A modified biuret method suitable for protein determination of corn-based products was developed by introducing a combination of an alkaline reagent with sodium dodecyl sulfate (reagent A) and heat treatments. The method was tested on seven corn-based samples. The results showed mostly good agreement (P>0.05) as compared to the Kjeldahl values. The proposed method was found to enhance the accuracy of prediction on zein content using bovine serum albumin as standard. Reagent A and sample treatment were proved to effectively improve protein solubilization for the thermally-dried corn-based products, e.g. corn gluten meal. The absorbance was stable for at least 1-h. Moreover, the whole measurement of protein content only needs 15-20min more than the traditional biuret assay, and can be performed in batches. The findings suggest that the proposed method could be a timesaving alternative for routine protein analyses in corn processing factories.

Fabrication and characterization of Pickering emulsions and oil gels stabilized by highly charged zein/chitosan complex particles (ZCCPs).

Herein, we reported a facile method to fabricate ultra-stable, surfactant- and antimicrobial-free Pickering emulsions by designing and modulating emulsions' interfaces via zein/chitosan colloid particles (ZCCPs). Highly charged ZCCPs with neutral wettability were produced by a facile anti-solvent procedure. The ZCCPs were shown to be effective Pickering emulsifiers because the emulsions formed were highly resistant to coalescence over a 9-month storage period. The ZCCPs were adsorbed irreversibly at the interface during emulsification, forming a hybrid network framework in which zein particles were embedded within the chitosan network, yielding ultra-stable food-grade zein/chitosan colloid particles stabilized Pickering emulsions (ZCCPEs). Moreover, stable surfactant-free oil gels were obtained by a one-step freeze-drying process of the precursor ZCCPEs. This distinctive interfacial architecture accounted for the favourable physical performance, and potentially oxidative and microbial stability of the emulsions and/or oil gels. This work opens up a promising route via a food-grade Pickering emulsion-template approach to transform liquid oil into solid-like fats with zero trans-fat formation.

Ultrasound-based protein determination in maize seeds.

The need for a simple and accurate method for protein estimation in alcoholic extracts led to the reexamination of the optimum conditions of a colorimetric assay based on the biuret reaction. Sonication time and the other experimental parameters were optimized after kinetics study on the extraction of either zein or total proteins. Zein extraction and purity were investigated by (1)H and (13)C NMR spectroscopy, SDS-PAGE electrophoresis, and UV-visible spectrophotometry (UV-vis). A zein assay was proposed, which involves the reaction of copper ions in copper phosphate powder with zein extracted in ethanolic solutions under strong alkaline environment. Furthermore, we extended this procedure to determine total proteins in maize samples simultaneously with their ultrasonic-assisted (US) extraction with an alkaline-alcoholic solution. Proteins in both types of extracts were well characterized by UV-vis spectroscopy. However, the 545 nm absorbance of the violet-colored supernatants which is proportional to the protein content was found to be the key parameter of the improved biuret-based protein assay. Comparison of values obtained by this procedure and by Micro-Kjeldahl method was in excellent agreement. A scaled-down procedure agreed well with the standard procedure. Enhanced accuracy and repeatability was found in protein determination in maize using the modified biuret method. The optimization of reagent concentrations and incubation times were studied as well.

A simple, fast and highly sensitive colorimetric detection of zein in aqueous ethanol via zein-pyridine-gold interactions.

Formation of gold nanoparticles in aqueous ethanol in the presence of pyridine-functionalized single-chain nanoparticles allows for the fast and highly sensitive colorimetric detection of zein corn protein.

Development of a high-performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight-mass spectrometry methodology for the determination of three highly antihypertensive peptides in maize crops.

The simultaneous quantification of three highly antihypertensive peptides (LRP, LSP, and LQP) in six maize crops using novel HPLC-ESI-Q-ToF methodology is presented. The method included the extraction of α-zein proteins from maize, their purification by acetone precipitation, digestion with thermolysin and HPLC separation in a fused-core column. Several MS parameters were optimized to increase sensitivity and reduce spontaneous fragmentation of peptide ions into the ESI source. The ions with m/z 193.1315, 385.2558 (for LRP), 316.1867 (for LSP), and 357.2132 (for LQP) were monitored in the optimization and characterization of the method. In order to improve the repeatability, sensitivity, and the stability of peptides in the sample, the removal of urea was required. The use of two solid-phase extraction methods to remove urea from digested extract was evaluated. For the first time filter-aided sample preparation approach for the study of bioactive peptides in foodstuffs has been proposed. The optimized HPLC-ESI-Q-ToF method was characterized by the evaluation of linearity, LOD, LOQ, precision, and recovery. A study on the existence of matrix interferences was also performed. The developed method was applied to the quantification of LRP, LQP, and LSP peptides in maize lines using the standard addition method. The results showed the highest yield of LSP peptide in EZ11A line and LRP and LQP peptides in A632 line.

Technical note: quantification of zeins from corn, high-moisture corn, and corn silage using a turbidimetric method: comparative efficiencies of isopropyl and tert-butyl alcohols.

Zeins are corn endosperm storage proteins that encapsulate starch granules into a protein matrix, which can act as a barrier to starch accessibility and digestion. Laboratory methods to quantify zein are seldom used because they are considered arduous and time-consuming. A recently published rapid turbidimetric method (mTM) was reinvestigated by changing the solution originally used for the zein solubilization step. In particular, the aim was to explore whether, and to what extent, the use of tert-butyl alcohol (t-BuOH-mTM) in lieu of isopropyl alcohol (i-PrOH-mTM) was able to improve the quantification of zeins from dry corn, high-moisture corn, and corn silage samples. The nature of the alcohol influenced the zein extraction values, and t-BuOH-mTM gave higher zein values in corn (3.6 vs. 3.3 g/100 g of dry matter) and corn silage samples (1.2 vs. 0.9 g/100 g of dry matter) compared with i-PrOH-mTM. In contrast, similar zein extraction values were obtained for high-moisture corn (2.1 vs. 1.9 g/100 g of dry matter, respectively). Sodium dodecyl sulfate-PAGE analysis revealed no contamination by nonzein proteins with the use of tert-butyl alcohol. Overall, these findings indicated that tert-butyl alcohol has a greater ability to solubilize zein compared with isopropyl alcohol and thus the t-BuOH-mTM allowed greater extraction of zeins. Considering the growing interest of animal nutritionists in zein proteins, such results should provide useful information for routine laboratory analysis.

Analysis of migrated hydrogel used for breast augmentation revealed prolamin (a cereal seed storage protein).

BACKGROUND: We report on a case of leakage and migration to the upper abdomen of an unknown injected material that was used for breast augmentation. It was revealed to be prolamin by Fourier transform infrared (FTIR) analysis and pyrolysis gas chromatography/mass spectrometry (PY-GC/MS). METHODS: A 35-year-old woman who had undergone mammary augmentation by transaxillary injection 8 years previously presented with a decreased size of her left breast and a palpable mass in the left upper quadrant (LUQ). Mammogram and ultrasonography showed multiple dense masses and several hypoechoic areas, respectively. Abdominal ultrasound showed a hypoechoic lesion between the subcutaneous layer and the abdominal wall muscles. When the left breast and the lump in the LUQ were explored, 90 and 160 cc of yellow, sticky, granular gel gushed out. FTIR analysis and PY-GC/MS were used to investigate the component of the removed gel. RESULTS: When this gel was analyzed by FTIR with the transmittance mode, intensity bands appeared at 3295.2 (NH2), 2927.2 (CH), 1650 (C=C), 1544.6 (C-C), and 1403.1 (C-N) cm(-1). The result showed a 93.84% match with purified zein, a 91.19% match with zein from corn, and a 90.27% match with poly(N-methyl acrylamide). FTIR with the attenuated total reflectance (ATR) mode revealed that the gel matched with wheat gluten flour. Based on the result of PY-GC/MS, the gel was suspected to be protein. CONCLUSION: This is the first such report on performing chemical analysis of a leaked injected gel from human breast implantation. The removed gel from the breast augmentation was revealed to be prolamin, which is a cereal seed storage protein. We think FTIR might be a useful tool for analyzing and confirming extracted materials that were previously injected to the body.

Hardness methods for testing maize kernels.

Maize is a highly important crop to many countries around the world, through the sale of the maize crop to domestic processors and subsequent production of maize products and also provides a staple food to subsistance farms in undeveloped countries. In many countries, there have been long-term research efforts to develop a suitable hardness method that could assist the maize industry in improving efficiency in processing as well as possibly providing a quality specification for maize growers, which could attract a premium. This paper focuses specifically on hardness and reviews a number of methodologies as well as important biochemical aspects of maize that contribute to maize hardness used internationally. Numerous foods are produced from maize, and hardness has been described as having an impact on food quality. However, the basis of hardness and measurement of hardness are very general and would apply to any use of maize from any country. From the published literature, it would appear that one of the simpler methods used to measure hardness is a grinding step followed by a sieving step, using multiple sieve sizes. This would allow the range in hardness within a sample as well as average particle size and/or coarse/fine ratio to be calculated. Any of these parameters could easily be used as reference values for the development of near-infrared (NIR) spectroscopy calibrations. The development of precise NIR calibrations will provide an excellent tool for breeders, handlers, and processors to deliver specific cultivars in the case of growers and bulk loads in the case of handlers, thereby ensuring the most efficient use of maize by domestic and international processors. This paper also considers previous research describing the biochemical aspects of maize that have been related to maize hardness. Both starch and protein affect hardness, with most research focusing on the storage proteins (zeins). Both the content and composition of the zein fractions affect hardness. Genotypes and growing environment influence the final protein and starch content and, to a lesser extent, composition. However, hardness is a highly heritable trait and, hence, when a desirable level of hardness is finally agreed upon, the breeders will quickly be able to produce material with the hardness levels required by the industry.

Nanoscalar structures of spray-dried zein microcapsules and in vitro release kinetics of the encapsulated lysozyme as affected by formulations.

Sustained release of antimicrobials may be a viable solution to enhance the bioavailability during the shelf life of food products. In this work, spray-drying was used to encapsulate a model antimicrobial of lysozyme in corn zein. The effects of zein/lysozyme (20:1 to 4:1) and zein/thymol (1:0 to 4:1) ratios on the microstructures of microcapsules and in vitro release profiles of the encapsulated lysozyme were investigated. In all cases, less lysozyme was released at higher pH, resulting from stronger molecular attraction between zein and lysozyme. Nanoscalar matrix structures of microcapsules were correlated with release characteristics of the encapsulated lysozyme. At intermediate zein/lysozyme (10:1) and zein/thymol (50:1) ratios, microcapsules had a continuous matrix structure and showed sustained release (11.1-65.3%) of lysozyme at pH 6 over 49 days. This work may be developed into practical food grade delivery systems of antimicrobials.

Electrospun zein fibers as carriers to stabilize (-)-epigallocatechin gallate.

In this study, a method was developed for continuous electrospinning of ultrafine corn zein protein fibers with diameters ranging from 150 to 600 nm. Fiber-forming solutions with various zein concentrations (10% to 30%, w/w) and aqueous ethanol concentrations (60% to 90%, w/w) were electrospun at 15 and 20 kV. Scanning electron microscopy results showed that the morphology of zein fibers was affected by aqueous ethanol concentration, zein concentration, and the applied voltage. The optimal condition for forming bead-less fibers was found to be 20% protein, 70% alcohol, and 15 kV. The zein fibers resisted solubilization in water, although swelling and plasticization were apparent after the water treatment. The efficacy of zein fibers was tested for stabilization of a green tea polyphenol, (-)-epigallocatechin gallate (EGCG), by incorporating the EGCG in zein fiber-forming solutions. Freshly spun fibers were less effective at immobilizing the EGCG upon immersion in water (82% recovery) as compared to fibers that were aged at 0% relative humidity for at least 1 d (>98% recovery) before water immersion. Fourier transform infrared spectroscopy studies demonstrated that hydrogen bonding, hydrophobic interactions, and physical encapsulation are the major contributors to the stabilization of EGCG in zein fibers in water.

Ultrarapid quantitation of maize proteins by perfusion and monolithic reversed-phase high-performance liquid chromatography.

The main objective of this study was to develop a new methodology alternative to the classical Kjeldahl analysis for determining maize proteins in maize products and seeds. For that purpose, two different chromatographic methodologies using perfusion and monolithic stationary phases, both enabling rapid separations of maize proteins, were investigated. Due to the difficulty to find suitable standards for this type of analysis, three different maize products were initially tested as proteins standards: zein F4000, corn gluten meal, and maize flour. Different figures of merit (i.e., linearity, correlation coefficient, precision, limits of detection and quantitation), as well as the presence of matrix inferences, were investigated. The results obtained for the different chromatographic stationary phases and protein standards were compared in order to select the most suitable analytical conditions. Despite both perfusion and monolithic methodologies resulting, in general, as appropriate for the quantitation of maize proteins, the highest reduction of analysis time and lowest detection and determination limits provided by perfusion methodology enabled to select this one as the method of choice for the quantitation of maize proteins. Regarding the different protein standards studied in this work, in general the best results were obtained using the zein standard. Compared to Kjeldahl methodology, perfusion chromatography yields total protein contents in shorter analysis time while enabling the separation of the different kinds of proteins. Due to the high diversity and complexity of industrial maize products, the proposed chromatographic method could be a very useful tool for their routine analysis.

Technical note: a method to quantify prolamin proteins in corn that are negatively related to starch digestibility in ruminants.

Compared with floury or high-moisture corns, dry corn with a greater percentage of vitreous endosperm has been demonstrated to be negatively related to starch digestibility and milk yield of lactating dairy cows. Starch granules in corn are encapsulated by hydrophobic prolamin proteins that are innately insoluble in the rumen environment. Corn prolamin proteins are named zein, and laboratory methods to quantify zein exist but are seldom employed in ruminant nutrition because of their arduous nature. In this study, advances in cereal chemistry were combined with rapid turbidimetric methods yielding a modified turbidimetric zein method (mTZM) to quantify zein in whole corn. Ten dry corns containing unique endosperms were evaluated using the mTZM. Corns with flint, dent, floury, or opaque endosperms were found to contain 19.3, 11.3, 5.8, and 4.9 g of zein/100 g of starch, respectively. The ability of mTZM to differentiate corn endosperm types as defined by least significant difference was 2.6 g of zein/100 g of starch. Ten high-moisture corns of varying moisture content were also evaluated using the mTZM. Zein content of high-moisture corns as defined by mTZM ranged from 8.3 to 2.8 g of zein/100 g of starch with a least significant difference of 1.2 g of zein/100 g of starch. The mTZM determined that zein contents of high-moisture, floury, and opaque corns were markedly less than those of flint and dent dry corns, indicating that mTZM has the ability to quantify starch granule encapsulation by hydrophobic prolamin proteins in whole corn.

Transgenic maize endosperm containing a milk protein has improved amino acid balance.

In order to meet the protein nutrition needs of the world population, greater reliance on plant protein sources will become necessary. The amino acid balance of most plant protein sources does not match the nutritional requirements of monogastric animals, limiting their nutritional value. In cereals, the essential amino acid lysine is deficient. Maize is a major component of human and animal diets worldwide and especially where sources of plant protein are in critical need such as sub-Saharan Africa. To improve the amino acid balance of maize, we developed transgenic maize lines that produce the milk protein alpha-lactalbumin in the endosperm. Lines in which the transgene was inherited as a single dominant genetic locus were identified. Sibling kernels with or without the transgene were compared to determine the effect of the transgene on kernel traits in lines selected for their high content of alpha-lactalbumin. Total protein content in endosperm from transgene positive kernels was not significantly different from total protein content in endosperm from transgene negative kernels in three out of four comparisons, whereas the lysine content of the lines examined was 29-47% greater in endosperm from transgene positive kernels. The content of some other amino acids was changed to a lesser extent. Taken together, these changes resulted in the transgenic endosperms having an improved amino acid balance relative to non-transgenic endosperms produced on the same ear. Kernel appearance, weight, density and zein content did not exhibit substantial differences in kernels expressing the transgene when compared to non-expressing siblings. Assessment of the antigenicity and impacts on animal health will be required in order to determine the overall value of this technology.

CE-MS of zein proteins from conventional and transgenic maize.

In this work, an original CE-MS method has been developed to analyze the complex zein protein fractions from maize. A thorough optimization of: (i) zein protein extraction, (ii) CE separation, and (iii) electrospray-MS (ESI-MS) detection is carried out in order to obtain highly informative CE-MS profiles of this fraction. The developed CE-MS method provides good separation of multiple zein proteins based on their electrophoretic mobilities as well as adequate characterization of these proteins based on their M(r). Zein proteins with small M(r) differences (below 100 Da) were easily separated and successfully analyzed by CE-MS. Thus, apart of the so-called 15-kDa-beta-zein and 16-kDa-gamma-zein, which are demonstrated to be formed by a heterogeneous group of proteins, numerous alpha-zeins belonging to the 19- and 22-kDa fraction were also identified for the first time in this work. The usefulness of this CE-MS method was corroborated by comparing the zein-protein fingerprints of various maize lines including transgenic and their corresponding nontransgenic isogenic lines cultivated under the same conditions.