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.

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.

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.

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.

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.

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.

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.

Allelic variation and differential expression at the 27-kilodalton zein locus in maize.

Allelic variation between inbred lines at the 27-kilodalton zein gene locus in maize has been used to study gene expression in developing endosperm. The inbred lines W22 and W23 contain two genes for this protein within two tandem repeats; the individual genes are virtually identical, with 99.9% homology in the 5'-flanking regions. Using gene-specific oligonucleotide probes, we have shown that transcripts of the downstream gene are found at a 2.5-fold-higher level than those of the upstream gene. Another inbred line, BSSS53, has one copy of the gene which is a recombinant of the duplicated genes at the 3'-flanking region. This line has been used in reciprocal crosses to demonstrate dosage effects for the overexpression of the downstream gene and to show that the overexpression of mRNA is reflected in a corresponding increase in the protein level. The accumulation of the protein through development does not, however, always correspond to the difference in mRNA levels.

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.

Physical and biochemical properties of maize hardness and extrudates of selected hybrids.

Protein and starch determinants of maize kernel hardness and extruded products were characterized to better define the role of endosperm texture during extrusion. Maize physical properties were correlated with total proteins and zein subclasses (p < 0.01). The extrusion process significantly altered protein solubility and increased protein fragmentation as measured by RP-HPLC and size exclusion chromatography. Harder grits and extrudates demonstrated higher amylose content, lower degree of starch damage, and fragmentation at different screw speeds than softer grits and extrudates. Differences in extrudate expansion ratio, water absorption index, water solubility index, oil absorption capacity, and breaking stress between harder and softer hybrids were related to protein aggregation and fragmentation as well as starch damage and fragmentation.

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.

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.

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.

Basic study of corn protein, zein, as a biomaterial in tissue engineering, surface morphology and biocompatibility.

The zein films, were prepared for culturing human liver cells (HL-7702) and mice fibroblast cells (NIH3T3), while the Corning microplate and polylactic acid (PLA) were chosen as controls. The surface morphology of zein films prepared by two different methods was studied by scanning electron microscope (SEM), which revealed that the zein films were composed of particles of diameter 100-500 and 500-2500 nm, respectively. The biocompatibility of zein films was assessed by attachment, extensibility and proliferation of cells on them. Our study indicated that over 60% of both HL-7702 cells and NIH3T3 cells could attach to the Corning microplate, zein films and PLA at 3h after seeding. The concentration and particle sizes for preparing zein films did not seem to affect the proliferation of the cells tested. There were no significant differences in the proliferation of both HL-7702 cells and NIH3T3 cells between the Corning microplate and two kinds of zein films, except that the zein film composed of smaller particles at the lowest concentration exhibited a very good ability for proliferation of both the cells, while PLA was a poor matrix in the latter period of the cell proliferation. This preliminary study demonstrates that zein is a promising biomaterial with good biocompatibility for the development of tissue engineering.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of zeins in mature maize kernels.

A high-throughput method has been developed to allow rapid analysis of maize seed storage proteins by matrix-assisted laser desorption time-of-flight mass spectrometry. The extraction solution containing an organic solvent, a reducing agent, and a volatile base has been optimized to enable extraction of all classes of zein proteins (alpha-, beta-, gamma-, and delta-). A near-saturating concentration of matrix, 2-(4-hydroxyphenylazo)benzoic acid, was necessary to obtain strong peaks for the most lipophilic zeins, the alpha-zeins. Zein proteins with small mass differences, difficult to separate by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were resolved through this analysis. Mass signals corresponding to the 10-kDa delta-, 15-kDa beta-, 16-kDa gamma-, 27-kDa gamma-, and several 19 and 22-kDa alpha-zeins were detected. The zein identities were further confirmed by the association of the number of cysteine residues in each zein MS peak, as determined by iodoacetamide derivatization, with the number predicted from its coding sequence. The relative zein abundance in the zein MS peaks was also correlated with the relative zein EST abundance among endosperm EST libraries. This method was utilized to examine the zein composition of a number of corn inbred lines and opaque mutants.

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.

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.

Isolation and quantitation of zeins in waxy and amylose-extender and wild flint and dent maize endosperm using a new solvent sequence for protein extraction.

Protein distribution in endosperm of maize grains differing by their texture, flint or dent, and by their genotype, wild or waxy or amylose-extender, was examined by the successive use of 0.5 M NaCl, 0.5 M NaCl plus 0.6% 2-mercaptoethanol (2ME) at neutral and then alkaline pH, and 55% 2-propanol plus 0.6% 2ME as extractants. Proteins extracted in the presence of 2ME were characterized by their size polymorphism and amino acid composition. Proteins isolated with NaCl plus 2ME at neutral pH corresponded with a mixture of gamma-zein (27 kDa) and glutelin-like proteins. Proteins isolated with NaCl plus 2ME at pH 10 were a mixture of gamma-zeins (27 and 16 kDa) and beta-zeins (14 kDa). Alcohol-soluble proteins consisted of alpha-, beta-, and delta-zeins, alpha subunits being predominant. Zein quantitation was improved by weighing the nitrogen percentage of extracts by their zein content, as estimated from the data on amino acid composition. The data reported by Wolf et al. (Cereal Chem. 1975, 52, 765) were integrated to the results of this work to suggest the occurrence of an inverse correlation between amylose in starch and zeins in proteins.