Comparison of the characteristics of prolamins among foxtail millet varieties with different palatability: Structural, morphological, and physicochemical properties.

Recently, there are considerable interests in the influence of prolamins on eating quality of grains. To inquire the potential effect of prolamins on the palatability of foxtail millet, prolamin characteristics under its raw (PR) and post-cooked (PC) state among three typical varieties with high (Zhonggu, ZG), medium (Zhaonong, ZN), and low (Hongmiao, HM) palatability were compared. The distinctive differences in amino acid composition, molecular structure, physicochemical properties were found in PRs and PCs, especially for HM variety. HM-PR recorded the lowest hydrophobic amino acids and surface hydrophobicity while having the superior hydration properties. The lowest denaturation temperature was found in HM-PR, which also had the highest denaturation enthalpy (ΔH). Nevertheless, HM-PR exhibited irregularly spherical protein body with the largest mean diameter. Evidenced by the highest random coil and lower α-helix and ß-sheet content, a less stable secondary structure of HM-PR was found, corresponding to the most intensified disulfide cross-linking and protein aggregations in HM upon cooking. Overall, HM-PR was presumed to greatly affect the hydro-thermal utilization efficiency of starch granules during cooking, given the steric-hindrance effect of prolamins on granules in endosperm. The Present study provided new insights into the role of prolamins on foxtail millet palatability.

Isolation of Oat (Avena sativa L.) Total Proteins and Their Prolamin Fractions for 2D Electrophoresis.

Appropriate sample preparation is essential to obtaining good results of two-dimensional gel electrophoresis (2-DE). For various reasons (particularly phenolic compounds, proteolytic enzymes, and cell-wall mucilages) the extraction of proteins from plant material, among them oat proteins, is difficult. During isolation all soluble substances that may interfere with the analysis (especially isoelectric focusing) are removed, and proteins of interest are separated from the remains. However, the applied procedure of isolation cannot be too extensive, because additional stages cause loss of the proteins.In this chapter, we describe a simple procedure for the isolation of oat total proteins and their prolamin fractions prior to 2-DE, without necessity of considerable purification. It can be used for oat protein fractionation, measurement of oat protein concentration, and their 2-DE analysis, with particular reference to prolamin fractions. The presented routine includes modified methods of plant seed proteins extraction and sequential Osborne extraction, based on oat protein solubility differences.

Formulation of Eco-friendly Medicated Chewing Gum to Prevent Motion Sickness.

An attempt was made to formulate medicated chewing gum to prevent motion sickness using natural gum base for faster onset of action and easy administration, anywhere and anytime, without access to water. To avoid the discard issue of gum cud, natural gum base of Triticum aestivum (wheat grain) was explored because of its biodegradable and biocompatible nature and easy availability. Prolamin, extracted from wheat, showed good chewing capacity, elasticity, high water retention capacity, antifungal activity, and compatibility with the drug. Formulations were prepared based on a two-factor and three-level factorial design. Amount of calcium carbonate (texturizer) and gum base were selected as independent variables. Elasticity and drug release were considered as the dependent variables. All batches were evaluated for the content uniformity, elasticity study, texture study, in vitro drug release study, and chewiness study. Results revealed that medicated chewing gum containing 80 mg of calcium carbonate and 500 mg of gum base showed good elasticity and more than 90% drug release within 16 min. Thus, this study suggested that both good elasticity and chew ability and abundant availability of wheat grain can act as a potential gum base for medicated chewing gum.

Purification of barley dimeric α-amylase inhibitor-1 (BDAI-1) and avenin-like protein-a (ALP) from beer and their impact on beer foam stability.

Foam stability is a key factor of beer quality for consumers and brewers. Recent beer proteome analyses have suggested that barley dimeric α-amylase inhibitor-1 (BDAI-1) and avenin-like protein-a (ALP) derived from barley are important for beer foam stability. In this study, BDAI-1 and ALP were purified from a Japanese commercial beer sample using salt precipitation and column chromatography. The purification level was verified using two-dimensional gel electrophoresis, mass spectrometry, and database searches. Purified BDAI-1 and ALP were added to a beer sample to compare the foam stability to that of a control beer sample. As a result, beer foam stability was significantly improved by BDAI-1 but not by ALP, thereby suggesting that BDAI-1 affects beer foam stability whereas ALP does not.

Accuracy of ELISA detection methods for gluten and reference materials: a realistic assessment.

The determination of prolamins by ELISA and subsequent conversion of the resulting concentration to gluten content in food appears to be a comparatively simple and straightforward process with which many laboratories have years-long experience. At the end of the process, a value of gluten, expressed in mg/kg or ppm, is obtained. This value often is the basis for the decision if a product can be labeled gluten-free or not. On the basis of currently available scientific information, the accuracy of the obtained values with commonly used commercial ELISA kits has to be questioned. Although recently several multilaboratory studies have been conducted in an attempt to emphasize and ensure the accuracy of the results, data suggest that it was the precision of these assays, not the accuracy, that was confirmed because some of the underlying assumptions for calculating the gluten content lack scientific data support as well as appropriate reference materials for comparison. This paper discusses the issues of gluten determination and quantification with respect to antibody specificity, extraction procedures, reference materials, and their commutability.

Amino acid profiles and quality from lotus seed proteins.

BACKGROUND: Protein composition, amino acid profile and nutritional value of the lotus seed and its Osborne fractions were investigated. The seed was rich in protein with 19.85%, and showed well balanced amino acid composition compared with FAO/WHO pattern, Its nutritive properties were similar to those observed in the reference soybean protein. Phenylalanine, tyrosine, leucine and lysine were the limiting amino acids in the seed proteins. The albumin and globulin were the main protein fraction, the amino acid profile and nutritional value were close to the seed protein. RESULTS: Changes in transition temperature and thermal stability were observed through different solvent extractions. Albumin possessed the predominant thermal stability (81.4 °C) followed by globulin (74.49 °C), prolamin (69 °C) and glutelin (65.6 °C). So, solvent compositions influence the profile of AAs and their nutritive value, and aqueous solvent with 0.1 mol L⁻¹ NaCl was an efficient protein solubiliser. CONCLUSION: The results indicated that the extraction processes influenced the lotus seed protein quality and thermal stability. Overall, the study revealed that the lotus seed protein was nutritionally well-balanced protein and might be of significant importance in the formulation of diets for humans.

Separation and identification of rice prolamins by two-dimensional gel electrophoresis and amino acid sequencing.

There are difficulties in detecting and separating rice prolamin polypeptides by 2D-PAGE analysis because prolamin polypeptides are insoluble, and the amino acid sequences show high homology among them. In this study, we improved the prolamin extraction method and the 2D-PAGE procedure, and succeeded in separating prolamin polypeptide species by 2D-PAGE and in identifying major prolamin polypeptide sequences.

What is in a beer? Proteomic characterization and relative quantification of hordein (gluten) in beer.

The suite of prolamin proteins present in barley flour was characterized in this study, in which we provide spectral evidence for 3 previously characterized prolamins, 8 prolamins with only transcript evidence, and 19 genome-derived predicted prolamins. An additional 9 prolamins were identified by searching the complete spectral set against an unannotated translated EST database. Analyses of wort, the liquid extracted from the mashing process during beer production, and beer were undertaken and a similar suite of prolamins were identified. We have demonstrated by using tandem mass spectrometry that hordeins are indeed present in beer despite speculation to the contrary. Multiple reaction monitoring (MRM) mass spectrometry was used for the rapid analyses of hordein in barley (Hordeum vulgare L.) beer. A selection of international beers were analyzed and compared to the results obtained with hordein deletion beers. The hordein deletion beers were brewed from grains carrying mutations that prevented the accumulation of either B-hordeins (Risø 56) or C-hordeins (Risø 1508). No intact C-hordeins were detected in beer, although fragments of C-hordeins were present in wort. Multiple reaction monitoring analysis of non-barley based gluten (hordein)-free beers targeting the major hordein protein families was performed and confirmed the absence of hordein in several gluten-free commercial beers.

Preparation and characterization of enzymatically hydrolyzed prolamins from wheat, rye, and barley as references for the immunochemical quantitation of partially hydrolyzed gluten.

Celiac disease (CD) is a permanent gastrointestinal disorder characterized by the intolerance to a group of proteins called gluten present in wheat, rye, barley, and possibly oats. The only therapy is a strict lifelong gluten-free diet. The standard method for gluten determination in foods produced for CD patients is the R5-enzyme-linked immunosorbent assay (ELISA) as proposed by the recent Codex Alimentarius Draft Revised Standard. This test is based on the determination of prolamins, the alcohol-soluble proteins of gluten, and is available as a sandwich ELISA for intact proteins and as a competitive ELISA for gluten-derived peptides. While the suitability of the sandwich ELISA including a wheat prolamin (gliadin) reference for calibration has been shown by various studies and a ring test, the competitive ELISA still lacks a convenient reference for the quantitation of gluten peptides in fermented cereal foods (e.g., sourdough products, starch syrup, malt extracts, beer). Therefore, the aim of the present study was to prepare a suitable reference for the quantitation of partially hydrolyzed gluten in fermented wheat, rye, and barley products. The prolamin fractions from barley (hordein) and rye (secalin) were isolated from corresponding flours by means of a modified preparative Osborne fractionation. The prolamin fraction from wheat was obtained as reference gliadin from the Prolamin Working Group. The prolamin fractions were successively digested by pepsin and trypsin or pepsin and chymotrypsin procedures, which have been used for CD-specific toxicity tests on cereal storage proteins for many years. The protein/peptide content (N x 5.7) of the prolamin fractions and digests, which was the basis for the calculation of the gluten content by means of ELISA, varied between 67.1% and 96.0%. The prolamin fractions and enzymatic digests were then tested for their response in both sandwich and competitive assays. Intact prolamins responded similarly in both ELISA showing no important differences between the cereals. In the case of digested proteins, however, the sandwich ELISA was considerably less sensitive than the competitive ELISA. The former provided approximately 40% and the latter 70% of the signal intensity obtained with the intact prolamins. Thus, the combination of the competitive ELISA and the enzymatic digests of prolamin fractions as reference was considered to be an adequate system for the analysis of partially hydrolyzed gluten. The limit of detection using a peptic-tryptic hordein digest as reference was 2.3 microg prolamin equivalent per milliliter, and the limit of quantitation was 6.7 microg prolamin equivalent per milliliter. This system was applied for the determination of gluten equivalents in five commercial beverages based on fermented cereals.

Processing scale-up of sicklepod (Senna obtusifolia L.) seed.

Sicklepod (Senna obtusifolia L.) is an invasive weed species especially of soybean and other field crops in the southeastern United States. The seeds contain a small amount (5-7%) of a highly colored fat as well as various phenolics, proteins, and galactomannans. The color of sicklepod seed oil is such that the presence of a small amount of the weed seed in a soybean crush lowers the quality of the soybean oil. Sicklepod is very prolific, and even volunteer stands yield >1000 lb of seed per acre, and prudence calls for tapping the potential of this weed as an alternative economic crop in the affected region. Pursuant to this, we have shown in laboratory-scale work the feasibility of separating the components of sicklepod seed. However, at kilogram and higher processing quantities, difficulties arise leading to modification of the earlier approach in order to efficiently separate components of the defatted seed meal. In a version for cleanly separating the proteins, the defatted meal was extracted with 0.5 M NaCl solution to remove globular proteins. Prolamins were extracted from the pellet left after salt extraction using 80% ethanol, and glutelins were then obtained in 0.1 N alkali from the residual solids left from ethanol treatment. In a pilot-scale version for water-soluble polysaccharides, the defatted meal was stirred with deionized water (DI) and centrifuged. The pooled centrifugates were heated to 92 degrees C (20-25 min), filtered, cooled to room temperature, and passed through a column of Amberlite XAD-4 to separate the polysaccharides from the anthraquinones. Senna obtusifolia L. is a one-stop-shop of a seed (from food components to medicinals).

Diversity of monomeric prolamins in triticale cultivars determined by capillary zone electrophoresis.

Capillary zone electrophoretic (CZE) analysis of monomeric prolamins (wheat gliadins and rye secalins) covered 28 hexaploid triticale ( Triticosecale x Wittm.) cultivars. The ethanol-soluble proteins were separated on an uncoated fused-silica capillary using the isoelectric 60 mM iminodiacetic (IDA) buffer in conjunction with 20% (v/v) acetonitrile and 0.075% (w/v) polyvinylpyrrolidone (PVP). For each separation, dynamic coating of the capillary wall with a buffer containing 0.1 M IDA and 0.05% (w/v) hydroxypropylmethylcellulose (HPMC) was performed. Separations of prolamins provided very good resolution and high reproducibility (<0.8% RSD). Prolamin profiles of all analyzed cultivars showed both qualitative and quantitative differences, including number of peaks, presence or absence of peaks, and area of peaks. The number of prolamin peaks detected in particular triticale cultivars varied from 22 to 28; in total, 56 components were distinguished. The CZE electropherograms of prolamins showed five main groups of protein peaks, in order of mobility alpha-prolamins, beta-prolamins, gamma-prolamins, omega1-prolamins, and omega2-prolamins, with migration times of 6.8-7.7, 7.8-10.4, 10.5-12.2, 12.3-17.4, and 17.5-25.6 min, respectively. Triticale seeds in comparison with wheat contained fewer alpha-prolamins and higher quantity of omega-prolamins. Hierarchical clustering of the investigated cultivars was based on Bhattacharyya distances calculated from the CZE data. The cultivars grouped in four main clusters. The obtained CZE results were compared with A-PAGE data.