Proteins from Blackberry Seeds: Extraction, Osborne Isolate, Characteristics, Functional Properties, and Bioactivities.

Blackberry fruit contains high levels of nutrients and phenolic compounds. Blackberry pomace accounts for 20~30% of its whole fruit during processing and is generally treated as fertilizer. Blackberry pomace has many seeds that contain carbohydrates, polyphenols, flavonoids, pectin, protein, and other bioactive nutrients. However, its functional properties and seed protein compositions have not been reported. We used a single-factor experiment, response surface, and Osborne isolate method to extract protein isolate, albumin, globulin, glutelin, and prolamin from blackberry seeds for the first time and evaluated their characteristics and functional properties. Glutelin and protein isolate showed good water-holding capacity, emulsification, and foaming capacity, while albumin and globulin showed good oil-holding capacity and thermal stability. They were found to have good antioxidant activities that might be good DPPH free radical scavengers, especially prolamin, which has the lowest IC50 value (15.76 µg/mL). Moreover, globulin had the lowest IC50 value of 5.03 µg/mL against Hela cells, 31.82 µg/mL against HepG2 cells, and 77.81 µg/mL against MCF-7 cells and a high selectivity index (SI), which suggested globulin had better anti-cervical, antihepatoma, and anti-breast activity but relatively low cytotoxicity. These seed proteins may have great prospects for the development and application of food and drugs in the future.

Flexible processing technology of coix seed prolamins by combined heat-ultrasound: Effects on their enzymatic hydrolysis characteristics and the hypoglycemic activities of derived peptides.

The self-assembled structures of coix seeds affected the enzymatic efficiency and doesn't facilitate the release of more active peptides. The influence of heating combined with ultrasound pretreatment (HT + US) on the structure, enzymatic properties and hydrolysates (CHPs) of coix seed prolamin was investigated. Results showed that the structural of coix seed prolamins has changed after HT + US, including increased surface hydrophobicity, reduced α-helix and random coil content, and a decrease in particle size. So that, leads to changes in thermodynamic parameters such as an increase in the reaction rate constant and a decrease in activation energy, enthalpy and enthalpy. The fractions of <1000 Da, degree of hydrolysis and α-glucosidase inhibitory were increased in the HT + US group compared to single pretreatment by 0.68%-17.34%, 12.69%-34.43% and 30.00%-53.46%. The peptide content and α-glucosidase inhibitory activity of CHPs could be maintained at 72.21 % and 57.97 % of the initial raw materials after in vitro digestion. Thus, the findings indicate that HT + US provides a feasible and efficient approach to can effectively enhance the enzymatic hydrolysis efficiency and hypoglycaemic efficacy of CHPs.

Characterization of rye flours and their potential as reference material for gluten analysis.

The safety of gluten-free products relies on accurate gluten analysis, most commonly using ELISA. These test kits are calibrated to gliadins or wheat gluten, because there is no reference material (RM) for rye. Our aim was to select representative samples out of 32 rye cultivars for use as RM. All cultivars were characterized by RP-HPLC, gel permeation HPLC and R5 and G12 ELISA. The protein and gluten content ranged from 5.5 to 11.2 g/100 g and 3.0 to 7.8 g/100 g, respectively. The average protein distribution was 40% albumins/globulins, 23% γ-75k-secalins, 17% γ-40k-secalins, 14% ω-secalins and 6% high-molecular-weight-secalins. The mean prolamin/glutelin ratio was 4.4 for rye and this translates to an estimated conversion factor from rye prolamins to gluten of 1.2, instead of the usual factor of 2. Seven cultivars were selected for RM production based on cluster analysis, geographical origin and availability to comprehensively cover the diversity of rye.

Purified Clinoptilolite-Tuff as an Efficient Sorbent for Gluten Derived from Food.

Various gluten-related diseases (celiac disease, wheat allergy, gluten sensitivity) are known and their incidence is growing. Gluten is a specific type of plant storage protein that can impair the health of gluten-prone persons following consumption, depending on the origin. The most severe effects are induced by wheat, barley, and rye. The only treatment is based on the absolute avoidance of those foods, as even traces might have severe effects on human well-being. With the goal of binding gluten impurities after ingestion, an in vitro setting was created. A special processed kind of zeolite, purified clinoptilolite-tuff (PCT), was implemented as an adsorber of gluten derived from different origins. Zeolites are known for their excellent sorption capacities and their applications in humans and animals have been studied for a long time. Tests were also performed in artificial gastric and intestinal fluids, and the adsorption capacity was determined via a certified validated method (ELISA). Depending on the kind of gluten source, 80-130 µg/mg of gluten were bound onto PCT. Hence, purified clinoptilolite-tuff, which was successfully tested for wheat, barley, and rye, proved to be suitable for the adsorption of gluten originating from different kinds of crops. This result might form the basis for an expedient human study in the future.

Quinoa protein: Composition, structure and functional properties.

Quinoa (Chenopodium quinoa willd.) is an annual herbaceous flowering plant showing appropriate nutritional and functional properties due to its high quality protein with a wide amino acid spectrum, particularly rich in lysine. The mature quinoa seed predominantly consists of 11S-type globulin called chenopodin, comprising about 37% of the total protein, and also 2S albumin accounting for 35% of the seed protein both stabilized through disulfide bridges. Moreover, quinoa seed contains low concentration of prolamins (0.5-7% of total protein) making it suitable for patients with celiac disease. Different enzymatic, chemical and physical modification methods also can influence the structural and finally nutritional and functional properties of protein isolate. Consequently, considering appropriate nutritional and functional properties of quinoa protein, it can be considered as a good candidate to supply human food products.

Speciation of Selenium in Brown Rice Fertilized with Selenite and Effects of Selenium Fertilization on Rice Proteins.

Foliar Selenium (Se) fertilizer has been widely used to accumulate Se in rice to a level that meets the adequate intake level. The Se content in brown rice (Oryza sativa L.) was increased in a dose-dependent manner by the foliar application of sodium selenite as a fertilizer at concentrations of 25, 50, 75, and 100 g Se/ha. Selenite was mainly transformed to organic Se, that is, selenomethionine in rice. Beyond the metabolic capacity of Se in rice, inorganic Se also appeared. In addition, four extractable protein fractions in brown rice were analyzed for Se concentration. The Se concentrations in the glutelin and albumin fractions saturated with increasing Se concentration in the fertilizer compared with those in the globulin and prolamin fractions. The structural analyses by fluorescence spectroscopy, Fourier transform infrared spectrometry, and differential scanning calorimetry suggest that the secondary structure and thermostability of glutelin were altered by the Se treatments. These alterations could be due to the replacements of cysteine and methionine to selenocysteine and selenomethionine, respectively. These findings indicate that foliar fertilization of Se was effective in not only transforming inorganic Se to low-molecular-weight selenometabolites such as selenoamino acids, but also incorporating Se into general rice proteins, such as albumin, globulin glutelin, and prolamin, as selenocysteine and selenomethionine in place of cysteine and methionine, respectively.

Rheological characterisation of gluten from triticale (x Triticosecale Wittmack).

BACKGROUND: Triticale gluten still remains very poorly characterised rheologically. In this study the mechanical spectra of gluten isolated from four triticale cultivars were registered and fitted with Cole-Cole functions yielding the visco-elastic plateau parameters. Master spectra were calculated. A retardation test was performed and used to calculate the composite mechanical spectra and the width of visco-elastic plateau l. Protein fractional composition of triticale flour and gluten was studied using capillary zone electrophoresis. RESULTS: Differentiated HMW-GS/SS compositions were identified in the triticale cultivars studied. The rheological parameters reached the following values: JN0 1.05·10-3 to 2.69·10-3 Pa-1 , GN0 372 to 956 Pa, ω0 0.003 to 0.06 rad s-1 , l 169 to 3121, Je0 1.57·10-3 to 5.03·10-3 Pa-1 , Ge0 199 to 637 Pa and η0 1.06·107 to 3.93·107 Pa s. CONCLUSIONS: Visco-elastic properties of triticale gluten correspond to the lower end of medium visco-elasticity shown by common wheat gluten. Master spectra and the composite mechanical spectra prove that four triticale glutens exhibit practically an identical type of visco-elastic behaviour of a biopolymeric visco-elastic liquid similar to wheat gluten. The visco-elastic plateau parameters GN0 , JN0 , ω0 and l appeared significantly correlated with the contents of prolamins and secaloglutenins in triticale flours and glutens. © 2017 Society of Chemical Industry.

Assessment of Avenins from Different Oat Varieties Using R5-Based Sandwich ELISA.

Gluten derived from wheat and related triticeae cereals possesses distinct amino acid sequences that provoke the immunopathogenic features of celiac disease (CD) in genetically susceptible individuals. However, the role of oat-derived gluten, or avenins, in CD pathogenesis remains a disputed matter, as evidenced by a lack in harmonized legislation regarding gluten classification in relation to gluten-free labeling. In this study, we have analyzed a panel of pure oat cultivars using a sandwich ELISA based on the R5 monoclonal antibody (mAb), which binds to canonical epitopes occurring within celiagenic peptides present in triticeae-derived gluten but reportedly not present in avenins. We have identified three varieties of oats that reproducibly bind R5 antibodies and levels indicating the presence of gluten at more than the 20 ppm gluten regulatory threshold. Nested assessment using Western blot analysis and alternative gluten detection systems corroborated these results. Collectively, these data suggest that select oat varieties may prove problematic to patients with CD and to food companies and regulatory agencies and will extend our basic understanding of current gluten detection systems.

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.

2-DE Separation and Identification of Oat (Avena sativa L.) Proteins and Their Prolamin Fractions.

At present two-dimensional polyacrylamide gel electrophoresis (2-DE) is the most widely used proteomic tool, which enables simultaneous separation of even thousands of proteins with a high degree of resolution. The quality of 2-DE separation depends on the type of biological material used as a protein source. The presence of interfering compounds (e.g., phenols, as it is the fact in plant material including oat seeds) impedes 2-DE run. With the use of this technique it is possible to analyze the complex protein mixtures, characteristic protein fractions, as well as individual proteins.The purpose of this chapter is to describe the 2-DE technique (the separate stages of the first and the second dimension) for determining the oat protein composition (oat seed proteome), separation and preliminary identification of oat prolamin fractions. Electrophoretically separated proteins are identified on the basis of pI markers (identifying the location of both ends of an IPG strip) and on 2D SDS-PAGE standards. The gel images of oat proteins are analyzed with the help of ImageMaster 2D Platinum 6.0 program (Amersham Bioscience, part of GE Healthcare, Uppsala, Sweden). It allows finding unique spot identifiers for the occurrence of oat prolamin fractions in oat total proteins. The characteristic spots of similar shape and intensity (anchoring spots) and characteristic groups of spots can be searched for the purpose of identification.

Comparative analysis of prolamin and glutelin fractions from wheat, rye, and barley with five sandwich ELISA test kits.

The safety of gluten-free foods is essential for celiac disease (CD) patients to prevent serious complications. Enzyme-linked immunosorbent assays (ELISAs) are recommended for gluten analysis to monitor the compliance of gluten-free products to the Codex threshold of 20 mg gluten/kg. However, due to the specific features of each gluten ELISA test kit, the results often deviate systematically and largely depend on the characteristics of the antibody. This comprehensive study assessed the specificities and sensitivities of three monoclonal (R5, G12, and Skerritt) and two polyclonal antibodies to the alcohol-soluble prolamin and alcohol-insoluble glutelin fractions of gluten from wheat, rye, and barley, all of which harbor CD-active epitopes. Reversed-phase high-performance liquid chromatography served as independent reference method to quantify gluten protein concentrations and allow comparisons of different gluten fractions within one kit and between kits. Wheat prolamins were detected quite accurately by all antibodies, but high variability between antibody specificities and sensitivities was observed for rye and barley prolamins and rye glutelins, and the largest discrepancies were found for wheat and barley glutelins. The gluten content (sum of prolamins and glutelins) was either overestimated up to six times (rye) or underestimated up to seven times (barley). Overestimation of gluten contents may unnecessarily limit the availability of gluten-free products, but underestimation represents a serious health risk for CD patients. It is important to consider these differences between antibodies used in kits and consider what each kit is capable of measuring, especially with samples where the source of gluten is unknown.

Oat raw materials and bakery products - amino acid composition and celiac immunoreactivity.

BACKGROUND: The aim of this study was to compare the biochemical and immunochemical properties of avenins in some special oat raw materials and additionally the possibility of using them as a raw material for the gluten-free bakery products. METHODS: The compared oat raw materials were - oat flakes, commercial oat flours (including gluten-free oat flour) and residual oat flour, which is by-product of ß-glucan preparation. Biochemical characteristic included amino acid compositions and SDS-PAGE profiles of extracted avenins. The immunochemical reactivity with polyclonal anti-gluten and monoclonal anti-gliadin antibodies was evaluated qualitatively and quantitatively by immunoblotting and ELISA methods. Additionally, experimental bakery products made of examined raw materials were assessed according to their suitability for the celiac patients' diet. RESULTS: The highest protein content was measured in the ß-glucan preparation "Betaven" and gluten-free oat flour. Proteins of all materials are rich in glutamic and aspartic acid, leucine and arginine. Proportions of amino acids in avenins extracted from most of oat raw materials are similar, excluding gluten-free oat flour, which has a very low avenin content and proportions of individual amino acids are different. The SDS-PAGE protein pattern consisted of proteins with molecular weight of about 25-35 kDa. Polyclonal anti-gluten anti-body recognized all protein fractions of molecular weight higher than 20 kDa. Quantitative ELISA analysis shows that the majority of samples has a gliadin-like protein content within the range of 80-260 mg/kg, excluding gluten-free flours and corresponding bakery products. Altogether, ß-glucan preparation has extremely high level of gliadin-like proteins. CONCLUSIONS: In the examined oat raw materials and foods the contents of immunoreactive amino acid sequences exceeded the limit of 20 mg/kg (considered as gluten-free) except for gluten-free flours (oat and  the prepared mixture) and the bakery products based on gluten-free flours. Unfortunately, the rest of oat raw materials and products cannot be considered gluten-free.

Targeted modification of storage protein content resulting in improved amino acid composition of barley grain.

C-hordein in barley and ω-gliadins in wheat are members of the prolamins protein families. Prolamins are the major component of cereal storage proteins and composed of non-essential amino acids (AA) such as proline and glutamine therefore have low nutritional value. Using double stranded RNAi silencing technology directed towards C-hordein we obtained transgenic barley lines with up to 94.7% reduction in the levels of C-hordein protein relative to the parental line. The composition of the prolamin fraction of the barley parental line cv. Golden Promise was resolved using SDS-PAGE electrophoresis, the protein band were excised and the proteins identified by quadrupole-time-of-flight mass spectrometry. Subsequent SDS-PAGE separation and analysis of the prolamin fraction of the transgenic lines revealed a reduction in the amounts of C-hordeins and increases in the content of other hordein family members. Analysis of the AA composition of the transgenic lines showed that the level of essential amino acids increased with a concomitant reduction in proline and glutamine. Both the barley C-hordein and wheat ω-gliadin genes proved successful for RNAi-gene mediated suppression of barley C-hordein level. All transgenic lines that exhibited a reduction for C-hordein showed off-target effects: the lines exhibited increased level of B/γ-hordein while D-hordein level was reduced. Furthermore, the multicopy insertions correlated negatively with silencing.

Multiplex liquid chromatography-tandem mass spectrometry for the detection of wheat, oat, barley and rye prolamins towards the assessment of gluten-free product safety.

Celiac patients should feel confident in the safety of foods labelled or expected to be gluten-free. In this context, a targeted proteomic approach based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) technique was proposed to assess the presence of celiotoxic cereals, namely wheat, oats, barley and rye, in raw and processed food products. To this aim, unique marker peptides were properly selected in order to distinguish between the different cereal types. A revised cocktail solution based on reducing and denaturing agents was exploited for prolamin extraction from raw and processed food; in addition, defatting with hexane was carried out for sample clean-up, allowing to largely reduce problems related to matrix effect. Method validation on fortified rice flour showed good analytical performance in terms of sensitivity (limits of detection in the 2-18 mg kg(-1) range). However, poor trueness was calculated for self-made incurred bread (between 3 and 30% depending on the peptide), probably due to baking processes, which reduce gluten extractability. Thus, it is evident that in the case of processed foods further insights into sample treatment efficiency and reference materials for protein calibration are required to obtain accurate gluten determination. Finally, the developed method was applied for the analysis of market food products, offering the possibility to discriminate among cereals, with good agreement with labelled ingredients for gluten-containing foodstuffs.

Immunological characterization of the gluten fractions and their hydrolysates from wheat, rye and barley.

Gluten proteins in wheat, rye and barley cause celiac disease, an autoimmune disorder of the small intestine, which affects approximately 1% of the world population. Gluten is comprised of prolamin and glutelin. Since avoidance of dietary gluten is the only option for celiac patients, a sensitive gluten detection and quantitation method is warranted. Most regulatory agencies have set a threshold of 20 ppm gluten in foods labeled gluten-free, based on the currently available ELISA methods. However, these methods may exhibit differences in gluten quantitation from different gluten-containing grains. In this study, prolamin and glutelin fractions were isolated from wheat, rye, barley, oats and corn. Intact and pepsin-trypsin (PT)-digested prolamin and glutelin fractions were used to assess their immunoreactivity and gluten recovery by three sandwich and two competitive ELISA kits. The Western blots revealed varied affinity of ELISA antibodies to gluten-containing grain proteins and no reactivity to oat and corn proteins. ELISA results showed considerable variation in gluten recoveries from both intact and PT-digested gluten fractions among different kits. Prolamin fractions showed higher gluten recovery compared to their respective glutelin fractions. Among prolamins, barley exhibited higher recovery compared to wheat and rye with most of the ELISA kits used. Hydrolysis resulted in reduced gluten recovery of most gluten fractions. These results suggest that the suitability of ELISA for accurate gluten quantitation is dependent upon various factors, such as grain source, antibody specificity, gluten proteins and the level of their hydrolysis in foods.

Effect of rising atmospheric carbon dioxide concentration on the protein composition of cereal grain.

The present study investigates effects of rising atmospheric CO2 concentration on protein composition of maize, wheat, and barley grain, especially on the fractions prolamins and glutelins. Cereals were grown at different atmospheric CO2 concentrations to simulate future climate conditions. Influences of two nitrogen fertilization levels were studied for wheat and barley. Enriched CO2 caused an increase of globulin and B-hordein of barley. In maize, the content of globulin, α-zein, and LMW polymers decreased, whereas total glutelin, zein, δ-zein, and HMW polymers rose. Different N supplies resulted in variations of barley subfractions and wheat globulin. Other environmental influences showed effects on the content of nearly all fractions and subfractions. Variations in starch-protein bodies caused by different CO2 treatments could be visualized by scanning electron microscopy. In conclusion, climate change would have impacts on structural composition of proteins and, consequently, on the nutritional value of cereals.

Technical note: a modified method to quantify prolamin proteins in dry and high-moisture corn.

Numerous studies have evaluated laboratory methods to quantify prolamin proteins in dry corn; however, the utility of methods to quantify functional prolamins, which impede starch digestibility, in high-moisture corn (HMC) is less defined. As a result, a common rapid turbidimetric (rTM) laboratory procedure was modified (rapid Bradford method, rBM), extracting buffer-soluble proteins before prolamin solubilization in an effort to better quantify functional prolamins in HMC. Twenty samples of dry and HMC were evaluated by rTM and rBM procedures. Prolamin concentration in dry corn, as estimated by rTM or rBM methods, ranged from 6.12 to 2.20g/100 g of DM or 5.46 to 2.41g/100 g of DM, respectively. Dry corn mean prolamin concentrations, as estimated by rTM or rBM methods, were similar at 3.65 or 3.66g/100 g of DM. Prolamin concentration in HMC, as estimated by rTM, ranged from 4.99 to 3.24g/100 g of DM, with a mean prolamin concentration of 4.19g/100 g of DM, but estimation of prolamins in HMC by the rBM method resulted in lower mean (4.19 vs. 3.24g/100 g of DM) prolamin concentration. Prolamin concentration in dry and HMC measured by rTM was negatively related to peak absolute rates (PAR; mL/0.1 g of DM) of in vitro gas production. However, relationships between rTM prolamin concentration and PAR were not homogeneous and were different between dry and HMC. Prolamin proteins as determined by rBM were likewise negatively related to PAR, but corn type did not influence rBM prolamin concentration by PAR relationships. Data suggest that the rBM method defined more similar functional prolamin proteins, which impede starch degradability, in dry and HMC.

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.

Gliadin as a measure of gluten in foods containing wheat, rye, and barley-enzyme immunoassay method based on a specific monoclonal antibody to the potentially celiac toxic amino acid prolamin sequences: collaborative study.

The Working Group on Prolamin Analysis and Toxicity (WGPAT) organized a collaborative study to confirm whether the two R5 antibody-based ELISA test kits are able to detect gliadin in the lower mg/kg (ppm) level. Twenty laboratories investigated 12 blind-coded samples, spiked and naturally contaminated, to show the possibility of determining traces of gliadin in heat-treated or nonheat-treated foods by ELISA. It was shown that very small amounts of gliadin (below 100 ppm) could be detected by ELISA with a reproducibility RSD(R) (37%) and a repeatability RSD, (27%) common for ELISA under these conditions. The recovery of gliadin from the spiked samples was between 84 and 109%, based on the results of all laboratories, including those with poor performance. No false positives were found by the method (P < or =0.05), but one negative sample was contaminated during the bakery process. It is recommended that the method be accepted by AOAC as Official First Action.