Hordein Fractions Research Articles

Characterization of gliadin, secalin and hordein fractions using analytical techniques

Prolamins, alcohol soluble storage proteins of the Triticeae tribe of Gramineae family, are known as gliadin, secalin and hordein in wheat, rye and barley respectively. Prolamins were extracted from fifteen cultivars using DuPont protocol to study their physiochemical, morphological and structural characteristics. SDS-PAGE of prolamins showed well resolved low molecular weight proteins with significant amount of albumin and globulin as cross-contaminant. The β-sheet (32.72–37.41%) and β-turn (30.36–37.91%) were found higher in gliadins, while α-helix (20.32–28.95%) and random coil (9.05–10.28%) in hordeins. The high colloidal stability as depicted by zeta-potential was observed in gliadins (23.5–27.0 mV) followed secalins (11.2–16.6 mV) and hordeins (4.1–7.8 mV). Surface morphology by SEM illustrated the globular particle arrangement in gliadins, sheet like arrangement in secalins and stacked flaky particle arrangement in hordeins fraction. TEM studies showed that secalin and hordein fractions were globular in shape while gliadins in addition to globular structure also possessed rod-shaped particle arrangement. XRD pattern of prolamin fractions showed the ordered crystalline domain at 2θ values of 44.1°, 37.8° and 10.4°. The extracted prolamins fractions showed amorphous as well as crystalline structures as revealed by XRD and TEM analysis. Space saving hexagonal molecular symmetry was also observed in TEM molecular arrangement of prolamins which has profound application in development of plant-based polymers and fibres.

Fusarium tricinctum associated with Head Blight on barley in Argentina: pathogenicity and potential degradation of the different hordein fractions

The objective of the present work was to evaluate the pathogenicity of Fusarium tricinctum in barley and to determine the potential effects on grain quality, more specifically regarding the hordein fractions. For this purpose, Koch´s postulates were carried out to evaluate the pathogenicity of F. tricinctum in barley, under greenhouse conditions. Furthermore, the ability of F. tricinctum to degrade hordeins was tested by a sequential extraction method, and separated by polyacrilamide gel electrophoresis (SDS-PAGE). Finally, the relative abundance of each hordein fractions (D, C, and B-hordeins) was measured. The results demonstrated that F. tricinctum was pathogenic when barley spikes were inoculated, also showing that F. tricinctum could degrade barley C-hordeins by 21.80% of the total C-hordein content.

Effects ofFusarium graminearumandFusarium poaeon disease parameters, grain quality and mycotoxin contamination in barley (part II)

Barley is one of the most sown crops in the world, with multiple uses such as human consumption, animal feed and for the malting industry. This crop is affected by different diseases, such as Fusarium Head Blight (FHB), that causes losses in yield and quality. In the last years F. graminearum and F. poae were two of the most frequently isolated species in barley grains, so the aim of this study was to evaluate the interaction between these Fusarium species and the effects on disease parameters, grain quality and mycotoxin contamination on five barley genotypes under field conditions. Statistical differences between Fusarium treatments for some parameters depending mainly on the year/genotype were found. The results showed that the germination process was affected by both Fusarium species. As to grain quality and the different hordein fractions, it was observed that F. graminearum affects preferentially D and C-hordeins. Different concentrations of nivalenol, deoxynivalenol and their acetylated derivatives (3-acetyl deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol (15-ADON)) were detected. In the present work, no evidence of synergism between F. graminearum and F. poae were found regarding disease parameters and mycotoxin contamination. However, at least in the years with favorable climatic conditions to FHB development and depending on the barley genotype, a continuous monitoring is deemed necessary to prevent the negative impact on protein composition and germinative parameters © 2020 Society of Chemical Industry.

Effects of Ultrasonic-Microwave-Assisted Technology on Hordein Extraction from Barley and Optimization of Process Parameters Using Response Surface Methodology

We investigated the process intensification of ultrasonic-microwave-assisted technology for hordein extraction from barley. Response surface methodology was utilized to optimize the extraction conditions and to analyze the interaction between four selected variables: temperature, microwave power, ultrasonic power, and extraction time. The validated extraction yield of hordein reached 8.84% at 78°C, microwave power 298 W, and ultrasonic power 690 W after 20 min as optimum conditions. Compared with traditional water-bath extraction (4.7%), the ultrasonic-microwave-assisted technology effectively increased the hordein extraction yield and shortened the extraction time. According to the obtained quadratic model (R2 = 0.9457), ultrasonic power and extraction time were the first two significant factors. However, temperature limited the effects of other factors during extraction. SDS-PAGE and scanning electron microscopy were used to identify the hordein extract and to clarify the difference between the two hordein fractions extracted with new and traditional methods, respectively. Ultrasonic-microwave-assisted technology provided a new way to improve hordein extraction yield from barley and could be a good candidate for industrial application of process intensification.

Antioxidant capacities of fractionated barley hordein hydrolysates in relation to peptide structures

This study has revealed strong antioxidant activities of barley hordein hydrolysate fractions and investigated the relationships between antioxidant properties and structural features of hordein peptides. Barley hordein fractions separated based on molecular weight and surface hydrophobicity demonstrated strong antioxidant bioactivities, especially ferrous ion chelating capacity. Structural studies revealed the poly-l-proline helix, β-turn, and β-sheet as the dominant structural elements in the large molecular weight fraction, while most of the small-sized peptides assumed unordered random coil conformation accompanied by a large amount of ionized carboxyl groups produced as a result of partial deamidation of glutamine residues during hydrolysis. A study of antioxidant capacity in relation to peptide structure indicated the contribution of hydrophobic residues in radical scavenging and the positive effect of ionized carboxyl groups together with Lys and Met in metal chelation activity. QPYPQ was identified as the most repeated sequence in potent RP-HPLC fractions, which might be a particular structural motif that plays a key role in scavenging free radicals. Barley hordein with its unique sequence partitioning has potential to produce peptides with strong antioxidant bioactivities.

Dissecting the T‐cell response to hordeins in coeliac disease can develop barley with reduced immunotoxicity

Wheat, rye and barley prolamins are toxic to patients with coeliac disease. Barley is diploid with pure inbred cultivars available, and is attractive for genetic approaches to detoxification. To identify barley hordein fractions which activated the interferon-γ (IFN-γ) secreting peripheral blood T-cells from coeliac volunteers, and compare immunotoxicity of hordeins from experimental barley lines. To reactivate a T-cell response to hordein, volunteers underwent a 3-day oral barley challenge. Peripheral blood mononuclear cells (PBMC) were isolated from twenty-one HLA DQ2(+) patients with confirmed coeliac disease. IFN-γ ELISpot assays enumerated T-cells activated by purified prolamins and positive controls. Hordein-specific T-cells were induced by oral barley challenge. All prolamin fractions were immunotoxic, but D- and C-hordeins were most active. Barley lines lacking B- and C-hordeins had a 5-fold reduced hordein-content, and immunotoxicity of hordein extracts were reduced 20-fold compared with wild-type barley. In vivo oral barley challenge offers a convenient and rapid approach to test the immunotoxicity of small amounts of purified hordeins using fresh T-cells from patients in high throughput overnight assays. Barley lines that did not accumulate B- and C-hordeins were viable, yet had substantially reduced immunotoxicity. Creation of hordein-free barley may therefore be possible.

Relationship Between Malting Quality Traits and Hordeins as Affected by Timing of Nitrogen Fertilizer Application

ABSTRACTThe application of nitrogen (N) fertilizer in malting barley is necessary to obtain good yield, but it also influences kernel protein content, which affects malting quality. The negative correlation between kernel protein content and malting extract is due to the hordein proteins. The aim of this study was to determine the effect of N application timing on hordein fractions and how this influences malt quality. Reversed‐phase high‐performance liquid chromatography (RP‐HPLC) was used to determine the hordein fractions in the two‐ and six‐row barley parents and their doubled‐haploid progeny, for two seasons for one location, and two locations in the second season. There were no changes in hordein level in response to timing of N application over years and locations. The two‐row parent had the lowest hordein level and the six‐row progeny had the highest. Total hordein content of the two‐row progeny and six‐row parent were similar. The supply of additional N in the form of fertilizer rather than the timing of all N applications influenced the production of all hordein fractions. There were significant correlations between hordein fractions and malting quality that also were reported in other studies. Kernel plumpness showed significant negative correlations with D, C, and B hordeins and total hordein content when half of the fertilizer was applied at planting and the other half at the six‐leaf stage. Absorption showed significant positive correlation with C hordeins and a negative correlation with the B‐to‐C ratio, also when the second half of the N was given at the six‐leaf stage.

Antioxidative properties of partially purified barley hordein, rice bran protein fractions and their hydrolysates

Antioxidative properties of proteins from barley and rice bran and their hydrolysates were examined. Three major hordein fractions of barley, B, C and D hordeins, were partially purified by gel filtration. Albumin, globulin, prolamin and glutelin fractions of rice bran were fractioned by the Osborne method. Hydrolysates of these protein fractions were prepared by digesting with pepsin followed by trypsin. Antioxidant properties in terms of antioxidative activity against linoleic acid peroxidation and reducing activity without the lipid adjuvant were investigated. The globulin fraction from rice bran protein revealed the strongest antioxidative activity throughout the incubation time of 7 days ( p ≤ 0.05). The albumin fraction of rice bran protein showed the highest reducing activity (6964 μmol of Fe 2+) followed by globulin, prolamin, glutelin and hordein fractions with activities of 2904, 2017, 1809 and 1333 μmol of Fe 2+, respectively ( p ≤ 0.05). Partially purified C hordein exhibited the highest reducing activity compared with B and D hordeins. Protein hydrolysates obtained after digestion with pepsin and trypsin exhibited much greater antioxidative, as well as reducing, activities than those from before digestion.

A nearly non-functional mutant allele of the storage protein locus Hor2 in barley

The low content of the storage protein fraction hordein-2 in the high-lysine mutant Risø 56 is due to a mutation at or near the locus Hor2 coding for hordein-2 polypeptides. The mutant gene is recessive in its qualitative effect on the electrophoretic banding pattern of hordein-2, but co-dominant with respect to the relative amount of the hordein fractions. Homozygous mutant seeds were about 10 % smaller than normal seeds. Nd linkage was detected between the mutant gene and the translocation between chromosomes 2 and 5 in mutant 56

Relationship between hordein proteins and malt quality in barley cultivars grown in Croatia

The hordein proteins of ten barley (Hordeum vulgare L.) cultivars grown in region of eastern Croatia were studied for their contribution to the malting quality, particularly relating hordein influence on malt extract yield. The analysis of hordein proteins was carried out by reversed-phase high-performance liquid chromatography (RP-HPLC). The micromalting of investigated barley cultivars and malt analyses were done according to the EBC official methods. The quantitative analysis of hordein proteins has showed that B-hordein was major component, followed by C- and D-hordeins. Among investigated cultivars the malt extract yield varied from 76.8% (cv. Angora) to 82.6% (cv. Scarlett). Spring barleys had on the average higher malt extract yield in contrast to winter barleys. The significant negative correlation between the total hordein content and malt extract yield was found. Among hordein fractions the B-hordein and D-hordein significantly contributed to lower malt extract yield. RP-HPLC analysis of malt hordeins revealed that the amount of hordein degraded during malting significantly correlated with Kolbach index.

Effects of microwave irradiation on ruminal dry matter, protein and starch degradation characteristics of barley grain

This study evaluated effects of microwave irradiation (800 W) for 3, 5 and 7 min on dry matter (DM), crude protein (CP) and starch degradation characteristics of barley grain and to monitor the fate of its proteins in the rumen. Duplicate in situ bags of untreated or irradiated barley grain were incubated in the rumen of four non-lactating Holstein cows for up to 48 h. Proteins of untreated and irradiated bag residues were fractionated by gel electrophoresis. Irradiation for 3 min had no effect, but for 5 and 7 min decreased (P<0.05) the effective DM degradability. The washout fraction, and the degradation rate of CP, decreased (P<0.05) and the potentially degradable CP fraction increased (P<0.05) as irradiation period increased. Effective CP degradability of barley irradiated for 3, 5 and 7 min decreased (P<0.05) by 6%, 10% and 13%, respectively. Microwave irradiation increased (P<0.05) the washout fraction and decreased (P<0.05) the potentially degradable fraction and degradation rate of starch. Irradiation for 5 min had no effect on effective starch degradability, but for 3 and 7 min increased, and decreased it (P<0.05), respectively. From electrophoretic analyses, three major hordein fractions (B-, C- and D-hordein) occurred. In untreated barley grain, most hordein fractions had disappeared after 6 h of incubation, but in microwave irradiated barley, C- and D-hordein were not degraded until 12 h of incubation in the rumen. Microwave irradiation of barley grain for 3 min increased its starch degradability and, over 5 min, decreased the rate and extent of CP and starch degradation.

Protein and hordein content in barley seeds as affected by nitrogen level and their relationship to beta-amylase activity

Two barley cultivars differing in grain size and protein content were used to investigate the effects of nitrogen nutrition, cultivar and their interaction on grain protein content, hordein content and beta-amylase activity and the relationship between hordein content and beta-amylase activity during in vitro spike culture. The content of protein and hordein fraction, and beta-amylase activity in barley grains increased as the nitrogen level in culture solution increased. Grain protein content was significantly affected by nitrogen treatment and cultivar, and there was no significant interaction between nitrogen treatment and cultivar. Hordein content and beta-amylase activity were significantly affected by nitrogen treatment and cultivar as well as their interaction. Beta-amylase activity was positively correlated with grain protein and hordein contents, and the ratio of hordein B:C was negatively correlated with total protein content and beta-amylase activity.

Protein and hordein fraction content in barley seeds as affected by sowing date and their relations to malting quality

The effect of sowing date on grain protein, hordein fraction content and malting quality of two-rowed spring barley was investigated by using ten commercial cultivars with different grain protein content and the relationships among these traits were examined. The results showed that grain protein content and B hordein content increased as the sowing date postponed and were significantly affected by sowing date, while C and D hordein contents were less influenced by sowing date. There were significant differences in grain protein and hordein fraction content among the ten cultivars. The coefficient of variation of D hordein content was much larger than that of B and C hordein contents, suggesting its greater variation caused by different sowing dates. Beta-amylase activity and diastatic power were also significantly affected by sowing date, with malt extract being less affected. Significant differences in measured malt quality were found among the ten cultivars. Grain protein was significantly correlated with B hordein and malt extract positively and negatively, respectively. There was no significant correlation between beta-amylase activity or diastatic power and grain protein content. B hordein was negatively and significantly correlated with malt extract, but no significant correlations between C hordein, D hordein and malting quality traits.

Cloning and molecular characterization of B-hordeins from Hordeum chilense (Roem. et Schult.)

One of the main limitations of cereal breeding is the lack of genetic variability within cultivated crops. Hordeum chilense is a wild relative of Hordeum vulgare, which has been successfully used in the synthesis of amphiploids by crossing with Triticum spp. Among the agronomic traits of these new amphiploids, the allelic variation in the endosperm storage proteins and their influence on breadmaking and malting quality are of special interest. B-hordeins are sulfur rich prolamins, which account for 70-80% of the total hordein fraction in barley. In this work, rapid amplification of cDNA ends by PCR (RACE-PCR) has been used for the cloning of the full-length open reading frame (ORF) of six sequences of B3-hordeins from two lines of H. chilense. Two consensus sequences of 813 and 822 bp for the H1 and H7 lines, respectively, were determined by alignment of all the sequences generated. Between both lines, differences involving single base changes, which could correspond to single nucleotide polymorphisms (SNP), insertions and deletions were observed. Of these differences, only six out of the 13 within the ORF caused a change of amino acid. Two insertions/deletions of 9 and 12 bp were also observed between both lines. The derived amino acid sequences showed a similar structure to the B-hordeins from cultivated barley and other prolamins. The repetitive region is based on the repetition of the motif PQQPFPQQ. The copy number of the B3-hordeins was estimated as a minimum of nine and five copies for the H1 and H7 lines, respectively. The expression profile of the B-hordeins through the developing endosperm is also described in this work. This study of the storage proteins of H. chilense is a useful contribution to the knowledge of the genetic diversity available in wild relatives of cultivated barley. In addition, the origin of the different prolamins can be better understood with an in-depth knowledge of its wild equivalent.

Malting Behaviour of Barleys Grown in Canada and Spain as Related to Hordein and Enzyme Content

To continue our effort to analyse the genetic (varietal) and environmental (sites and years) effects on malting quality of barley, we have field-tested four barley varieties, two- and six-rowed, European and North American, in Spain and Canada in 1998 and 1999. The Spanish trials were autumn-sown whereas the Canadian ones were spring-sown. Barley grain was analysed for total protein and hordein contents and micromalted. Canadian-grown barleys had significantly lower contents of grain protein and all-three hordein fractions than the Spanish ones. They also had lower malt respiratory losses, wort β-glucan and viscosity but lower fine- and coarse-ground malt extract yield, friability, free amino nitrogen, Kolbach index, α-amylase and diastatic power. In other words, the Canadian-grown barleys, despite showing lower protein and hordein contents, produced malt of inferior quality than their Spanish counterparts, which, overall, produced higher quantities of degrading enzymes (amylolytic, proteolytic and cytolytic) during germination, thus being able to attain higher extract yield levels.

Effect of different heating conditions on the extractability of barley hordeins.

The extractability of hordeins from barley grains was investigated after wet and dry heating conditions. It was found that the amount of hordeins extractable with 55% 2-propanol decreased in a time-dependent manner after barley grains were steamed (wet heating), whereas hordeins showed no effect from heating in an oven at 100 degress C for up to 120 min (dry heating). The result of SDS-PAGE analysis revealed that B-hordein decreased time-dependently in extractability with wet heating and had almost completely disappeared by 60 min, but C-hordein remained unchanged until 120 min. With the use of the hordein fraction prepared from the nonheated barley grains, it was confirmed that B-hordein suspended in boiling water lost solubility in 55% 2-propanol. The insolubilized B-hordein was redissolved by the addition of 2-mercaptoethanol to 1%, which suggested that the intermolecular disulfide bonds would play a significant role in the loss of solubility. On the other hand, C-hordein did not lose solubility from being heated under the same conditions.

Relationships Between Barley Hordeins and Malting Quality in a Mutant of cv. Triumph I. Genotype by Environment Interaction of Hordein Content

The genetic (G), environmental (E) and G×E effects on total grain protein and hordein fractions, have been studied in a mutant (TL43) and its parent cultivar, the malting barley Triumph, in various seasons at Dundee (E Scotland) and Lleida (NE Spain). The grain protein content of TL43 was consistently about 2% higher than that of Triumph across environments. Differences in hordein composition were shown by SDS-PAGE electrophoresis. TL-43 had three B-hordein bands not present in Triumph which had a further three bands of the same hordein group not present in TL43. TL43 showed higher B-hordein content than Triumph in Scotland but lower in Spain, whereas the mutant showed consistently higher C- and D-hordein content than Triumph across environments, i.e. there was crossover G×E interaction for B-hordein and non-crossover for C- and D-hordein content. There were also differences in grain ultra-structure between the two lines, as TL43 showed a more dense protein matrix than Triumph, together with thinner pericarp, testa and aleurone layers. It was concluded that the mutation in TL43 had a significant effect on storage protein composition.

Analysis of Anti-Prolamin Monoclonal Antibody Reactivity Using Prolamin Fractions Purified by Preparative Electrophoresis

Coeliac disease (CD) is a gastrointestinal afliction triggered by the ingestion of prolamins from wheat, barley, rye and possibly oats. The only treatment for CD is a strict diet, free of the toxic components. Immunochemical methods are usually applied to certify foods aimed at coeliac patients. The characterization of a panel of four anti-prolamin monoclonal antibodies (MAbs) to be used to certify gluten-free products is described here. To this aim, purified gliadin, secalin and hordein fractions were obtained by preparative electrophoresis at acid pH. This procedure provides purified fractions not exposed to denaturing conditions. The specificity of the MAbs was tested by ELISA against purified fractions and ethanol extracts of wheat, barley, rye, oats, rice, maize, buckwheat, sorghum and soy. The four MAbs recognized only coeliac-toxic cereals. Each MAb reacted strongly with gliadins and showed differential reactivity against the different prolamin purified fractions. Some MAbs showed a broad pattern of recognition whereas others presented a more restricted one. The reactivity observed corresponded to structural homologies among gliadin, secalin and hordein fractions. It is remarkable that some fractions obtained by electrophoresis in the presence of sodium dodecylsulphate were not recognized by some MAbs, whereas the same components obtained by preparative A-PAGE showed high reactivity. This reinforces the suitability of the purification method employed in this study to isolate prolamin fractions. Using these purified prolamins, characterization of anti-prolamin MAb reactivity was achieved.

Mechanisms of Malt Extract Development in Barleys from Different European Regions: II. Effect of Barley Hordein Fractions on Malt Extract Yield

To examine environmental differences between southern and northern European malting barleys, particularly relating to protein and hordein influences on malt extract development, samples of the malting barley cultivar Alexis, from the Nordic countries and from the Iberian Peninsula, were used. Fractionation of hordeins was carried out on barley grams, prior to micromalting and malt analyses, using high performance liquid chromatography (HPLC). Different mechanisms for extract development, in malting barleys from the North and South of Europe, have been demonstrated. Barley protein content determined a significantly greater decrease in malt extract in the Nordic samples, compared with those from the Iberian Peninsula, while apparent final attenuation showed a more pronounced positive association with extract in the South than in the North. B-hordein was associated with a significanlty greater decrease in malt extract in the Nordic compared to the Mediterranean samples.

The Relationship Between D Hordein and Malting Quality in Barley

The contribution of grain protein to the malting quality of barley (Hordeum vulgareL.) was investigated by comparing the hordein composition and the malting quality in barley produced under a range of field conditions. Two malting cultivars, Schooner and Arapiles, and one feed cultivar, Galleon, were grown under five nitrogen regimes in each of two years. Hordein composition of the grain was determined at maturity using a combination of sodium dodecyl sulphate-polacrylamide gel electrophoresis and laser densitometry. Malt extract was determined on all samples after micromalting. Variation in growth conditions resulted in a wide range of grain protein contents and malt extract values, as well as variation in the proportions of the individual B, C and D hordeins in the grain. D hordein in particular varied over a 10-fold range. High levels of all protein fractions were associated with low malt extract. Total protein, as expected, displayed a strong, negative correlation with malt extract. The relationship was cultivar specific and separate regression lines were generated for each cultivar. Of the individual hordein fractions, D hordein displayed the strongest negative correlation with malt extract and its relationship to malt extract was independent of cultivar. A consistent relationship between D hordein and malt extract was observed across seasons, treatments and cultivars that was indicative of a causal relationship between D hordein and malting quality. D hordein therefore offers an alternative measurement to total protein for the prediction of malting quality over a wide range of environmental conditions and cultivars.