β-Glucan Assay Kit (Mixed Linkage)

Procedures for the measurement of starch, starch damage (gelatinised starch), resistant starch and the amylose/amylopectin content of starch, β-glucan, fructan, glucomannan and galactosyl-sucrose oligosaccharides (raffinose, stachyose and verbascose) in plant material, animal feeds and foods are described. Most of these methods have been successfully subjected to interlaboratory evaluation. All methods are based on the use of enzymes either purified by conventional chromatography or produced using molecular biology techniques. Such methods allow specific, accurate and reliable quantification of a particular component. Problems in calculating the actual weight of galactosyl-sucrose oligosaccharides in test samples are discussed in detail.

A collaborative study was conducted involving 8 laboratories (including the authors’ laboratories) to validate the streamlined enzymatic method for determination of β-D-glucan in barley and oats. In the method, the flour sample is cooked to hydrate and gelatinize β-glucan, which is subsequently hydrolyzed to soluble fragments with the lichenase enzyme. After volume and pH adjustments and filtration, the solution is treated with β-glucosidase, which hydrolyzes β-gluco-oligosaccharides to D-Glucose. D-Glucose is measured with glucose oxidase–peroxidase reagent. Other portions of lichenase hydrolysate are treated directly with glucose oxidase-peroxidase reagent to measure free glucose in test sample. If levels of free glucose are high, the sample is extracted first with 80% ethanol. For all samples analyzed, the repeatability relative standard deviation (RSD_r) values ranged from 3.1 to 12.3% and the reproducibility relative standard deviation (RSD_r) values ranged from 6.6 to 12.3%. The streamlined enzymatic method for determination of β-D-glucan in barley and oats has been adopted first action by the AOAC INTERNATIONAL.

An American Association of Cereal Chemists/AOAC collaborative study was conducted to evaluate the accuracy and reliability of an enzyme assay kit procedure for measurement of total starch in a range of cereal grains and products. The flour sample is incubated at 95 degrees C with thermostable alpha-amylase to catalyze the hydrolysis of starch to maltodextrins, the pH of the slurry is adjusted, and the slurry is treated with a highly purified amyloglucosidase to quantitatively hydrolyze the dextrins to glucose. Glucose is measured with glucose oxidase-peroxidase reagent. Thirty-two collaborators were sent 16 homogeneous test samples as 8 blind duplicates. These samples included chicken feed pellets, white bread, green peas, high-amylose maize starch, white wheat flour, wheat starch, oat bran, and spaghetti. All samples were analyzed by the standard procedure as detailed above; 4 samples (high-amylose maize starch and wheat starch) were also analyzed by a method that requires the samples to be cooked first in dimethyl sulfoxide (DMSO). Relative standard deviations for repeatability (RSD(r)) ranged from 2.1 to 3.9%, and relative standard deviations for reproducibility (RSD(R)) ranged from 2.9 to 5.7%. The RSD(R) value for high amylose maize starch analyzed by the standard (non-DMSO) procedure was 5.7%; the value was reduced to 2.9% when the DMSO procedure was used, and the determined starch values increased from 86.9 to 97.2%.

A commercially available enzymic method for the quantitative measurement of (1→3),(1→4)-β-glucan has been simplified to allow analysis of up to 10 grain samples in 70 min or of 100–200 samples by a single operator in a day. These improvements have been achieved with no loss in accuracy or precision and with an increase in reliability. The glucose oxidase/peroxidase reagent has been significantly improved to ensure colour stability for periods of up to 1 h after development. Some problems experienced with the original method have been addressed and resolved, and further experiments to demonstrate the quantitative nature of the assay have been designed and performed.

The major endosperm cell wall polysaccharide in barley and oats is a linear (1→3),(1→4)-β-D-glucan. In barley, it represents approximately 75% of the carbohydrate in endosperm cell walls. It is generally considered that the majority of the polysaccharide consists of two or three 1,4-β-linked D-glucosyl residues, joined by single 1,3-β-linkages. Barley flour contains mixed-linkage β-glucan fractions that vary in their ease of extraction. Methods employed for the extraction and purification of barley β-glucan are generally modifications of the procedure described by Preece and Mackenzie. Extraction efficiency may be related to the time and conditions of storage of the grain or flour or to conditions of pretreatment of the grain before extraction. Exhaustive extraction procedures have been applied to isolated endosperm cell wall preparations that are essentially devoid of starch and protein. This chapter describes a modification of the method of Preece and Mackenzie that allows the large scale, essentially quantitative extraction of mixed-linkage β-glucan from barley flour.

The major carbohydrate component of the endosperm cell walls of barley and oat grain is a mixed-linkage (1→3),(1→4)-β-D-glucan commonly termed barley β-glucan. Barley β-glucan forms highly viscous aqueous solutions and gelatinous suspensions. In the brewing industry it can lead to diminished rates of wort and beer filtration and to the formation of hazes, precipitates, and gels in stored beer. In an attempt to alleviate the problems caused by barley β-glucan in the brewing and animal feed industries, various approaches have been adopted including the breeding of barley varieties low in this component, the use of only well-modified malts in brewing, and the addition of enzymes active on barley β-glucan. None of these methods has been adopted as a standard procedure. Reasons for this include the lack of specificity or reliability of the assay or the tedious nature of the assay format that limits the number of samples that can be processed in a given time. This chapter describes an assay procedure that overcomes these limitations. In this assay, highly purified endo-1,3(4)-β-glucanase (lichenase) and β-glucosidase are employed. The glucan is depolymerized by lichenase to oligosaccharides, these oligosaccharides are quantitatively hydrolyzed by β-glucosidase to glucose, and this is specifically measured using glucose oxidase/peroxidase reagent. This method is suitable for the routine analysis of mixed-linkage β-glucan in cereal flours, malt, wort, and beer.

A method developed for the quantification of (1→3)(1→4)-β-D-glucan in barley flour has been modified to allow its use in the measurement of this component in malt, wort, beer and spent grain. For malt samples, free D-glucose was first removed with aqueous ethanol. Quantification of the polymer in wort and beer samples involved precipitation of the β-glucan with ammonium sulphate followed by washing with aqueous ethanol to remove free D-glucose. Spent grain was lyophilised and milled and then analysed by the method developed for malt. In all cases, the β-glucan was depolymerised with lichenase and the resultant β-gluco-oligosaccharides hydrolysed to D-glucose with β-D-glucosidase. The released D-glucose was then specifically determined using glucose oxidase-peroxidase reagent.

Mixed linkage β-glucane and pentosanes (mainly arabinoxylanes) are the major endosperm cell-wall polysaccharides of barley and wheat respectively. These polysaccharides, although minor components of the whole grain, significantly affect the industrial utilization of these cereals. The modification of barley corns during malting requires the dissolution of the β-glucan in the cell-wall of the starch endosperm. High β-glucane concentration in wort and beer effect the rate of filtration and can also lead to precipitate or gel formation in the final product. In a similar manner, pentosane is thought to cause filtration problems with wheat starch hydrolysates by increasing viscosity and by producing gelatinous precipitate which blocks filters. Ironically, it is this same viscosity building and water binding capacity which is considered to render pentosanes of considerable value in dough development and bread storage (anti-staling functions). In the current paper, some aspects of the beneficial and detrimental effects of pentosans and β-glucan in the industrial utilization of wheat and barley are discussed. More specifically, enzymic methods for the preparation, analysis and identification of these polysaccharides and for the removal of their functional properties, are described in detail.

A simple and quantitative method for the determination of (1→3) (1→4)-β-D-glucan in barley flour and malt is described. The method allows direct analysis of β-glucan in flour and malt slurries. Mixed-linkage β-glucan is specifically depolymerized with a highly purified (1→3) (1→4)-β-D-glucanase (lichenase), from Bacillus subtilis, to tri-, tetra- and higher degree of polymerization (d.p.) oligosaccharides. These oligosaccharides are then specifically and quantitatively hydrolysed to glucose using purified β-D-glucosidase. The glucose is then specifically determined using glucose oxidase/peroxidase reagent. Since barley flours contain only low levels of glucose, and maltosaccharides do not interfere with the assay, removal of low d.p. sugars is not necessary. Blank values are determined for each sample allowing the direct measurement of β-glucan in values are determined for each sample allowing the direct measurement of β-glucan in malt samples. α-Amylase does not interfere with the assay. The method is suitable for the routine analysis of β-glucan in barley samples derived from breeding programs; 50 samples can be analysed by a single operator in a day. Evaluation of the technique on different days has indicated a mean standard error of 0-1 for barley flour samples containing 3-8 and 4-6% (w/w) β-glucan content.

In this paper, examples of the use of enzymes in the modification, quantification and investigation of fine-structural details of mixed-linkage (1+3)(1+4)-β-D-glucans, xyloglucans, glucomannans and xanthan are presented and discussed.

Currently, feed enzymes are primarily obtained through fermentation of fungi, bacteria, and other microorganisms. Although the manufacturing technology for feed enzymes has evolved rapidly, the activities of these enzymes decline during the granulating process and the cost of application has increased over time. An alternative approach is the use of genetically modified plants containing complex feed enzymes for direct utilization in animal feedstuff. We co-expressed three commonly used feed enzymes (phytase, β-glucanase, and xylanase) in barley seeds using the Agrobacterium-mediated transformation method and generated a new barley germplasm. The results showed that these enzymes were stable and had no effect on the development of the seeds. Supplementation of the basal diet of laying hens with only 8% of enzyme-containing seeds decreased the quantities of indigestible carbohydrates, improved the availability of phosphorus, and reduced the impact of animal production on the environment to an extent similar to directly adding exogenous enzymes to the feed. Feeding enzyme-containing seeds to layers significantly increased the strength of the eggshell and the weight of the eggs by 10.0%–11.3% and 5.6%–7.7%, respectively. The intestinal microbiota obtained from layers fed with enzyme-containing seeds was altered compared to controls and was dominated by Alispes and Rikenella. Therefore, the transgenic barley seeds produced in this study can be used as an ideal feedstuff for use in animal feed.

Oat bran (OB) is a by-product of oat, which is rich in β-glucan. As a new food processing technology, ultrafine powder can improve the surface properties of samples. OB with different grinding times was prepared, and its functional components, physical properties, adsorption properties, and antioxidant properties were evaluated. Results showed that with increased grinding times, the average particle size of OB decreased significantly (p < .05). And the water-holding capacity, swelling capacity, and water solubility index of OB increased significantly (p < .05), whereas the animal and vegetable oil-holding capacities decreased. Oat bran could adsorb cholic acid and glucose, which was related to the time of superfine grinding. In addition, the antioxidant capacity of OB was improved after superfine grinding. Related analysis shows that there was significant positive relationship between β-glucan, polyphenols and soluble dietary fibers and antioxidant indicators (p < .05). The Fourier transform infrared (FTIR) results showed that the FTIR spectra of OB powder with different crushing times were similar. On the basis of the above analyses, it is suggested that OB prepared by superfine grinding for 5 min had good physical and chemical properties and antioxidant properties and is widely used in food.

Owing to its potential role in improving intestinal health, oat bran has received a lot of attention. In vitro digestion and fermentation were performed on oat brans that had been pretreated with steaming, microwave, and hot-air drying. The results showed that the dietary fiber after in vitro digestion and β-glucan content of pretreated oat brans changed insignificantly (p > 0.05). The abundance of Firmicutes and Proteobacteria varied significantly between samples (p < 0.05). The steaming oat bran was utilized by gut microbiota to produce acetate, propionate, and butyrate, the relative abundance of Faecalibacterium prausnitzii significantly increased, and Escherichia-Shigella significantly decreased. Furthermore, the genus level flora differed significantly between steaming, microwave, and hot-air drying oat bran. Overall, steaming improved oat bran fermentability, promoting increased SCFA production and bacterial shifts associated with health benefits. Our findings provide a solid foundation for further research into the role of oat bran and gut microbiota in host health.

This work aims to formulate shortbreads by partial substitution of wheat flour with brewers’ spent grains (BSGs), valorization strategy of this by-product. This study has underlined that replacing 30 % wheat flour with BSGs barley malt flour and BSG enriched with 30 % of oat flakes allows for a significant increase in fiber, particularly arabinoxylan, and protein, compared with the reference biscuit. The fiber content allows for a “high in fiber” label and meets the “gut health” EFSA claim. The obtained food products contribute to the recommended daily intake of bioactive compounds for health benefits, they do not show an increase in sugars and fats, compared to 100 % wheat flour shortbread, and have acceptable sensory characteristics. The formulation of shortbreads by partial substitution of wheat flour with BSGs can be an efficient and feasible strategy for the upcycling and valorization of this by-product throughout the production of functional food.

Roasting is the primary link in Chinese traditional whole oat food processing, but the role of roasting is rarely explored. This study aimed to clarify the effects of roasting treatment (160°C, 0-60 min) of oat kernels on the characteristics of oat flour from the aspects of micromorphology, constituents, gelation, structure, and starch digestibility. Results showed roasting treatment disrupted the structure of oat kernels, promoted the release of lipids, and caused the aggregation and wrapping of oat flour particles. After roasting treatment, starch and protein content of whole oat flour did not change significantly, but lipid content increased and β-glucan content decreased. The roasted samples had better water absorption (25°C, 2.41-2.69 g/g), swelling power (25°C, 2.44-2.78), and solubility (100°C, 25.40-28.11%), lower pasting viscosity (final viscosity, 1.84-3.61 Pa s), higher gel strength, and lower short-range order. Pasting viscosities and gel properties with roasting time conformed to the second-order polynomial model. Oat flour from roasting oat kernels for 40 min had the highest crystallinity (19.74%), the highest digestibility rate constant (k, 0.07), and lower product concentration (C_ꝏ, 77.74%). These results will contribute to the application of roasting technology in the modification of whole oats.

Arabidopsis (Arabidopsis thaliana) leaves possess a mechanism that couples the rate of nighttime starch degradation to the anticipated time of dawn, thus preventing premature exhaustion of starch and nighttime starvation. To shed light on the mechanism, we screened a mutagenized population of a starvation reporter line and isolated a mutant that starved prior to dawn. The mutant had accelerated starch degradation, and the rate was not adjusted to time of dawn. The mutation responsible led to a single amino acid change (S132N) in the starch degradation enzyme BETA-AMYLASE1 (BAM1; mutant allele named bam1-2D), resulting in a dominant, gain-of-function phenotype. Complete loss of BAM1 (in bam1-1) did not affect rates of starch degradation, while expression of BAM1(S132N) in bam1-1 recapitulated the accelerated starch degradation phenotype of bam1-2D. In vitro analysis of recombinant BAM1 and BAM1(S132N) proteins revealed no differences in kinetic or stability properties, but in leaf extracts, BAM1(S132N) apparently had a higher affinity than BAM1 for an established binding partner required for normal rates of starch degradation, LIKE SEX FOUR1 (LSF1). Genetic approaches showed that BAM1(S132N) itself is likely responsible for accelerated starch degradation in bam1-2D and that this activity requires LSF1. Analysis of plants expressing BAM1 with alanine or aspartate rather than serine at position 132 indicated that the gain-of-function phenotype is not related to phosphorylation status at this position. Our results strengthen the view that control of starch degradation in wild-type plants involves dynamic physical interactions of degradative enzymes and related proteins with a central role for complexes containing LSF1.

The quantification of β-glucan in oats is of immense importance for plant breeders and food scientists to develop plant varieties and food products with a high quantity of β-glucan. However, the chemical analysis of β-glucan is time consuming, destructive, and laborious. In this study, near-infrared (NIR) spectroscopy in conjunction with Chemometrics was employed for rapid and non-destructive prediction of β-glucan content in oats. The interval Partial Least Square (iPLS) along with correlation matrix plots were employed to analyze the NIR spectrum from 700–1300 nm, 1300–1900 nm, and 1900–2500 nm for the selection of important wavelengths for the prediction of β-glucan. The NIR spectral data were pre-treated using Savitzky Golay smoothening and normalization before employing partial least square regression (PLSR) analysis. The PLSR models were established based on the selection of wavelengths from PLS loading plots that present a high correlation with β-glucan content. It was observed that wavelength region 700–1300 nm is sufficient for the satisfactory prediction of β-glucan of hulled and naked oats with R²c of 0.789 and 0.677, respectively, and RMSE < 0.229.

The starch digestion processing of whole grain foods is associated with its health benefits in improving insulin resistance. This study modified the digestibility of whole quinoa flour (WQ) via heat-moisture treatment (HMT), HMT combined with pullulanase (HMT+P), HMT combined with microwave (HMT+M), and HMT combined with citric acids (HMT+A), respectively. Results showed that all the treatments significantly increased (p < 0.05) the total dietary fiber (TDF) content, amylose content, and resistant starch (RS) content, however, significantly decreased (p < 0.05) the amylopectin content and rapidly digestible starch (RDS) content of WQ. HMT+P brought the highest TDF content (15.3%), amylose content (31.24%), and RS content (15.71%), and the lowest amylopecyin content (30.02%) and RDS content (23.65%). HMT+M brought the highest slowly digestible starch (SDS) content (25.09%). The estimated glycemic index (eGI) was respectively reduced from 74.36 to 70.59, 65.87, 69.79, and 69.12 by HMT, HMT+P, HMT+M, and HMT+A. Moreover, a significant and consistent reduction in the heat enthalpy (ΔH) of WQ was observed (p < 0.05), after four treatments. All these effects were caused by changes in the starch structure, as evidenced by the observed conjunction of protein and starch by a confocal laser scanning microscope (CLSM), the decrease in relative crystallinity, and transformation of starch crystal.

The use of barley (Hordeum vulgare L.) in Morocco is still limited to food and feed despite the amplified demand by local industries for imported malt. This study aims to evaluate 36 barley elite lines for major grain physicochemical parameters and malt quality traits. Analysis of variance, Pearson correlation, principal component analysis (PCA), and hierarchical cluster analysis (HCA) were performed. The results showed significant genotypic variation among genotypes for individual grain and malt traits. High broad sense heritability was obtained for all traits except for plump grain percentage, malt friability, and germination capacity. Starch, malt extract, Kolbach index, grain area, and test weight correlated significantly and negatively with barley protein. Malt extract correlated positively with Kolbach index and starch, but a negative correlation with soluble protein and malt protein was found. Based on 12 characters, 77% of the total genotypic variation was explained by the three first principal components following PCA and four clusters were depicted based on HCA. Genotypes of high interest with desirable levels of quality standards were identified to be used as a malt quality traits donor while designing crossing programs.

The extent to which the quality and yield of plant varieties are influenced by the environment is important for their successful uptake by end users particularly as climatic fluctuations are resulting in environments that are highly variable from one growing season to another. The genotype-by-environment interaction (GEI) of milling quality and yield was studied using four winter oat varieties in multi-locational trials over 4 years in the U.K. Significant differences across the 22 environments were found between physical grain quality and composition as well as grain yield, with the environment having a significant effect on all of the traits measured. Grain yield was closely related to grain number m⁻² whereas milling quality traits were related to grain size attributes. Considerable genotype by environment interaction was obtained for all grain quality traits and stability analysis revealed that the variety Mascani was the least sensitive to the environment for all milling quality traits measured whereas the variety Balado was the most sensitive. Examination of environmental conditions at specific within-year stages of crop development indicated that both temperature and rainfall during grain development were correlated with grain yield and β-glucan content and with the ease of removing the hull (hullability).