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.

This study is aimed to produce and characterize a novel gluten-free ingredient from oat through sprouting at 18°C for 96 h. The nutritional and bioactive properties as well as key enzymatic activities were studied in sprouted oat powder and compared with those of oat grain powder (control). Sprouted oat powder was an excellent source of protein (10.7%), β-glucan (2.1%), thiamine (687.1 μg/100 g), riboflavin (218.4 μg/100 g), and minerals (P, K, Mg and Ca), and presented better amino acid and fatty acid compositions and levels of γ-aminobutyric acid (54.9 mg/100 g), free phenolics (507.4 mg GA/100 g) and antioxidant capacity (1744.3 mg TE/100 g) than control. Enhanced protease and α-amylase and reduced lipase activities were observed in sprouted oat powder, which are promising features to improve its nutritional, sensorial and health-promoting properties. These results support the use of sprouted oat powder as a promising gluten-free functional ingredient.

This study determined the effects of four thermal processing methods for germinated highland barley (GHB) on its nutritional composition, physicochemical properties, in vitro starch and protein digestibility, and in vitro prebiotic effects. The contents of total dietary fiber (TDF) and total phenols were significantly increased by steaming, microwave, baking and extrusion processing, while the contents of ash, starch and resistant starch were decreased. Except for baking, the other three methods improved the water hydration properties by increasing the water absorption index, water solubility index and swelling power. Thermally processed samples, especially those extruded, exhibited better thermal stability, pasting properties and in vitro protein digestibility, possibly because of the damage to the whole grain powder particles. The thermally processed digesta of GHB promoted the proliferation of Lactobacillus plantarum and L. delbrueckii in a dose-dependent manner, especially for those extruded, followed by those processed by steaming, microwave and baking. A Pearson correlation analysis showed that the prebiotic effect was positively correlated with the content of TDF in the different samples. Overall, thermal processing increased the quality and digestibility of GHB, with extrusion being the most suitable for industrial processing.

On two locations (Borovce, Slovak Republic and Karnobat, Republic of Bulgaria) 21 barley genotypes (Hordeum vulgare L.) were grown for two consecutive years (2017, 2018). Selected malting qualitative parameters were analysed in mature grains; the content of total starch, crude proteins, and β-D-glucan. The aim of the work was to analyse agroecological conditions affecting the content of selected quality parameters in the barley grain and to evaluate, select and exchange barley genotypes created with an added value in order to obtain suitable biological material usable in the changing environmental conditions of a country. The content of total starch in analysed samples was in the range 53.62% and 63.30%, crude proteins were between 11.50% and 15.46% and β-D-glucan was from 3.26% to 5.87%. Year, locality, genotype, and their interactions were factors affecting the content of all analysed parameters in the barley grain, however the content of proteins and β-D-glucan was relatively stable. In Slovak barley genotypes, higher levels of starch and lower levels of proteins and β-D-glucan were observed compared to Bulgarian genotypes. Higher amounts of starch were detected in the conditions of the Slovak Republic. In warmer conditions of the Republic of Bulgaria, higher amounts of proteins and β-D-glucan were observed. Statistically significant correlations (P≤0.05) were observed between total starch and crude proteins (r=-0.59) and between total starch and β-D-glucan (r=-0.40). The correlation between the content of proteins and β-D-glucan was positive (r=0.05), but in present experiment not statistically significant.

Feeding broiler chickens on diets based on cereal grains of high non-starch polysaccharides content such as wheat and barley can negatively impact their performance and gut health. Plant extracts can be used as a potential tool to alleviate these negative effects. The present study assessed the effects of dietary cereal type and the inclusion of a plant extract blend (PEB) on the growth performance, intestinal histomorphology, caecal microflora, and gene expression of selected biomarkers for gut integrity in broiler chickens in a 42-d experiment. Ross-308 male broilers were assigned into different dietary treatments and fed on two cereal types (corn- vs. wheat/barley-based) with/without added graded concentrations of a PEB (0, 250, 500, 1000, and 2000 mg/kg diet). There were no significant differences in the growth performance parameters, intestinal histomorphology, and caecal microflora due to the impact of dietary cereal type. However, lactobacilli count in the caecal microflora was increased in the group fed on a corn-based diet. The PEB supplementation especially at a level of 500 to 1000 mg/kg diet significantly increased the average BW and decreased the feed conversion ratio. It also increased the villi length of duodenum, jejunum, and ileum, decreased the duodenal crypt depth, and increased the villi length to crypt depth ratio in the duodenum, jejunum and ileum. Supplementation of the PEB decreased the total bacterial and coliform count and increased the lactobacilli count in a linear pattern. Gene expression of Occludin and Junction Adhesion Molecule was significantly increased in the PEB supplemented diets, whereby no influence was observed on mucin expression. In conclusion, supplementation of a PEB at levels of 500-1000 mg/kg can be used as a tool to improve broiler performance and gut health.

Barley grains contain a variable amount of biologically active compounds such as non-starch polysaccharides and phenol compounds. These compounds are important in nutrition due to their significant health benefits and technological role in food. We developed predictive models for β-glucans (BG), arabinoxylans (AX), bound phenols (BP), free phenols (FP), and anthocyanins (AN) based on near-infrared spectroscopy (NIRS) using two different NIRS instruments with different spectral range and spectral steps. Regressions of modified partial least squares (MPLS) and several combinations of scattering correction and derivative treatments were tested. The optimal calibration models generated high coefficients of determination for BG and BP, but not for AN content. The instrument with the highest resolution only gave better results for BG prediction models, and the addition of the visible range did not prove to be ostensibly advantageous to the determination of any of the active compounds of study, not even in the case of AN analysis.

Wheat bran consumption is associated with several health benefits, but its incorporation into food products remains low because of sensory and technofunctional issues. Besides, its full beneficial potential is probably not achieved because of its recalcitrant nature and inaccessible structure. Particle size reduction can affect both technofunctional and nutrition-related properties. Therefore, in this study, wet milling and cryogenic milling, two techniques that showed potential for extreme particle size reduction, were used. The effect of the milling techniques, performed on laboratory and large scale, was evaluated on the structure and physicochemical properties of wheat bran. With a median particle size (d₅₀) of 6 µm, the smallest particle size was achieved with cryogenic milling on a laboratory scale. Cryogenic milling on a large scale and wet milling on laboratory and large scale resulted in a particle size reduction to a d₅₀ of 28-38 µm. In the milled samples, the wheat bran structure was broken down, and almost all cells were opened. Wet milling on laboratory and large scale resulted in bran with a more porous structure, a larger surface area and a higher capacity for binding water compared to cryogenic milling on a large scale. The extensive particle size reduction by cryogenic milling on a laboratory scale resulted in wheat bran with the highest surface area and strong water retention capacity. Endogenous enzyme activity and mechanical breakdown during the different milling procedures resulted in different extents of breakdown of starch, sucrose, β-glucan, arabinoxylan and phytate. Therefore, the diverse impact of the milling techniques on the physicochemical properties of wheat bran could be used to target different technofunctional and health-related properties.

The objective of this study was to evaluate the effect of wheat–oat flour ratio on the physical properties and β-glucan characteristics of extrudates. Results showed that increasing the wheat–oat flour ratio resulted in a decrease in the water solubility index (r²=0.8567) and hardness (r²=0.9316), whereas the expansion ratio (r²=0.9307) and water absorption index (r²=0.9061) increased. Wheat flour generally caused an increase in L values from 57.81 to 62.94 providing bright samples. Few cells were observed at high wheat–oat flour ratios under a scanning electron microscope, and a smooth surface was noted. Meanwhile, the total (r²=0.9867) and soluble (r²=0.9848) β-glucan contents were inversely proportional to the wheat–oat flour ratio. Extrudates with added wheat flour had a high molecular weight, but wheat flour had no significant (P<0.05) effect on the viscosity of β-glucan extracts. Conclusively, incorporation of wheat flour at a wheat–oat flour ratio of 2.33 provides ready-to-eat food based on whole oat flour, on account of improving the texture and providing sufficient β-glucan contents (0.806 g/100 g) without significantly affecting β-glucan viscosity.

The present study was designed to characterize oat bran for their biological attributes. The results showed that bran of Avon variety contained high TDF, SDF, β-glucan and extractability of β-glucan than bran of oat variety Sargodha-81. The extrusion process exhibited the highest extractability of β-glucan (45.37%) followed by cooking (37.28%) and baking methods (32.45%). Moreover, the glucose level reduction was found significantly different when raw and processed oat bran diets fed to normal, hypercholesterolemic and diabetic rats. The highest reduction was recorded when fed on diet containing 30% processed oat bran. The processed oat bran exhibited more reduction as compared to raw oat bran. Furthermore, addition of 20% oat bran in wheat grits porridge was found to have significant effect (p < 0.05) on appearance, mouth feel and overall acceptability. Convincingly, it is recommended that processed oat bran may be introduced in diet-based remedy to rheostat lifestyle-related disorders.

Legumes are a popular grain-free alternative carbohydrate source in canine diets, however, information on their fermentative characteristics have not been established. Thus, the objectives of the present study were to 1) quantify the chemical compositions and 2) fermentative profile of select legumes using canine fecal inoculum. Five legume varieties, whole yellow peas (WYP), green lentils (GL), black bean grits (BBG), navy bean powder (NBP), and garbanzo beans, were analyzed and compared to a positive control, beet pulp (BP). Substrates were analyzed for gross energy (GE), dry and organic matter, crude protein (CP), acid hydrolyzed fat, and total dietary fiber (TDF) fractions, beta-glucans, starch-free, and hydrolyzed sugars, as well as fermentative characteristics: pH, short-chain fatty acids (SCFA), branched-chain fatty acids (BCFA), total gas, hydrogen, and methane. Substrates then underwent a two-stage in vitro digestion and subsequent fermentation using canine fecal inoculum for 0, 3, 6, 9, and 12 h. All test substrates contained approximately 8% to 9% moisture and 4.5 kcal/g GE. The highest CP content was observed in GL (27%). Analyzed TDF content of test substrates was greatest for WYP (32%) and GL (36%). Total starch content was greatest for GL (58%) and WYP (56%). Sucrose and stachyose were the most predominant free sugars and glucose was the most predominant hydrolyzed sugar among test substrates. After 3 and 6 h of fermentation, a net negative change in pH was observed among most substrates with a net negative change in all substrates after 9 and 12 h. Values for SCFA did not differ among substrates after 3 or 6 h of fermentation with BP and WYP among the greatest acetate (1,656 and 1,765 umol/g, respectively) and propionate production values (157.7 and 126.1, respectively) after 9 h. All substrates produced greater total gas volumes than WYP after 3 h, with no differences observed after any other time points. However, BP hydrogen production values were greater after 9 and 12 h (P < 0.0001; 726,042 and 394,675 ng/g, respectively) with greater methane production values after 12 h (P < 0.0001; 54,291 ng/g) than all test substrates. These data suggest that legumes offer a diverse macronutrient profile and appear to be a source of slowly fermentable fiber, which may have beneficial implications on the ratios of saccharolytic to proteolytic fermentation toward the distal colon.

The study was aimed at determining the effect of incorporating glucagel from 9 different hulless barley cultivars to wheat flour on the rheological properties of dough using Mixolab. The mixing properties were significantly affected as glucagel incorporation led up to 11.4% increase in dough consistency at a constant hydration of 56%. Addition of glucagel lowered both the dough stability and protein weakening by up to 18.85% and 10.81%, respectively. The peak viscosity on heating was up to 4.3% higher in samples containing glucagel. Moreover, the incorporation of glucagel to wheat flour caused lowering of shear thinning/breakdown by up to 76.8% which could be evident from the negative correlation (r = − 0.68, p < 0.05) of percent breakdown with the soluble β-glucan content. Retrogradation was up to 13.14% lower in the presence of glucagel. Higher levels of soluble β-glucan in the glucagel lowered retrogradation in the dough and a negative correlation of r = - 0.65 (p < 0.05) existed between the two parameters, demonstrating the pivotal role of glucagel in lowering of staling, therefore glucagel could be utilized in several bakery products.