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.

The proportion of soluble components in dietary fiber is an important factor affecting its physicochemical properties and physiological functions. The influence mechanisms of insoluble (IDF) and soluble dietary fiber (SDF) at different ratios on constipation were investigated. Results showed that SDF had higher active groups and water swelling capacity than IDF. The viscosity of chyme with SDF alone was the highest in oral and gastric phases. The gastric emptying rate and small intestine propulsion capacity increased significantly, especially when IDF/SDF was 1:1. IDF and a lower proportion of SDF (< 50 %) promoted gut microbiota diversity and short-chain fatty acids production. The contents of 5-hydroxytryptamine, acetylcholinesterase and gastrin reached the maximum value when the IDF ratio was 50 %. In conclusion, IDF could act synergistically with SDF to promote defecation and relieve constipation, and the effect was the best when the ratio of IDF to SDF was 1:1.

Previous studies have shown that fermented barley has a lower digestion rate. However, it remains unclear whether the antidigestibility of starch in fermented barley is affected by other nonstarch components. In this paper, the removal of protein, lipid, and β-glucan improved the hydrolysis rate of starch and the protein showed the greatest effect. Subsequently, the inhibitory mechanism of protein on starch digestion was elucidated from the perspective of starch physicochemical properties and structural changes. The removal of protein increased the swelling power of starch from 10.09 to 11.14%. The short-range molecular ordered structure and the helical structure content decreased. The removal of protein reduced the coating and particle size of the starch particles, making the Maltese cross more dispersed. In summary, protein in fermented barley enhanced the ordered structure of starch by forming a physical barrier around starch and prevented the expansion of starch, which inhibited the hydrolysis of starch.

Spent brewery grain (SBG) is a by-product of the brewery industry. The study aimed to investigate the prebiotic potential of SBG. The chemical composition and fermentation capacity of SBG were checked. The gut microbiota response to SBG was assessed in two in vitro models (batch fermentation and dynamic system). Substances with prebiotic properties, including arabinoxylans (16.7 g/100 g) and polyphenols (49.1 mg/100 g), were identified in SBG. Suitable growth and fermentation by probiotic bacteria were observed. The modulatory effect of gut microbiota depends on the in vitro system used. In batch fermentation, there was no stimulation of Bifidobacterium or lactic acid bacteria (LAB), but short-chain fatty acid (SCFA) and branched short-chain fatty acids (BCFA) synthesis increased. In dynamic, SBG exhibited a moderate bifidogenic effect, promoting Akkermansia and LAB growth while reducing Bacteroides and Escherichia-Shigella. SCFA stabilisation and reduction of BCFA content were noted. Moderate prebiotic effects were observed.

Highland barley non-starch polysaccharides (HBNP), particularly β-glucans, are known for their health-promoting effects, including modulation of glycemic response and enhancement of gut health. This study investigated the impact of different HBNP fractions on the properties and digestibility of high-glycemic index rice starch. HBNP was segmented into five fractions (HBNP-15, HBNP-30, HBNP-45, HBNP-60, and HBNP-75) using gradient ethanol precipitation, and these fractions exhibited varying molecular weights, monosaccharide compositions, and β-glucan contents. All fractions reduced rice starch's pasting viscosity, with 1 % HBNP-75 leading to a 99.1 % decrease in final viscosity. Morphological and size distribution analyses showed that HBNP fractions limited granule swelling and disrupted starch's continuous phase structure. HBNPs also reduced starch digestibility and increased the formation of resistant starch from 10 % to 28 %. These results suggest potential uses for HBNP fractions in developing low-glycemic starch-based foods.

The cage-like structure formed by proteins on starch surfaces is of considerable interest due to its digestion regulation. The structural, physicochemical and digestive properties of cage-like complexes formed by highland barley protein isolate (HBPI), prolamin (Pro), glutenin (Glu), and the combination of Pro and Glu (Pro+Glu) with highland barley starch (HBS) under spray drying (SD) were investigated. SD treatment significantly enhanced the inhibition of different proteins on HBS digestion, with the maximum RS content of 23.62% and the minimum C∞ of 78.54% observed in HBS with Glu. After SD, Pro was observed to significantly reduce the peak viscosity of HBS during pasting by 354 cP, while others significantly enhanced by 881.50–1615.00 cP. Low-field nuclear magnetic resonance results indicated that the strongly and weakly bound water in all gel samples partially transformed to free water after SD. The thermogravimetric analysis demonstrated that SD treatment resulted in a redshift of the thermal degradation peak at 150−450 °C for all samples. The structural properties of all samples revealed that SD treatment reduced short-range orderings, remained the A-type crystalline of HBS, and reduced the peak intensity of the diffraction peak at 20°. Furthermore, the microstructures of HBS with different proteins after SD exhibited distinct characteristics, with Glu forming a continuous cage-like structure on starch surfaces while Pro forming larger aggregates. This study offers methodological and theoretical support for targeted regulation of starch digestibility.