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.

BACKGROUND AND AIMS: Plant cell enlargement is unambiguously coupled to changes in cell wall architecture, and as such various studies have examined the modification of the proportions and structures of glucuronoarabinoxylan and mixed-linkage glucan in the course of cell elongation in grasses. However, there is still no clear understanding of the mutual arrangement of these matrix polymers with cellulose microfibrils and of the modification of this architecture during cell growth. This study aimed to determine the correspondence between the fine structure of grass cell walls and the course of the elongation process in roots of maize (Zea mays). METHODS: Enzymatic hydrolysis followed by biochemical analysis of derivatives was coupled with immunohistochemical detection of cell wall epitopes at different stages of cell development in a series of maize root zones. KEY RESULTS: Two xylan-directed antibodies (LM11 and ABX) have distinct patterns of primary cell wall labelling in cross-sections of growing maize roots. The LM11 epitopes were masked by mixed-linkage glucan and were revealed only after lichenase treatment. They could be removed from the section by xylanase treatment. Accessibility of ABX epitopes was not affected by the lichenase treatment. Xylanase treatment released only part of the cell wall glucuronoarabinoxylan and produced two types of products: high-substituted (released in polymeric form) and low-substituted (released as low-molecular-mass fragments). The amount of the latter was highly correlated with the amount of mixed-linkage glucan. CONCLUSIONS: Three domains of glucuronoarabinoxylan were determined: one separating cellulose microfibrils, one interacting with them and a middle domain between the two, which links them. The middle domain is masked by the mixed-linkage glucan. A model is proposed in which the mixed-linkage glucan serves as a gel-like filler of the space between the separating domain of the glucuronoarabinoxylan and the cellulose microfibrils. Space for glucan is provided along the middle domain, the proportion of which increases during cell elongation.

The relative molecular weight distribution of soluble barley beta-glucans (SBB) was monitored through the small intestine in pigs by analyzing water extracts of duodenal- and ileal digesta with HPLC-SEC. Variations among four diets, based on four different barley varieties, were documented as well as variations between animals fed the same diet. The results showed depolymerisation of the SBB throughout the whole small intestine independent of diet. The average molecular weight of the SBB was reduced to approximately 50% in duodenum in all the experimental animals.

The objective of the work was to evaluate the influence of genetic and mechanical removal of hulls from oat grains on their nutrient content. The studies included three cultivars and six lines of oat grains. In grain samples of hulled (5 samples), dehulled (5 samples) and naked (4 samples) oats, the following components were determined: chemical composition (ash, crude protein, crude fat, crude fibre and its components) and amino acids and fatty acid composition. The grain of naked and dehulled oats contained significantly more crude protein, crude fat and polyunsaturated fatty acids, and considerably less saturated fatty acids and crude fibre than hulled oats. In addition, the dietary fibre composition was more favourable than the naked oats. The coefficients of nutritional values of the protein (total essential amino acids, essential amino acid index and amino acids score) of naked oats were higher than hulled and dehulled oats. In all the tested oat grain samples, lysine was the most limiting amino acid. The study showed that genetic and mechanical reduction of the proportion of hulls in oat grains resulted in a significant decrease in dietary fibre content and a significant increase in nutrient content.

BACKGROUND: Barley rootlets, a malting by-product, are currently discarded or used as fodder. In this study, milled rootlets and Lactobacillus plantarum FST 1.7-fermented rootlets were incorporated into wheat bread. The objective was to formulate a high-nutrition alternative to wholemeal breads with improved technological attributes. RESULTS: Chemical analyses showed that rootlets contribute nutrients and bioactive compounds, including proteins, amino acids, fatty acids, carbohydrates, dietary fibre, polyphenols and minerals. Rootlets are particularly rich in essential amino acids, especially lysine, the typically limiting essential amino acid of cereals. Additionally, rootlets offer potential dietary fibre health benefits such as protection against cardiovascular disease, cancers and digestive disorders. CONCLUSION: Breads prepared with a (fermented) rootlet inclusion level of up to 10% compared favourably with wholemeal breads from nutritive, technological and textural perspectives. Furthermore, they were well accepted by sensory panellists. Using rootlets as a food ingredient would have the added benefit of increasing this malting by-product's market value.

The scheme of flour milling in mill can be expressed in diagram as a milling flow. There can be described a weight part or percentage of flour from every stage of breaking, scratch and reduction. The similar flow diagram can be drawn expressing the ash content in every of flour streams of a whole milling flow. A milling flow of barley is considerably different from that of wheat and to some part also from rye mill flow. Currently, high-yielding naked barley cultivars are preferred in the Western world, and they can be used in products where outstanding starch or non-starch polysaccharide properties are required. The aim of this work was to assess the milling results with regard to the yield of single streams in connection with their chemical composition, especially β-glucans (fiber) content. Barley sample was naked barley of Czech origin of crop 2010. The balance tables showing the yield of β-glucans and ash in single streams were compiled. Resulting data were judged in comparison to the milling flow with the purpose to recommend the parameters and best streams of milling flow as a source of special nutritionally lucrative products.

A rapid method for determination of the soluble and total amount of β-glucan was investigated. β-Glucan was extracted from the samples with the modified extraction method and then incubated with lichenase and β-glucosidase. The optimum incubation parameters were determined by using pure β-glucan solutions as follows: lichenase concentration, 0.04 international units (IU) ml^-1; β-glucosidase concentration, 0.125 IU ml^-1; reaction time, 20 min; reaction temperature, 50°C; reaction acidity, pH 6. After enzymatic incubation, free D-glucose produced by the action of enzymes on soluble and total β-glucans was measured with an amperometric glucose electrode. The linearity of the β-glucan assay was determined as 8.0% (w/w) under the considered enzymatic reaction condition. In the last part of the study, soluble and total β-glucan contents of certain samples were determined by both the improved method and the AOAC Official Method. A high correlation (R2: 0.992) between the results of these two methods was observed.

An experiment was conducted to study the effects of inclusion of a wheat cultivar (high in nonstarch polysaccharides) and xylanase supplementation of the diet on productive performance, pH of the gastrointestinal tract, nutrient retention, and intestinal enzyme activity of Hy-Line W-36 laying hens from 25 to 47 wk of age. The experiment was completely randomized with 8 treatments arranged factorially with 4 levels of wheat (0, 23, 46, and 69%) that corresponded to a dietary arabinoxylan content of 3.0, 3.3, 3.6, and 3.9%, with or without xylanase supplementation. Each treatment was replicated 5 times. For the entire experimental period, egg weight (P < 0.05) and egg mass (P < 0.01) were reduced and the feed conversion ratio was hindered (P < 0.05) with increased levels of wheat in the diet, but ADFI and egg production were not affected. Xylanase supplementation improved egg production (P < 0.05), egg mass (P < 0.01), and the feed conversion ratio (P < 0.01). Diet did not affect egg quality at any age, except for shell thickness at 47 wk that was improved with xylanase supplementation (P < 0.05). Digesta pH of the different organs of the gastrointestinal tract was not affected by wheat inclusion or xylanase supplementation. Ileal viscosity increased (P < 0.001) with wheat inclusion and decreased (P < 0.001) with xylanase supplementation at all ages. Fat digestibility (P < 0.001) decreased with increased levels of wheat but AME_n content of the diets (P < 0.05) and nitrogen retention were not affected. Wheat inclusion increased (P < 0.001) amylase (33 wk), lipase (33 wk), and aminopeptidase (47 wk) activity in the duodenum as well as lipase activity in the jejunum at 47 wk of age. However, xylanase supplementation did not affect the activity of any of the enzymes studied. It is concluded that most of the negative effects of wheat inclusion in the diet were reduced or even disappeared with xylanase supplementation. Wheat with a high nonstarch polysaccharide content (Pishtaz cultivar) can be used at levels of up to 69% in laying-hen diets without negatively affecting bird performance, provided that feeds are supplemented with xylanase.

The contents of fat, starch, pentosan, fructan, β-glucan and several mono- and oligosaccharides in grain were evaluated to find out the possible effects of the Wx-D1 gene of bread wheat using two sets of near-isogenic waxy and non-waxy lines and two low-amylose mutant lines with a common genetic background of Kanto 107. These materials have two non-functional Wx-A1b and Wx-B1b alleles in common. Waxy near-isogenic lines with a non-functional Wx-D1d allele showed consistently increased contents of fat, total fructan, β-glucan, glucose, fructose, sucrose, 1-kestose, 6-kestose, neokestose, nystose and bifurcose compared with non-waxy lines with a functional Wx-D1a allele throughout three growing/harvest seasons. Starch and total pentosan contents were inconsistently influenced by the allelic status of the Wx-D1 locus, while water-soluble pentosan and raffinose contents were not affected. The compositional changes of a low-amylose mutant line with an almost non-functional Wx-D1f allele were closely similar to those of waxy near-isogenic lines, while significantly different changes were barely observed in another low-amylose mutant line with a partly functional Wx-D1g allele in two seasons. These results showed that the Wx-D1 gene has pleiotropic effects on the fat and saccharide contents of bread wheat grain.

This study aimed to quantify the changes caused by varying germination conditions on the contents of some bioactive compounds in barley and oats. Samples of the two grains were germinated at temperatures between 10 and 20°C for a period of 2–6 days, using a two-dimensional central composite design. The germination temperature had only minor effect in comparison with the germination time. Slight changes in the mineral content of the malts were observed, mainly caused by steeping. Phytate has been seen as an anti-nutritional compound, as it complexes minerals and lowers their bioavailability. The phytate content in barley malts was considerably lower than in the native kernels. Variations in the germination conditions did not have a significant effect on phytate content. In oats, degradation of phytate was significantly enhanced by prolonging the germination period. It was possible to retain the amounts of soluble dietary fibre, when short germination periods were applied. However, long germination periods caused an extensive breakdown of soluble dietary fibre, especially beta-glucan. The content of insoluble fibre, however, was increased by applying long germination periods for oat malts.

Brewers' spent grain (BSG) is a readily available, high volume low cost byproduct of brewing and is a potentially valuable resource for industrial exploitation. The variation in BSG composition and the implications for microbiological spoilage by a resident microflora might affect the potential to use BSG as a reliable food-grade industrial feedstock for value-added downstream processing. Fresh samples of BSG from a range of 10 breweries have been analysed for their microbial and chemical composition. The results show that a resident microflora of mainly thermophilic aerobic bacteria (<10⁷ g^-1 fresh weight) persists on BSG. This population is susceptible to rapid change but at the point of production BSG can be considered microbiologically stable. Chemically, BSG is rich in polysaccharides, protein and lignin. Residual starch can contribute up to 13% of the dry weight and BSG from lager malts has higher protein content than that from ale. In general, at the point of production, BSG is a relatively uniform chemical feedstock available for industrial upgrading. Differences between breweries should not present problems when considering BSG for industrial exploitation but susceptibility to microbial colonisation is identified as a potential problem area which might restrict its successful exploitation.

Background: Epidemiological studies show inverse relationship between intake of wholegrain cereals and several chronic diseases. Components and mechanisms behind possible protective effects of wholegrain cereals are poorly understood. Objective: To characterise commercial rye bran preparations, compared to wheat bran, regarding structure and content of nutrients as well as a number of presumably bioactive compounds. Design: Six different rye brans from Sweden, Denmark and Finland were analysed and compared with two wheat brans regarding colour, particle size distribution, microscopic structures and chemical composition including proximal components, vitamins, minerals and bioactive compounds. Results: Rye brans were generally greener in colour and smaller in particle size than wheat brans. The rye brans varied considerably in their starch content (13.2–28.3%), which reflected variable inclusion of the starchy endosperm. Although rye and wheat brans contained comparable levels of total dietary fibre, they differed in the relative proportions of fibre components (i.e. arabinoxylan, β-glucan, cellulose, fructan and Klason lignin). Generally, rye brans contained less cellulose and more β-glucan and fructan than wheat brans. Within small variations, the rye and wheat brans were comparable regarding the contents of tocopherols/tocotrienols, total folate, sterols/stanols, phenolic acids and lignans. Rye bran had less glycine betaine and more alkylresorcinols than wheat brans. Conclusions: The observed variation in the chemical composition of industrially produced rye brans calls for the need of standardisation of this commodity, especially when used as a functional ingredient in foods.

Brewers’ spent grain (BSG), a high-volume coproduct from the brewing industry, primarily contains proteins, barley cell wall carbohydrates, and lignin. To create new possibilities for the exploitation of this large biomass stream, the solubilization of BSG by the combined action of carbohydrases (Depol 740 and Econase) and peptidase (Alcalase and Promod 439) was explored. Hydrolysis protocols were optimized with respect to temperature (influencing both microbial contamination and rate of enzymatic hydrolysis), pH, enzyme dose, order of enzyme addition, and processing time. On the basis of this approach, one- and two-step protocols are proposed taking 4−8 h and yielding combined or separate fractions of hydrolyzed oligosaccharides and liberated hydrolyzed protein. Optimized procedures resulted in the solubilization of >80% of the proteinaceous material, up to 39% of the total carbohydrates, and up to 42% of total dry matter in BSG. Of the original xylan present in BSG, 36% could be solubilized. Sequential and simultaneous treatments with the two enzyme types gave similar results. In sequential processes, the order of the carbohydrase and peptidase treatments had only minor effects on the outcome. Depol 740 released more pentoses than Econase and gave slightly higher overall dry matter solubilization yields.

Content of dietary fiber and dietary fiber components in whole-grain rye (n = 18) were analyzed. The average total content, when fructan was included, was for dietary fiber 19.9% (range of 18.7−22.2%) and for extractable dietary fiber 7.4% (range of 6.9−7.9%). Arabinoxylan was the main dietary fiber component, with an average total content of 8.6%, followed by fructan (4.1%). During baking of whole-grain rye bread, only small changes in total content of arabinoxylan, arabinogalactan, and β-glucan occurred, while the content of resistant starch increased and the content of fructan decreased in a baking-method-dependent manner. The molecular-weight distribution of extractable arabinoxylan in the flour was analyzed with a new method and ranged from 4 × 10⁴ to 9 × 10⁶ g/mol, with a weight average molecular weight of about 2 × 10⁶ g/mol. During crisp bread making, only a limited degradation of arabinoxylan molecular weight was detected, while a notable degradation was observed in sour-dough bread. The molecular weight of extractable β-glucan in the whole-grain rye flour ranged from 10⁴ to 5 × 10⁶ g/mol, with a weight average molecular weight of 0.97 × 10⁶ g/mol. During bread making, the molecular weight of the β-glucan was substantially degraded.