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.

Traditional noodle samples (erişte) were supplemented with hull-less barley and lentil flours as the source of β-glucan and protein at different ratios and their cooking quality, phenolic content, antioxidant capacity and estimated GI values were evaluated. The estimated GI of control erişte produced from wheat flour was the highest (74.7), while GI of those supplemented with 15%, 30%, 45% barley or lentil flour were 68.7%, 66.0%, 61.2% and 67.5%, 63.8%, 60.6%, respectively. GI values of mixtures of barley and lentils flours (Mix-1-4 samples) were lower (58.9-61.0). All noodles supplemented with barley and/or lentil flours had medium GI values. The erişte samples supplemented with 45% hull-less barley flour and Mix erişte samples meet the requirements of FDA health claim (0.75 g β-glucan per serving). Protein content of control sample was 16.30%, while those supplemented with lentil flour had higher protein contents (18.15%-22.36%). Hence, noodle samples supplemented with 30% and 45% lentil flour can be labeled as “high protein” and all other noodle samples can be labeled as “source of protein” according to EC Regulation because calories which can be received from proteins per serving are > 20% and > 12%, respectively. Significant increases were also observed in phenolic contents and antioxidant capacities of erişte samples supplemented with barley/lentil flours.

Oat bran addition to rice bread results in quality deterioration especially during storage, but its superfine particle size probably relieves this side effect. This study focuses on the gluten-free and rice starch-based bread. The effects of different particle sizes at medium (MOB, 359.67 μm), fine (FOB, 153.67 μm) and superfine (SOB, 35.77 μm) on the short-term storage quality of rice bread was confirmed. Oat bran addition affected pasting properties of rice flour, leading to a significant decrease in values of final viscosity and setback, which was intensified much more by the smaller particle sizes. The decrease in particle size of oat bran also led to an increase in brightness and whiteness values of rice bread. Gas-holding capacity of rice bread was reduced by MOB and FOB, including specific volume, gas cell diameter and volume, but SOB reversed this reduction and recovered to the same level as control bread. SOB mitigated the increase in bread firmness during storage and led to the highest springiness while the lowest baking loss. The water-binding capacity of rice bread with SOB was much higher than that with MOB and FOB, according to the moisture migration analysis. The inhibition of water evaporation by SOB during storage further delayed the amylopectin retrogradation and starch recrystallization. Additionally, SOB prevented the rice starch hydrolysis, exhibited an increase in RS (35.39 %), which was much higher than those of MOB (23.30%) and FOB (25.30%). These findings supported the efficient inhibition of SOB on rice bread staling.

This study aimed to assess the effects of partial and complete replacement of synthetic vitamin E (Vit E) with grape pomace extract, rich in polyphenols, and wine lees derivatives, abundant in β-glucans, on broiler performance, meat quality, health biomarkers, and intestinal integrity. There was a control group (CON) fed a basal diet with commercially recommended Vit E levels, while a second group (GP) was fed the same basal diet included zin-bacitracin as an antimicrobial growth promoter. The remaining three groups (W25, W50, W100) had 25%, 50%, and 100% of Vit E replaced by 25, 50, and 100 mg polyphenols as GAE/kg feed from grape pomace extract, respectively, supplemented also with 150 mg β-glucans/kg feed. The W25 group tended to have lower body weight compared to the CON and GP groups (2565 g vs. 2762 g and 2727 g, respectively; p = 0.056), while feed intake was significantly higher in the CON group (p = 0.021). Malondialdehyde concentrations in breast and thigh muscles were elevated in the W100 group at 24- and 72-hours post-mortem (p = 0.039 and p = 0.030, respectively). Glutathione reductase levels in blood plasma were significantly reduced (p = 0.022) in the GP, W50, and W100 groups compared to CON. Additionally, CLDN1 expression in the jejunal mucosa was significantly increased in W100 compared to the CON group (p = 0.045). These findings suggest that partial substitution of Vit E with grape pomace and wine lees derivatives by 50% could be a feasible commercial dietary strategy since growth performance, oxidative status, and gene expression were not impaired.

The prevalence of ulcerative colitis (UC) increases with unhealthy eating habits. Both surgery and medication have the potential to treat the condition, but they may also have more negative effects. This study investigated the anti-inflammatory mechanism of 20% and 40% doses of different highland barley (HB) components (whole grain, peeled, and bran) in a 2% dextran sulfate sodium induced UC mouse model. The results showed that supplementation with a 20% dose of peeled HB restored body weight, disease activity index, colon length, serum interleukin-1β and interleukin-10 levels, liver glutathione peroxidase content, and superoxide dismutase activity to normal levels in mice compared to UC mice. Moreover, the damage caused by UC to the mice's colon was significantly reduced, and the relative expression levels of interleukin-1β, interleukin-6, and tumor necrosis factor-α were all significantly downregulated. Additionally, it increased the abundance of Bacteroidota and Firmicutes, improving the balance of gut microbiota.

Plant-based milk contains high nutritional value with enriched vitamins, minerals, and essential amino acids. This study aimed to enhance the biochemical and biological properties of rice milk through yeast fermentation, using the novel fermenting strain Saccharomyces cerevisiae RSO4, which has superb fermenting ability for an innovative functional beverage. An integrated omics approach identified specific genes that exhibited genetic variants related to various cellular processes, including flavor and aroma production (ARO10, ADH1-5, and SFA1), whereas the proteomic profiles of RSO4 identified key enzymes whose expression was upregulated during fermentation of cooked rice, including the enzymes in glycogen branching (Glc3), glycolysis (Eno1, Pgk1, and Tdh1/2), stress response (Hsp26 and Hsp70), amino acid metabolism, and cell wall integrity. Biochemical and metabolomic analyses of the fermented rice milk by the RSO4 strain using the two rice varieties, Homali (Jasmine) white rice or Riceberry colored rice, detected differentially increased levels of bioactive compounds, such as β-glucan, vitamins, di- and tripeptides, as well as pleasant flavors and aromas. The results of this study highlight the importance of selecting an appropriate fermenting yeast strain and rice variety to improve property of plant-based products as innovative functional foods.