Extraction and Physicochemical Characterization of Rye β‐Glucan and Effects of Barium on Polysaccharide Molecular Weight

Use of saturated Ba(OH)₂ to extract rye β‐glucan led to a depolymerized product. Similar depolymerization of β‐glucan was observed when oat bran was extracted with this reagent. Isolated oat β‐glucan, detarium xyloglucan, guar galactomannan, and wheat and rye arabinoxylan were also depolymerized by treatment with the barium reagent. The degree of depolymerization was related to time of contact with, and concentration of, the barium. Rye β‐glucan of two different molecular weights (MW) were isolated and characterized. The structure of rye β‐glucan, as evaluated from the ratio of (1→3)‐linked cellotriosyl to (1→3)‐linked cellotetraosyl primary structural units, most closely resembles barley β‐glucan. Analytical variability of this ratio is discussed. A freshly prepared solution (2%) of the higher MW sample showed shear thinning behavior typical of cereal β‐glucans. The lower MW sample at 2% was not shear thinning, but on further purification, after storage for seven days, a 6% solution had gelled as shown by the mechanical spectrum.     

Molecular Weight Distribution of β‐Glucan in Oat‐Based Foods

Oats, different oat fractions as well as experimental and commercial oat‐based foods, were extracted with hot water containing thermostable α‐amylase. Average molecular weight and molecular weight distributions of β‐glucan in extracts were analyzed with a calibrated high‐performance size‐exclusion chromatography system with Calcofluor detection, specific for the β‐glucan. Oats, rolled oats, oat bran, and oat bran concentrates all had high Calcofluor average molecular weights (206 × 10⁴ to 230 × 10⁴ g/mol) and essentially monomodal distributions. Of the oat‐containing experimental foods, extruded flakes, macaroni, and muffins all had high average molecular weights. Pasteurized apple juice, fresh pasta, and teacake, on the other hand, contained degraded β‐glucan. Calcofluor average molecular weights varied from 24 × 10⁴ to 167 × 10⁴ g/mol in different types of oat bran‐based breads baked with almost the same ingredients. Large particle size of the bran and short fermentation time limited the β‐glucan degradation during baking. The polymodal distributions of β‐glucan in these breads indicated that this degradation was enzymatic in nature. Commercial oat foods also showed large variation in Calcofluor average molecular weight (from 19 × 10⁴ g/mol for pancake batter to 201 × 10⁴ g/mol for porridge). Boiling porridge or frying pancakes did not result in any β‐glucan degradation. These large differences in molecular weight distribution for β‐glucan in different oat products are very likely to be of nutritional importance.     

Factors Influencing β‐Glucan Levels and Molecular Weight in Cereal‐Based Products

The beneficial role of soluble dietary fiber in human nutrition is well documented and has lead to a growing demand for the incorporation of β‐glucan, particularly from oats and barley, into foods. β‐Glucan with high solubility and high molecular weight distribution results in increased viscosity in the human intestine, which is desirable for increased physiological activity. Molecular weight, level, and solubility of β‐glucan are affected by genotype, environment, agronomic input, and the interactions of these factors and food processing methods. Available literature reveals that the level of β‐glucan in a finished product (e.g. bread, cake, muffins) depends upon several factors in the production chain, whereas food processing operations are major factors affecting molecular weight and solubility of β‐glucans. Therefore, to avail themselves of the natural bioactive compounds, food manufacturers must pay attention not only to ensure sufficient concentration of β‐glucan in the raw material but also to the processing methods and functional properties of β‐glucan, minimizing enzymatic or mechanical breakdown of the β‐glucans in end‐product and optimizing processing conditions. This review discusses the different sources of β‐glucan for use in human functional foods and factors affecting the levels and the molecular weight of β‐glucan at various pre‐ and postharvest operations.     

Development of an Orange‐Flavored Barley β‐Glucan Beverage

Barley β‐glucan concentrate shows great potential as a functional food ingredient, but few product applications exist. The objectives of this study were to formulate a functional beverage utilizing barley β‐glucan concentrate, and to make a sensory evaluation of beverage quality in comparison to pectin beverages and to assess shelf stability over 12 weeks. Three beverage treatments containing 0.3, 0.5, and 0.7% (w/w) barley β‐glucan were developed in triplicate. Trained panelists found peely‐ and fruity‐orange aroma and sweetness intensity to be similar (P > 0.05) for all beverages tested. Beverage sourness intensity differed among beverages (P ≤ 0.05). Panelists evaluated beverages containing 0.3% hydrocolloid as similar (P > 0.05), whereas beverages with 0.5 and 0.7% β‐glucan were more viscous (P ≤ 0.05) than those with pectin at these levels. Acceptability of beverages was similar according to the consumer panel. Shelf stability studies showed no microbial growth and stable pH for all beverages over 12 weeks. Colorimeter values for most beverages decreased (P ≤ 0.05) during the first week of storage, mostly stabilizing thereafter. With an increase in concentration, β‐glucan beverages became lighter in color (P ≤ 0.05) and cloudier, but these attributes for pectin beverages were not affected (P > 0.05). β‐Glucan beverages exhibited cloud loss during the first three weeks of storage. β‐Glucan can therefore be successfully utilized in the production of a functional beverage acceptable to consumers.     

Extraction of β‐Glucan from Oat Bran in Laboratory Scale

Effects of various enzymes and extraction conditions on yield and molecular weight of β‐glucans extracted from two batches of commercial oat bran produced in Sweden are reported. Hot‐water extraction with a thermostable α‐amylase resulted in an extraction yield of ≈76% of the β‐glucans, while the high peak molecular weight was maintained (1.6 × 10⁶). A subsequent protein hydrolysis significantly reduced the peak molecular weight of β‐glucans (by pancreatin to 908 × 10³ and by papain to 56 × 10³). These results suggest that the protein hydrolyzing enzymes may not be pure enough for purifying β‐glucans. The isolation scheme consisted of removal of lipids with ethanol extraction, enzymatic digestion of starch with α‐amylase, enzymatic digestion of protein using protease, centrifugation to remove insoluble material, removal of low molecular weight components using dialysis, precipitation of β‐glucans with ethanol, and air‐drying.     

Release of β‐Glucan from Cell Walls of Starchy Endosperm of Barley

β‐Glucan can be solubilized from barley by warm water, with increasing solubilization as the temperature is increased. Substantially less glucan is extracted if the barley is dehusked using sulfuric acid, particularly if the dehusked barley is denatured. This indicates that enzymes capable of solubilizing glucan are present in barley. Various purified enzymes promote the solubilization of glucan from denatured and dehusked barley. Apart from endo‐β‐(1→3)(1→4)‐glucanase, these enzymes include endo‐xylanases, arabinofuranosidase, xyloacetylesterase, and feruloyl esterase. Ferulic acid and, probably, acetyl groups are esterlinked to arabinoxylan, not β‐glucan, in the cell walls of barley starchy endosperm, so the ability of the esterases, xylanases, and arabinofuranosidase to solubilize glucan indicates the pentosan component of the cell wall can restrict the extraction of glucan.     

Effect of Processing on Viscosity and Molecular Weight of (1,3)(1,4)‐β‐Glucan in Western Australian Oat Cultivars

Six Australian milling oat cultivars grown over two growing seasons were characterized for differences in (1,3)(1,4)‐β‐glucan (β‐glucan) viscosity, solubility, molecular weight (M_w), and the effect of processing. Oat cultivars grown in 2012 had significantly higher extracted β‐glucan viscosity from oat flour than the same oat cultivar grown in 2011 (P < 0.05, mean 137 and 165 cP, respectively). Noodle β‐glucan mean viscosity for 2012 (147 cP) was significantly higher than for 2011 (128 cP). β‐Glucan from ‘Williams’ and ‘Mitika’ oats had the highest viscosity (P < 0.05) in flour (5.92 and 5.25%, respectively) and noodles (1.64 and 1.47%, respectively) for both years, compared with the other oat cultivars. β‐Glucan (M_w) of Williams for 2012 and ‘Kojonup’ for both years were the least affected by processing, with an average drop of 33% compared with a maximum of 63% for other cultivars. Therefore, Williams showed superior β‐glucan properties to other oat cultivars studied, and can potentially provide improved health benefits. High and low β‐glucan M_w populations were found in the same elution peak after processing. Oat cultivars chosen for processing should be those with β‐glucans that are more resistant to processing, and that maintain their physiochemical properties and, therefore, bioactivity.     

Extractability,Structure and Molecular Weight of β‐Glucan from Canadian Rye (Secale cereale L.) Whole Meal

Oat and barley (1→3)(1→4)‐β‐d ‐glucans (β‐glucan) are readily extracted by hot water but rye β‐glucan is resistant to such extraction. This poor extractability might be due to entrapment within a matrix of arabinoxylan (AX) cross‐linked through phenolic constituents. AX are the major nonstarch polysaccharides of the rye kernel. In this study, several approaches were compared in an effort to determine optimum conditions for extraction of high yields, high molecular weight (MW), and high purity of β‐glucan from Canadian rye whole meal. Variables investigated included sodium hydroxide concentrations, extraction time, sample prehydration, extraction under low temperature, and prior extraction of AX with barium hydroxide. There was a linear relationship between the strength of NaOH and amount of β‐glucan extracted and because MW was essentially the same up to 1.0N NaOH, this extraction agent, at room temperature for 90 min, was selected to isolate rye β‐glucan. The β‐glucan was then purified and structure and molecular weight distribution studied.     

Distribution of β‐Glucan in the Grain of Hull‐less Barley

Nine hull‐less barley (HB) containing waxy (0–7% amylose), normal (≈25% amylose), or high amylose (≈42% amylose) starch with normal or fractured granule make‐up and 4–9% (1→3)(1→4)‐β‐d ‐glucans (β‐glucan) were pearled to remove 70% of the original grain weight in 10% intervals. The pearled fractions were analyzed for β‐glucan distribution within HB grain. Protein content of the pearled fractions indicated that the three outermost fractions contained pericarp and testa, aleurone, and subaleurone tissues, respectively. For all HB, β‐glucan and acid‐extract viscosity were very low in the outermost 20% of the kernel. For low β‐glucan HB, β‐glucan content was the greatest in the subaleurone region and declined slightly toward inner layers. For high β‐glucan HB, however, more than 80% of grain β‐glucan was distributed more evenly throughout the endosperm. Acid extract viscosity was significantly (P < 0.01) correlated with total (r = 0.75) and soluble (r = 0.87) β‐glucan content throughout the kernel of all HB. Growing conditions, location and year, had significant effects on the concentration of protein, starch and β‐glucan. However, protein, starch, and β‐glucan distribution patterns were not affected by growing conditions. The difference in β‐glucan distribution between low and high β‐glucan HB may explain the difference in milling performance of HB with low or high β‐glucan.     

Determination of β‐Glucan Molecular Weight Using SEC with Calcofluor Detection in Cereal Extracts

A high‐performance size‐exclusion chromatography system (HPSEC) was set up with detection based on the specific binding of Calcofluor to β‐glucan for determination of amount and molecular weight of β‐glucan in different cereal extracts. To calibrate the HPSEC system, a purified β‐glucan was fractionated into narrow molecular weight ranges and the average molecular weight was determined before analysis on the HPSEC system. The detector response was similar for β‐glucans from oats and barley and appeared to be independent of molecular weight. Four different methods for extraction of β‐glucan from different cereal products were tested: two alkaline, one with hot water and added α‐amylase, and one with water and added xylanase. Inactivation of endogenous β‐glucanase was crucial for the stability of the extracts, even when extracting at high temperature or pH. Yields varied widely between the different extraction methods but average molecular weight and molecular weight distribution were similar. Extraction with sodium hydroxide generally gave a higher yield and molecular weight of β‐glucan in the extracts.     

Glycemic Response to Oat Bran Muffins Treated to Vary Molecular Weight of β‐Glucan

Oat bran muffins, containing 4 or 8 g of β‐glucan per two‐muffin serving, were prepared with or without β‐glucanase treatment to produce a range of β‐glucan molecular weights from 130,000 to just over 2 million. Following an overnight fast, the glycemic responses elicited by the untreated and treated muffins was measured in 10 healthy subjects and compared with a control whole wheat muffin. Taken all together, the 4‐g β‐glucan/serving muffins reduced blood glucose peak rise (PBGR) by 15 ± 6% compared with the control. The 8‐g β‐glucan/serving muffins had a significantly greater effect (44 ± 5% reduction compared with the control, P < 0.05). The efficacy of the muffins decreased as the molecular weight was reduced from a 45 ± 6% reduction in PBGR (P < 0.05) for the untreated muffins (averaged of both serving sizes) to 15 ± 6% (P < 0.05) for muffins with the lowest molecular weight. As the molecular weight was reduced from 2,200,000 to 400,000, the solubility of the β‐glucan increased from a mean of 44 to 57%, but as the molecular weight was further decreased to 120,000, solubility fell to 26%. There was a significant correlation (r² = 0.729, P < 0.001) between the peak blood glucose and the product of the extractable β‐glucan content and the molecular weight of the β‐glucan extracted.     

Preparation and Characterization of Films Using Barley and Oat β‐Glucan Extracts

Films for potential food use were prepared from aqueous solutions of β‐glucan extracted from hulled barley, hull‐less barley, and oats. The extracts (75.2–79.3% β‐glucan) also contained proteins, fat, and ash. Glycerol was used as a plasticizer. The films were translucent, smooth, and homogeneous in structure on both sides. Water vapor permeability of films prepared from 4% solutions of β‐glucan extracts were higher than those from 2% solutions, despite similar values for water vapor transmission rate. Mechanical properties were influenced by both β‐glucan source and concentration. The oat β‐glucan films showed higher tensile strength and water solubility, and lower color, opacity, and deformation values than those of barley. Films prepared from hull‐less barley cv. HLB233 remained intact upon immersion in water for 24 hr.     

β-Glucanase Activity and Molecular Weight of β-Glucans in Barley After Various Treatments

β-Glucanase activity interferes with molecular characterization of mixed-linkage (1→3)(1→4)-β-d -glucans (β-glucans). Reductions in β-glucanase activity were determined after barley cvs. Azhul, Waxbar, and Baronesse were treated with autoclaving (120°C, 45 min), calcium chloride (0.05M, 1 hr), 70% ethanol (80°C, 4 hr), hydrochloric acid (0.1N, 1 hr), oven heating (120 and 140°C, 40 min), sodium hydroxide (0.0025M, 1 hr), and 5% trichloroacetic acid (TCA) (40°C, 1 hr). High-performance size-exclusion chromatography (HPSEC) of α-amylase-treated aqueous extracts was used to demonstrate the effects of treatments on the molecular weights of β-glucans. The HPSEC system included multiple-angle, laser light scattering, refractive index, and fluorescence detectors. β-Glucanase activities, ranging from 52 to 65 U/kg of barley, were reduced by autoclaving (50–75%), hot alcohol (67–76%), oven heating (40–96%), CaCl₂ (75–95%), NaOH (76–89%), and TCA (92–96%). Some malt β-glucanase activity remained after most treatments. HCl and TCA treatments reduced extraction and molecular weights of β-glucans. Weight-average molecular weights (M_w) for β-glucans extracted with water at 23°C were low (most <8 × 10⁵). Base treatment (pH 9) and extraction at 100°C for 2.5 hr resulted in the greatest extraction of β-glucans and highest M_w. As a result, the conditions seem appropriate for measurement of physical characteristics of β-glucans in cereal products.     

Extraction of β‐Glucan from Oats for Soluble Dietary Fiber Quality Analysis

Extraction protocols for β‐glucan from oat flour were tested to determine optimal conditions for β‐glucan quality testing, which included extractability and molecular weight. We found mass yields of β‐glucan were constant at all temperatures, pH values, and flour‐to‐water ratios, as long as sufficient time and enough repeat extractions were performed and no hydrolytic enzymes were present. Extracts contained about 30–60% β‐glucan, with lower proportions associated with higher extraction temperatures in which more starch and protein were extracted. All commercial starch hydrolytic enzymes tested, even those that are considered homogenous, degraded β‐glucan apparent molecular weight as evaluated by size‐exclusion chromatography. Higher concentration β‐glucan solutions could be prepared by controlling the flour‐to‐water ratio in extractions. Eight grams of flour per 50 mL of water generated the highest native β‐glucan concentrations. Routine extractions contained 2 g of enzyme‐inactivated flour in 50 mL of water with 5mM sodium azide (as an antimicrobial), which were stirred overnight, centrifuged, and the supernatant boiled for 10 min. The polymer extracted had a molecular weight of about 2 million and was stable at room temperature for at least a month.     

High‐Starch and High‐β‐Glucan Barley Fractions Milled with Experimental Mills

This study focused on the performance of two hulless barley cultivars (Doyce and Merlin) and one commercial husked (hulled) sample using experimental milling. The purpose was to use experimental milling as a preliminary indicator of the milled streams with potential use for fuel ethanol production and fractions that could be used in food products. Experimental mills designed for flour production evaluation from wheat were Chopin CD1 Auto, Quadrumat Sr, Buhler, and an experimental Ross roller mill walking flow. Results indicate that the shorts had the highest levels of β‐glucan from all the mills. However, the β‐glucan content in the break flours was highest with the roller mill walking flow and the Chopin CD1 for the hulless cultivars. The lowest β‐glucan content in the break flour was found with the Buhler for Doyce. Break flour and, to a slightly lesser extent, reduction flour from all cultivars tested on all mills contained the highest starch content (up to 83%) and are therefore most appropriate for use as feedstock for fuel ethanol production. Conversely, bran and shorts from all cultivars and mills were lowest in starch (as low as 25%), making them ideal as low‐starch food ingredients.     

Structure of (1→3)(1→4)‐β‐d‐Glucan in Waxy and Nonwaxy Barley

The endosperm cell walls of barley are composed largely of a (1→3)(1→4)‐β‐d ‐glucan commonly known simply as β‐d ‐glucan (Wood 2001). There has been much research into the characteristics of barley β‐glucan because of the influence of this polysaccharide on performance of barley in malting and subsequent brewing of beer, and in feed value, especially for young chicks (MacGregor and Fincher 1993). The potential for β‐glucan to develop high viscosity is a problem in these uses, but from the perspective of human nutrition, this characteristic may be an advantage. The glycemic response to oat β‐glucan is inversely related to (log)viscosity (Wood et al 1994a) and there is evidence to suggest that the lowering of serum cholesterol levels associated with oat and barley products (Lupton et al 1994; Wood and Beer 1998) is at least in part due to the β‐glucan (Braaten et al 1994) and probably also its capacity to develop viscosity in the gastrointestinal tract (Haskell et al 1992).     

Viscosity and Solubility of β‐Glucan Extracted Under In Vitro Conditions from Barley β‐Glucan‐Fortified Bread and Evaluation of Loaf Characteristics

Fortifying bread with β‐glucan has been shown to reduce bread quality and the associated health benefits of barley β‐glucan. Fortification of bread using β‐glucan concentrates that are less soluble during bread preparation steps has not been investigated. The effects of β‐glucan concentration and gluten addition on the physicochemical properties of bread and β‐glucan solubility and viscosity were investigated using a less soluble β‐glucan concentrate, as were the effects of baking temperature and prior β‐glucan solubilization. Fortification of bread with β‐glucan decreased loaf volume and height (P ≤ 0.05) and increased firmness (P ≤ 0.05). Gluten addition to bread at the highest β‐glucan level increased height and volume (P ≤ 0.05) to values exceeding those for the control and decreased firmness (P ≤ 0.05). β‐Glucan addition increased (P ≤ 0.05) extract viscosity, as did gluten addition to the bread with the highest β‐glucan level. Baking at low temperature decreased (P ≤ 0.05) β‐glucan viscosity and solubility, as did solubilizing it prior to dough formulation. Utilization of β‐glucan that is less soluble during bread preparation may hold the key to effectively fortifying bread with β‐glucan without compromising its health benefits, although more research is required.     

Prediction of β‐Glucan Concentration Based on Viscosity Evaluations of Raw Oat Flours from High β‐Glucan and Traditional Oat Lines

Rheological properties of raw oat flour slurries were determined in experimental high β‐glucan (≤7.8%) and traditional oat lines (4–5% β‐glucan) grown in two consecutive years. Three different media were used to disperse oat flours: deionized water, silver nitrate solution (to inactivate endogenous enzymes), and alkali solution (to solubilize both water‐soluble and water‐insoluble β‐glucans). Significant correlations (P < 0.05) between viscosity of slurries and β‐glucan concentration obtained in either deionized water (r = 0.833), silver nitrate (r = 0.940), or alkali (r = 0.896) solutions showed that β‐glucans were the main contributor to oat extract viscosity. The highest correlation was obtained in silver nitrate solution, suggesting that inactivating endogenous enzymes is important to obtain high correlations. Predictive models of oat β‐glucan concentration based on the viscosity profile were developed using partial least squares (PLS) regression. Prediction of β‐glucan concentration based on viscosity was most effective in the silver nitrate solution (r = 0.949, correlation coefficient of predicted vs. analyzed β‐glucans) and least effective in the alkali solution (r = 0.870). These findings demonstrate that the β‐glucan in oat could be predicted by measuring the viscosity of raw flours in silver nitrate solution, and this method could be used as a screening tool for selective breeding.     

Effects of β‐Glucan Content and Pearling of Barley in Diet‐Induced Obese Mice

The effects of the β‐glucan content and pearling of barley on abdominal obesity and the proinflammatory state were investigated in diet‐induced obese mice. Male C57BL/6J mice were randomly divided into four groups and fed either a high‐fat diet containing high‐β‐glucan barley (Beau Fiber [BF]) or a high‐fat diet containing β‐glucan‐free barley (Shikoku‐hadaka 84(bgl ) [BGL]) as whole grain flour or 60% pearled flour for 12 weeks. The weights of mesenteric fat, serum total and low density lipoprotein cholesterol levels, serum insulin and fasting glucose levels, oral glucose tolerance test results, and messenger RNA (mRNA) expression of proinflammatory markers in epididymal fat in both BF groups were significantly lower than those of both BGL groups. The abundance of Bacteroides in both BF groups was significantly higher than that in both BGL groups, whereas the abundance of Clostridium clusters in both BF groups was significantly lower than that in both BGL groups. No significant differences between the whole grain and pearled flours were observed. These results suggest that high‐β‐glucan barley attenuates the progression of abdominal obesity and the proinflammatory state in diet‐induced obese mice compared with β‐glucan‐free barley, possibly by modifying insulin secretion and the microbiota.     

Content and Molecular Weight Distribution of Oat β‐Glucan in Oatrim,Nutrim, and C‐Trim Products

The soluble fiber, β‐glucan, in oat products is an active hypolipidemic component that is responsible for lowering plasma lipids. Quantitative analysis of β‐glucan in oat hydrocolloids such as Oatrim, Nutrim, and C‐Trim was performed to measure the total β‐glucan content and molecular weight distribution. For the measurement of total β‐glucan content, both modified flow‐injection analysis (FIA) method and the standard AACC enzymatic method were employed. FIA method uses the enhanced fluorescence produced when β‐glucan forms complexes with Calcofluor. Experimental results of both the modified FIA method and the standard AACC enzymatic method revealed very good coincidence with each other. This result confirms the applicability of either technique for the quantitative evaluation of β‐glucan in hydrocolloids. Molecular weight (MW) distribution of β‐glucan was determined by size‐exclusion chromatography with postcolumn detection. Experimental results revealed that the molecular weight of β‐glucan in the Trim products was decreased during the manufacturing process. This result was ascribed to the rigorous processing condition of jet‐cooking.