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.     

In Vitro Binding of Bile Acids by Rice Bran,Oat Bran,Barley and β‐Glucan Enriched Barley

The in vitro bile acid binding by rice bran, oat bran, dehulled barley, and β‐glucan enriched barley was determined using a mixture of bile acids at a duodenal physiological pH of 6.3. Six treatments and two blank incubations were conducted testing substrates on an equal protein basis. The relative in vitro bile acid binding of the cereal brans on an equal total dietary fiber (TDF) and insoluble dietary fiber (IDF) basis considering cholestyramine as 100% bound was rice bran 45 and 49%; oat bran 23 and 30%; dehulled barley 33 and 57%; and β‐glucan enriched barley 20 and 40%, respectively. Bile acid bindings on equal protein basis for the respective cereals were 68, 26, 41, and 49%. Bile acid binding by rice bran may account to a great extent for its cholesterol‐lowering properties, while bile acid binding by oat bran suggests that the primary mechanism of cholesterol lowering by oat bran is not due to the bile acid binding by its soluble fiber. Bile acid binding was not proportional to the soluble fiber content of the cereal brans tested. Except for dehulled barley, bile acid binding for rice bran, oat bran, and β‐glucan enriched barley appear to be related to their IDF content. Highest relative bile acid binding values for rice bran and β‐glucan enriched barley were observed on an equal protein basis, whereas highest values for dehulled barley were based on IDF. Data suggest that of all four cereals tested, bile acid binding may be related to IDF or protein anionic, cationic, physical and chemical structure, composition, metabolites, or their interaction with active binding sites.     

Effect of Heat Treatments on Microbial Load and Associated Changes to β‐Glucan Physicochemical Properties in Whole Grain Barley

Health claims for barley β‐glucan (BG) have prompted the development of food products containing barley; however, some new products (such as milled grain used without a cook step, as in a smoothie) do not use any form of heat treatment during processing or prior to consumption, which could affect microbial safety and potential health benefits. The aims of this research were to evaluate current commercial barley products for microbial counts and BG characteristics and to determine the effects of different heat treatments on these attributes in whole grain barley samples. Three heat treatments (micronization, roasting, and conditioning) were performed on three cultivars of barley (CDC Rattan, CDC McGwire, and CDC Fibar). The microbial quality was measured with standard plate count (SPC), mold and yeast count (MYC), and coliforms or Escherichia coli . Only four of the 17 commercial barley products tested met acceptable microbial limits used in this study. All three heat treatments applied to the barley samples in this study reduced SPC, MYC, and coliforms to an acceptable level. BG was extracted with an in vitro digestion method to determine its viscosity, molecular weight (MW), and solubility. All three heat treatments produced BG extracts with high viscosity and MW compared with untreated barley. Overall, heat treatments improved both the safety and the potential health benefits from soluble BG in whole grain barley.     

Gastrointestinal Release of β‐Glucan and Pectin Using an In Vitro Method

The release of soluble dietary fiber is a prerequisite for viscous effects and hence beneficial health properties. A simple in vitro method was adapted to follow the release during gastrointestinal digestion, and the percentage of solubilized fiber was measured over time. β‐Glucan from oat bran was mainly released during gastric digestion while the release of pectin from sugar beet fiber continued in the small intestine. Unmilled fractions of sugar beet fiber released more soluble fiber than oat bran flakes, probably due to the porous structure of sugar beet fiber as a result of manufacturing processes, but also due to differences in source. Milling to smaller fiber particles significantly improved releasability (from 20 to 55% released β‐glucan and from 50 to 70% released pectin, respectively, after digestion). When milled fibers were included in individual food matrices, the release was reduced by protein and starch matrices (5% β‐glucan and 35% pectin released, respectively) and slowed by fat (45% β‐glucan and 60% pectin released). This may result in a too low or too late release in the upper small intestine to be able to interfere with macronutrient uptake. The method may be suitable for predicting the gastrointestinal release of soluble dietary fibers from food matrices in the development of healthy food products.     

Effect of Health Information on Consumer Acceptability of Bread Fortified with β‐Glucan and Effect of Fortification on Bread Quality

Fortifying bread with β‐glucan reduces bread sensorial properties, though fortification using β‐glucan concentrates of low solubility under the conditions of dough preparation has not been investigated. This study investigated the consumer acceptability and purchase intent of bread fortified with a less soluble β‐glucan concentrate at levels corresponding to 0, 0.75, and 1.5 g β‐glucan/serving bread in relation to the provision of health information, gender, and whole wheat bread consumption. The effect of β‐glucan concentration on the physical properties of the bread produced under pilot plant settings was also investigated. β‐Glucan addition decreased (P < 0.05) loaf volume, increased firmness, and produced a darker, redder bread (P < 0.05), though fortification at 1.5 g β‐glucan/serving bread decreased height as well (P < 0.05). Consumer evaluation (n = 122) revealed that health information increased liking of appearance, flavor, and overall acceptability of the 1.5 g/serving bread to levels similar to or exceeding that of the control. Liking of the 1.5 g β‐glucan/serving bread appearance increased more in women than in men and for consumers who regularly consumed whole wheat bread for perceived health benefits when β‐glucan health information was provided. The provision of β‐glucan health information may be the key to increasing consumer acceptability of bread fortified with β‐glucan.     

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.     

Wet Processing of Barley Grains into Concentrates of Proteins, β‐Glucan,and Starch

An improved wet method was developed to process barley into fractions concentrated in protein, (1‐3)(1‐4)‐β‐d ‐glucan (BG), starch, or other carbohydrates (CHO). Alkaline concentration, solvent to barley flour ratio (SFR), and extraction temperature were evaluated for their effects on concentration and recovery of protein, BG, starch, oil, ash, and other CHO in each fraction type. Results show that the three parameters and their interactions all had significant effects, resulting in varying nutrient concentrations and recovery rates in each type of fractions. For protein fractions, protein content varied from 37.7 to 75.2%, protein recovery from 8.5 to 75.7%, and increasing alkaline concentration and SFR improved nutrient recovery. For BG fractions, BG content ranged from 21.5 to 87.0%, BG recovery from 28.6 to 78.0%, and increasing alkaline concentration decreased BG content but increased its recovery significantly. For starch fractions, starch content varied from 76.9 to 93.9%, starch recovery from 33.6 to 63.9%, and all parameters had little effect on the nutrient concentrations, but alkaline concentration and SFR improved recovery of starch, other CHO, and mass. Overall, the improved wet method was effective in concentrating the major nutrients from barley into their respective fractions, but process optimization through manipulating the three parameters is necessary to achieve a maximum concentration or recovery rate of a nutrient of interest in a specific fraction.     

Dough Properties and Bread Quality of Wheat–Barley Composite Flour as Affected by β‐Glucanase

Barley is rich in nutritionally positive compounds, but the quality of bread made of wheat–barley composite flours is impaired when a high percentage of barley is used in the mixture. A number of enzymes have been reported to be useful additives in breadmaking. However, the effect of β‐glucanase on breadmaking has scarcely been investigated. In this paper, the influence of different levels (0.02, 0.04, 0.06, and 0.08%, based on composite flour) of β‐glucanase (100,000 U/g) on the properties of dough and bread from 70% wheat, 30% barley composite flour were studied. Although dough development time, dough stability, and protein weakening value decreased after β‐glucanase addition, dough properties such as softness and elasticity as well as bread microstructure were improved compared with the control dough. β‐Glucanase also significantly improved the volume, texture, and shelf life of wheat–barley composite breads. The use of an optimal enzyme concentration (0.04%) increased specific volume (57.5%) and springiness (21%), and it reduced crumb firmness (74%) and staling rate. Bread with added β‐glucanase had a better taste, softness, and overall acceptability of sensory characteristics compared with the control bread. Moreover, the quality of wheat–barley composite bread after addition of 0.04% β‐glucanase was nearly equal to the quality of pure wheat bread. These results indicate that dough rheological characteristics and bread quality of wheat–barley composite flour can be improved by adding a distinct level of β‐glucanase.     

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.     

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.     

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.     

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.     

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.     

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.     

Effect of Extraction Conditions on Yield,Composition, and Viscosity Stability of Barley β-Glucan Gum

Cereal β-glucan can function as a thickener, but endogenous β-glucanase enzymes of the grain cleave β-glucan, reducing its viscosity. Although different extraction techniques have been developed, the viscosity stability of β-glucan gum has not been reported. The objective of this study was to investigate the effect of extraction treatments on the yield, purity, and viscosity stability of barley β-glucan (BBG) gum. A regular barley cultivar, Condor, and a waxy cultivar blend were extracted at pH 7–10 and 55°C for 0.5 hr. Four extraction conditions were evaluated: 1) extraction at high pH with no additional heat treatment; 2) boiling of extract; 3) prior refluxing of flour with 70% ethanol; and 4) treatment of extract with thermostable α-amylase for purification. Viscosity of extracts was monitored for ≥24 hr at 25°C. The highest β-glucan purities were achieved with a boiled Condor extract at pH 7 (81.3% db, 4.1% yield) and with refluxed waxy barley extracted at pH 8 and treated with α-amylase and (79.3% db, 5.1% yield). Gums extracted without subsequent heat treatment or prior refluxing of flour had high protein (>17%) and starch (>24%) impurities, respectively. The viscosity of gums obtained without heating was unstable. Prior refluxing treatment was not sufficient to stabilize final extracts. Boiling extracts resulted in stable but low viscosity. Reflux followed by purification treatment produced the highest stable viscosity for 0.5% solutions of both Condor (64 mPa sec^-1, pH 7) and waxy (48.8 mPa sec^-1, pH 8) extracts. Stable BBG gum with high viscosity can be obtained using thermal treatments in combination with high pH. The potential use of such gums as thickeners in food systems needs to be assessed.     

Isolation,Fractionation, and Structural Characteristics of Alkali‐Extractable β‐Glucan from Rye Whole Meal

The main nonstarch polysaccharide of rye is arabinoxylan (AX), but rye contains significant levels of (1→3)(1→4)‐β‐d ‐glucan, which unlike oat and barley β‐glucan, is not readily extracted by water, possibly because of entrapment within a matrix of AX cross‐linked by phenolics. This study continues objectives to improve understanding of factors controlling the physicochemical behavior of the cereal β‐glucans. Rye β‐glucan was extracted by 1.0N NaOH and increasing concentrations of ammonium sulfate were used to separate the β‐glucan from AX and prepare a series of eight narrow molecular weight (MW) distribution fractions. Composition and structural characteristics of the isolated β‐glucan and the eight fractions were determined. High‐performance size‐exclusion chromatography (HPSEC) with both specific calcofluor binding and a triple detection (light scattering, viscometry, and refractive index) system was used for MW determination. Lichenase digestion followed by high‐performance anion exchange chromatography of released oligosaccharides, was used for structural evaluation. The overall structure of all fractions was similar to that of barley β‐glucan.     

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.     

Effect of Dough Processing Conditions and DATEM on Norwegian Hearth Bread Prepared from Frozen Dough

The objective of this study was to examine treatments that directly influence Norwegian lean doughs destined to be frozen. Therefore a strip-block experimental design with four dough treatment factors (wheat flour blend, diacetyl tartaric acid esters of monoglycerides [DATEM], water absorption, and dough temperature) and two storage factors (frozen storage time and thawing time) was used. Four levels were selected for frozen storage time and two levels were selected for the remaining factors. After frozen storage (2–70 days), the doughs were thawed and baked. Principal component analysis showed that to obtain a high loaf volume and bread score after freezing, a high dough temperature after mixing (27°C) was essential. The highest form ratio (height/width) level was obtained after 28 days of frozen storage and with a short thawing time (6 hr). Analysis of variance (ANOVA) of dough treatments showed that an increase in dough temperature from 20 to 27°C after mixing resulted in a significant increase in loaf volume (1,653 to 2,264 mL), form ratio (0.64 to 0.69), and bread score (1.7 to 3.2), and a reduction in loaf weight (518.4 to 512.5 g) and crumb score (7.9 to 5.9, i.e., a more open bread crumb). Also, the addition of DATEM significantly increased loaf volume (1,835 to 2,081 mL), form ratio (0.64 to 0.69), and bread score (2.2 to 2.6). Frozen dough storage time significantly affected loaf volume, loaf weight, bread score, and crumb score. Increasing thawing time from 6 to 10 hr significantly increased loaf volume (1,855 to 2,121 mL), and reduced the form ratio (0.69 to 0.63) and loaf weight (516.8 to 511.4 g). ANOVA of the interaction between dough treatment and frozen storage time showed that decreasing water absorption significantly increased the loaf volume.     

Molecular Weight of β‐Glucan Affects Physical Characteristics,In Vitro Bile Acid Binding,and Fermentation of Muffins

Muffins containing different amounts and molecular weights (MW) of β‐glucan were evaluated for the effect of β‐glucan on the physical characteristics of the muffins and on in vitro bile acid binding and fermentation with human fecal flora. Wheat flour muffins were prepared with the addition of β‐glucan extracts with high‐, medium‐, or low‐MW. For oat flour muffins, the native oat flour contained high‐MW β‐glucan; the oat flours were treated to create medium‐ and low‐MW β‐glucan within the prepared muffin treatments. For each 60‐g muffin, the amounts of β‐glucan were 0.52, 0.57, and 0.59 g for high‐, medium‐, and low‐MW β‐glucan wheat flour muffins, and 2.38, 2.18, and 2.23 g for high‐, medium‐, and low‐MW β‐glucan oat flour muffins, respectively. The lower the MW of the β‐glucan in muffins, the lower the height and volume of the muffins. The oat flour muffins were less firm and springy than the wheat flour muffins as measured on a texture analyzer; however, MW had no effect on muffin texture. The oat flour muffins bound more bile acid than did the wheat flour muffins. The muffins with high‐MW β‐glucan bound more bile acid than did those with low‐ and medium‐MW β‐glucan. Muffin treatment affected the formation of gas and total short‐chain fatty acids (SCFA) compared with the blank without substrate during in vitro fermentation. There were no differences in pH changes and total gas production among muffin treatments. The high‐MW β‐glucan wheat flour muffins produced greater amounts of SCFA than did the wheat flour muffin without β‐glucan and the oat flour muffins; however, there were no differences in SCFA production among muffins with different MW. In general, the β‐glucan MW affected the physical qualities of muffins and some potential biological functions in humans.