Preparation and characterization of molecular weight standards of low polydispersity from oat and barley (1→3)(1→4)-β- -glucan

Purified (1→3)(1→4)-β- -glucans (β-glucans) from oat and barley with broad molecular weight (MW) distribution were separated into seven fractions using gradient precipitation with ammonium sulfate (NH₄)₂SO₄. The MW of each fraction decreased consecutively with the concentration of (NH₄)₂SO₄ at which it was precipitated. The MW distribution of each fraction was much narrower compared to the parent sample and is comparable to commercially available pullulan MW standards. To determine whether the fractionation process was separating sub-fractions of different structure, the original β-glucan sample and each fraction were hydrolyzed by a (1→3)(1→4)-D-β-glucan-4-glucanohydrolase (lichenase, E.C.3.2.1.73) and the liberated oligosaccharides were analyzed by high performance anion exchange chromatography. The analysis revealed no differences in oligosaccharide pattern (DP 2–9) derived from each fraction and the parent sample. In particular, the tri/tetra oligosaccharide ratio remained constant for all fractions, indicating no fractionation based on structural features had taken place. The effect of starting β-glucan concentration on the fractionation process was studied. The results showed that it was possible to achieve good separation at overlapping parameter c[η] lower than 3.5. Further increase in starting β-glucan concentration hindered clear separation of the fractions. Temperature also affected the fractionation efficiency. The higher the temperature, the lower the amount of (NH₄)₂SO₄ that was necessary to precipitate the samples of same MW. A Mark Houwink relationship was derived from the measured MW and intrinsic viscosity for fractions from oat and barley, respectively.     

Physicochemical change and protein oxidation in porcine longissimus dorsi as influenced by different freeze–thaw cycles

Effects of different freeze–thaw cycles (0, 1, 3 and 5) on physicochemical change and protein oxidation in porcine longissimus dorsi were investigated. When the number of freeze–thaw cycles increased, the thawing losses, cooking loss and b*-value increased (P < 0.05), a*-value decreased (P < 0.05). The cutting forces of pork increased after one cycle of freeze–thaw (from 28.3 N to 40.4 N) (P < 0.05), but the further increase of freeze–thaw cycles would lead to decrease of cutting force. The decreases in Ca²⁺- and K⁺-ATPase activity and sulfhydryl group (P < 0.05) content with concomitant increases in carbonyl content and thiobarbituric acid-reactive substances (TBARS) value (P < 0.05) showed that multiple freeze–thaw could cause the porcine protein and fat oxidation, especially for the pork subjected to five freeze–thaw cycles. Gel electrophoresis patterns of porcine muscle showed that multiple freeze–thaw cycles could cause cross-linking of protein in myofibril. Overall, the freeze–thaw process has a detrimental effect on the quality of pork.     

Effect of partially hydrolyzed oat β-glucan on the weight gain and lipid profile of mice

Oat β-glucan hydrolysates with different molecular weights were prepared and their physicochemical, hypocholesterolemic, and weight-reducing characteristics were evaluated. The enzymatic hydrolysis by cellulase caused a decrease in the molecular weight of oat β-glucan (1450–370 × 10³ g/mol), which also affected swelling power and bile acid/fat binding capacities. In addition, mice were fed high-fat diet supplemented with β-glucans with three different molecular weights (1450, 730, 370 × 10³ g/mol). The diets treated with β-glucans significantly reduced the body weight of the mice. However, the molecular weight of β-glucans did not appear to significantly affect the serum lipid profile.     

Stability and rheology of egg-yolk-stabilized concentrated emulsions containing cereal β-glucans of varying molecular size

The effects of barley and oat β-glucans on rheological and creaming behaviour of concentrated egg-yolk-stabilized model emulsions were investigated. Four polysaccharide preparations were used, two from each cereal; one sample with high and one with low molecular weight, i.e. the molecular weights were alike in pairs (110×10³ and 40×10³, respectively). In order to elucidate the mechanism of action of β-glucans in emulsions, Tween 20-stabilized emulsions were also examined. Tween 20 enhances neither the continuous phase viscosity nor the interactions between the droplets, so the changes could be easily attributed to β-glucans. It appeared that the low Mw β-glucan samples stabilize emulsions against creaming by means of network formation in the continuous phase while their high molecular weight counterparts enhance the viscosity of the continuous phase. Comparison of dynamic rheological tests between a reference emulsion without β-glucans and emulsions containing β-glucans showed that the polysaccharides largely affects the viscoelastic behaviour of the emulsion. Ageing of β-glucan-containing emulsions did not affect significantly the viscoelastic properties except for the emulsions containing low Mw β-glucans extracted from oat. Interestingly, all emulsions containing β-glucans creamed approximately the same after 30 days of storage regardless which preparation was used. The egg yolk constituents seemed to play a dominant role on the viscoelastic and the creaming behaviour of the emulsions, i.e. the viscoelastic behaviour was further enhanced and this could not only be attributed to the presence of the β-glucans but also to the stronger interactions between the oil droplets. Ageing did not affect the viscoelastic properties of β-glucan-containing emulsions while the reference emulsion, prepared only with egg yolk, showed a decrease in the value of storage modulus. The former could be interpreted as a steady consistency of the product during storage independent of the creaming behaviour. The creaming behaviour varied among the samples with the high molecular weight β-glucans from oat showing the highest stability.     

Structure and physicochemical properties of β-glucans and arabinoxylans isolated from hull-less barley

The molecular characteristics of β-glucans and arabinoxylans from selected hull-less barleys were examined following their sequential extraction with water (45 and 95 °C), Ba(OH)₂, Ba(OH)₂/H₂O, and NaOH which resulted in five distinct fractions designated as WE45, WE95, Ba(OH)₂, Ba(OH)₂/H₂O, and NaOH, respectively. Both water-extractable fractions consisted mainly of β-glucans but up to 20% of arabinoxylans were found in WE45 of normal and high amylose barley varieties. Analysis of the water-extractable β-glucans, indicated differences in molecular features (weight average molecular weight (M_w), β-(1→4) to β-(1→3) linkage ratios, and amounts of cellulosic regions along the β-glucan chain) between WE45 and WE95, and among β-glucans from different cultivars. The variation observed in the viscoelastic behaviour of WE45 and WE95 fractions confirmed differences in the molecular weight among water-extractable β-glucans derived from various barley cultivars.Differences in the structure of arabinoxylans from the various fractions and cultivars were also observed. Water-extractable arabinoxylans (WE45 and WE95 fractions) had significantly lower xylose to arabinose (Xylp/Araf) ratios (1.47 and 1.52) than those extracted with Ba(OH)₂ (1.70), implying a greater degree of branching in the more readily soluble fractions. Small varietal differences in molecular weight (M_w), root mean square radius (R_g), and polydispersity (M_w/M_n) among Ba(OH)₂ fractions correlated with the substitution pattern of the xylan backbone.     

Preparation of a Molecular Library of Branched β-Glucan Oligosaccharides Derived from Laminarin

To study the structure of β-glucans, we developed a separation method and molecular library of β-glucan oligosaccharides. The oligosaccharides were prepared by partial acid hydrolysis from laminarin, which is a β-glucan of Laminaria digitata. They were labeled with the 2-aminopyridine fluorophore and separated to homogeneity by size-fractionation and reversed phase high-performance liquid chromatography (HPLC). Branching structures of all isomeric oligosaccharides from trimers to pentamers were determined, and a two-dimensional (2D)-HPLC map of the β-glucan oligosaccharides was made based on the data. Next, structural analysis of the longer β-glucan oligosaccharide was performed using the 2D-HPLC map. A branched decamer oligosaccharide was isolated from the β-glucan and cleaved to smaller oligosaccharides by partial acid hydrolysis. The structure of the longer oligosaccharide was successfully elucidated from the fragment structures determined by the 2D-HPLC map. The molecular library and the 2D-HPLC map described in this study will be useful for the structural analysis of β-glucans.     

Influence of Heat Pretreatments of Oat Grain on the Viscosity of Flour Slurries

Heat treatment of oat grain had significant effects on the viscosity of flour slurries. Steamed oats produced highly viscous flour slurries, whose viscosity increased with time, whereas the viscosity of flour slurries produced from raw or roasted (104°C for 120 min) oats was much lower and degraded rapidly. Slurry viscosity was correlated with (1→3), (1→4)-β-D -glucan concentration in the flours and treatment of slurries with lichinase lowered viscosity significantly. Effects of steaming were partially reversed by roasting treatment and vice versa. Mixtures of equal amounts of raw and steamed flour resulted in slurries more viscous than either flour alone, but that viscosity degraded after 3 h to less than the mean viscosity of the steamed and raw controls. Water-soluble extracts from steamed flour had about twice the viscosity of raw or roasted flour extracts, but contained only 80% of the (1→3), (1→4)-β-D -glucan present in those extracts. Molecular weight analysis of soluble carbohydrates from raw, roasted and steamed soluble extracts indicated the molecular weight of β-glucans in these extracts was similar. However, if extracts were made from slurries that had incubated for 3 h, extensive degradation of β-glucans was evident in raw and roasted samples. It is likely that enzymic degradation of (1→3),(1→4)-β-D -glucans is responsible for much of the decreased raw and roasted flour slurry viscosity over extended time periods, but different heat treatments appear to also affect (1→3), (1→4)-β-D -glucan polymer interaction. © 1997 SCI.     

Structural modification and characterization of rice starch treated by Thermus aquaticus 4-α-glucanotransferase

Rice starch was modified using Thermus aquaticus 4-α-glucanotransferase (TAαGTase) in this study. The changes in the molecular structure and the effect on the starch retrogradation by TAαGTase treatment were investigated on isolated rice starch. By treating TAαGTase, molecular weight profile of amylopectins shifted to higher elution time from 1.0 × 10⁸ to 2.4 × 10⁷ or 0.8 × 10⁷, depending on the level of enzyme dosage. Meanwhile, there were huge increases in the proportions of content corresponding to amylose size and even smaller molecules. On treating with TAαGTase, short branch chains (DP 1–8) increased, and longer branch chains (>DP 19) increased significantly as well, with a broader distribution up to DP 46 compared to the control rice starch. Amylose content decreased from 30.0 to 21.8–23.7%. This indicated that the amylose could be transferred to the amylopectin branch chain by the disproportionation of TAαGTase, resulting in lowering the amylose content and the formation of amylopectin with a broader branch-chain length distribution. TAαGTase modified rice starch showed that X-ray diffraction pattern of the B-type crystalline even before cold storage, and that a variety of cyclic glucans (DP 5–19) were produced by enzymatic reaction. In particular, the accelerated rate of starch retrogradation was clearly observed compared to the control due to an overall increase in the number of elongated long-branch chains, decrease in the amount of amylose–lipid complex, and the possible synergistic effects of these factors.     

Structural characterization of barley β-glucan extracted using a novel fractionation technique

A barley β-glucan concentrate prepared according to a novel technology was further purified and subjected to detailed structural characterization by NMR spectroscopy. β-Glucan was hydrolysed with β-glucan-4-glucanohydrolase (lichenase). Fractions of hydrolysate were collected using an HPLC-fraction collector. Intact β-glucan and the major fractions collected were subjected to MALDI-TOF–MS and NMR analyses. The two major oligosaccharides produced by lichenase hydrolysis of purified barley β-glucan were identified as β-d-Glc p-(1 → 4)- β-d-Glc p-(1 → 3)-β-d-Glc p and β-d-Glc p-(1 → 4)-β-d-Glc p-(1 → 4)-β-d-Glc p-(1 → 3)-β-d-Glc p based on ¹³C and ¹H NMR data. Spectrums were similar to those documented for barley β-glucan in the literature.     

Gelling properties of kefiran, a food-grade polysaccharide obtained from kefir grain

The physicochemical and gelling properties of kefiran, a water-soluble glucogalactan with probed health-promoting properties, were investigated. Gel permeation chromatograms revealed a single distribution of molecular weight corresponding to 10⁷ Da. Intrinsic viscosity of kefiran determined using Huggins extrapolations was 6.0 dl/g and using Kramer approximations was 5.95 dl/g.Kefiran has a Newtonian behaviour in diluted solutions, which becomes pseudoplastic at higher concentrations. Rheological behaviour of the solution before and after freeze drying was evaluated by small deformation oscillatory rheological measurements. The mechanical spectrum of solution corresponded to an entangled network behaviour. After freeze–thaw treatment of the solution, a rheological behaviour transition from a liquid-like system to a gel was observed. The storage modulus (G′) in cryogels was 35 times higher than the value obtained for the solution. Rheological characteristics of the cryogel were influenced by kefiran concentration. As the polymer concentration increased, higher number of interactions was evident for the increment in both moduli (G′ and G″). The behaviour of kefiran cryogels about 37 °C determines its ability to melt at mouth temperature. These results suggest that kefiran cryogels could be an interesting alternative for its application in food formulations.     

Concurrent phase separation and gelation in mixed oat β-glucans/sodium caseinate and oat β-glucans/pullulan aqueous dispersions

The phase separation behavior of mixed oat β-glucans/sodium caseinate and oat β-glucans/pullulan aqueous dispersions at 20 °C has been studied. The concentration of β-glucans required for induction of phase separation and the physical state of the separated phases, as revealed by visual observations and dynamic rheometry, depended on the molecular weight of β-glucans and the initial polymeric composition. For β-glucans with apparent molecular weights (M_w) 35 and 65 × 10³ the β-glucan concentration at which thermodynamic incompatibility occurred decreased from about 2–2.5% (w/w) at low concentrations (∼0.2%) of sodium caseinate or pullulan to about 1–1.5% (w/w) β-glucans at high levels (up to 7.5% w/w) of the second biopolymer; these bi-phasic systems consisted of an upper liquid phase and a lower gel-like phase. For β-glucans with M_w of 110 × 10³, a bi-phasic system with two liquid phases appeared above a certain β-glucan concentration, which decreased from approximately 4% to 1% (w/w) with increasing sodium caseinate levels in the range of 0.2–7.5% (w/w). With further increase in β-glucan concentration, the lower phase turned into a gel, and at even higher β-glucan concentrations, the polymer demixing process was ‘arrested’ by chain aggregation events, leading to a macroscopically single gel phase. Generally, the aggregation of β-glucans seemed to interfere with the phase separation phenomenon resulting in an increase of β-glucan concentration in the lower phase between 5% and 110% and only a slight increase of sodium caseinate or pullulan concentration in the upper phase (<10%), due to kinetic entrapment of the polymeric components into a highly viscous medium.     

Kiwifruit protease extract injection reduces toughness of pork loin muscle induced by freeze–thaw abuse

Kiwifruit juice injection was used to tenderize freeze–thaw abused porcine meat, and the enzyme activity in relation to meat tenderness was determined. Whole pork loins (Longissimus lumborum) were subjected to 5 freeze–thaw cycles (–29 °C ↔ 4 °C). Non-frozen control and freeze–thaw treated loins were either non-injected or injected with 10% water or diluted kiwifruit juice (meat weight basis). After 48 h of incubation, samples were analyzed for pH, proteolysis, cooking loss, and Warner-Bratzler shear force. Shear force of meat increased 2-fold (P < 0.05) after 1 freeze–thaw cycle but decreased when loins were subjected to additional freeze–thaw treatments. The kiwifruit juice injection lowered the pH of meat from 5.6 to 5.2 and increased the cooking loss from 21% to 30% (P < 0.05). However, as indicated by the shear force reduction, tenderness of meat improved more than 2-fold (P < 0.05) after kiwifruit juice injection, in agreement with the SDS–PAGE patterns that showed substantial degradation of myosin in samples treated with kiwifruit juice. The actinidin assay confirmed the existence of significant proteolytic activity in kiwifruit juice extract and in injected loin samples, i.e., 4.65 units/mL and 1.27 units/g, respectively.     

Study of conformational properties of cereal β-glucans by computer modeling

Molecular modelling was used to study the conformational properties of cereal β-glucans and compared with experimental results. Followed the steps of exploring global minima of β 1-4 and β 1-3 linked disaccharides and building up the repeating units, the consecutive cellotriosyl units, which are considered as reaction sites of β-glucan chains, were created and then characterized as a three fold helix with a pitch of 41.35 Å. Mostly importantly, the 3D molecular model of cereal β-glucans was created and the moderately extended sinuous chain conformation was first visualized. The conformational parameters of cereal β-glucans were calculated by RMMC simulation, which are in good agreement with experimental results. The calculated parameters also revealed that the chain stiffness of β-glucans increased with the tri/tetra ratio.     

Molecular weight and structure of water soluble (1→3), (1→4)-β-glucans affect pasting properties of oat flours

Seven experimental oat lines with high (5.9% to 7.2%), medium (5.3% to 5.5%), and low (4.4%) β-glucan concentrations were evaluated for the effects of β-glucan molecular weight (MW) and structure on viscosities of oat-flour slurries. The MW of β-glucans was determined by size-exclusion high-performance liquid chromatography. The structural features of β-glucans were measured by using fluorophore-assisted capillary-electrophoresis after complete hydrolysis with lichenase. The oat-slurry viscosities were measured on a Rapid Visco Analyser under 4 conditions: (1) without starch (amylolysis, removal of starch by α-amylase); (2) without β-glucan (removal of β-glucan by lichenase); (3) natural action of enzymes (autolysis, in sodium buffer); and (4) inhibition of enzymes (in silver nitrate solution). Excluding one line (regression outlier), significant correlations (P < 0.05) between peak MW of β-glucan and viscosities of oat slurries were obtained under inhibition. The ratio of degree of polymerization (DP) 3/DP4 negatively correlated with viscosity under amylolysis, autolysis, and inhibition (P < 0.05). The amount of DP ≥ 5 negatively correlated with pasting final viscosity after β-glucan removal by lichenase (P < 0.05). Positive correlations (P < 0.05) between the ratio of β-(1→4)/β-(1→3) linkages and viscosities under autolysis and inhibition were found. Overall, these findings demonstrated that the peak MW, ratio of DP3/DP4, amount of DP ≥ 5, and ratio of β-(1→4)/β-(1→3) linkages of β-glucans impacted pasting properties of oat-flour slurries.     

THE EFFECT OF β-GLUCAN MOLECULAR WEIGHT ON THE SENSITIVITY OF DYE BINDING ASSAY PROCEDURES FOR β-GLUCAN ESTIMATION

An assay procedure has been developed for quantifying β-glucan, based upon its reaction with the dye Congo Red. The sensitivity of this method to changes in β-glucan molecular weight has been determined using β-glucans prepared from standard material by acid hydrolysis and comparison has been made with a Calcofluor-based method for quantifying β-glucans. The Congo Red assay was found to be optimally sensitive to β-glucans with molecular weights of approximately 2.5 × 10⁵ Daltons, whereas the Calcofluor assay was most sensitive to β-glucans with molecular weights in excess of 5 × 10⁴ Daltons. The purity of the β-glucan used during this investigation was determined using an enzyme-based procedure in which the polysaccharide was degraded to glucose by the addition of β-glucanase. The β-glucanase employed was partially-purified from Trichoderma viride cellulase using a novel batch ion-exchange method which is also described.     

Processing and formulation effects on rheological behavior of barley β-glucan aqueous dispersions

A freeze–thaw cycling process applied to barley β-glucan aqueous dispersions resulted in network structure development, which remained intact after thawing in both acidic and neutral conditions. The effects of freeze–thaw cycling were milder in the presence of sucrose where β-glucans were less prone to cryogel formation, giving weaker cryostructurates. The effect of three operating factors (β-glucan content, sucrose content and salt content) and their interactions on viscosity at shear rates of 10, 50 and 125 s⁻¹ was studied using response surface methodology. Significant positive linear effects were identified for all the factors on the first two models, while salt was not significant for the response at 125 s⁻¹. The β-glucan also exhibited significant negative quadratic effects for all three responses. Significant negative interactions were observed between β-glucan and sucrose, and β-glucan and salt for the viscosity at 10 and 50 s⁻¹, while for the viscosity at 125 s⁻¹, only the interaction between β-glucan and sucrose was significant. Response surface methodology and a second order regression model were used for the study of three factors (pH, temperature and time) influencing the viscosity of β-glucan dispersions of a low and a high molecular weight sample, following acid hydrolysis. Significant linear effects on viscosity of the low and high molecular weight samples were obtained for all the factors. A significant positive interaction effect was observed between pH and time for the viscosity of the lower molecular weight sample, while time exhibited a significant negative quadratic effect for the response of the high molecular weight sample. Acid hydrolysis had a stronger impact on viscosity reduction of the high molecular weight sample.     

Comparison of the solution properties of (1→3),(1→4)-β-d-glucans extracted from oats and barley

The solution properties of β-glucan isolated from whole grains of oats and barley were studied by viscosity and oscillatory measurements both in water and in aqueous cuoxam, an ammoniacal Cu(OH)₂/CuCl-solution. The viscosity measurements of water solutions show stronger shear thinning for oat than for barley β-glucans. The crossover points of the moduli G′ and G″ in cuoxam solutions were different for the two β-glucans, i.e. 10 Hz for oat β-glucan and 1.5 Hz for barley β-glucan. The molecular weights of the β-glucans were similar. Therefore, the difference in solution properties seems to be related to the difference in structure. This difference is revealed by the ratio DP3:DP4 of the main building blocks of β-glucans, which was higher for barley than for oat β-glucan.     

Basic subsite theory assumptions may not be applicable to hydrolysis of cellooligosaccharides by almond β-glucosidase

Steady-state kinetic parameters for the hydrolysis of cellooligosaccharides by almond β-glucosidase were evaluated at pH 5.0 and 25°C in relation to the subsite theory (K. Hiromi, Biochem. Biophys. Res. Commun., 40, 1–6, 1970). The value of k₀/K_m decreased monotonously with increasing degree of polymerization (DP) of the substrates (DP = 2–6). Also, the K_m and k₀ values for cellotriose were smaller than those for cellobiose. These DP dependencies differ from those of most amylases and glucosidases studied so far, to which the subsite theory has been successfully applied. The subsite parameters could not be consistently obtained, which suggests that one or both of the two basic assumptions of the subsite theory might not be applicable to the hydrolysis of cellooligosaccharides by the enzyme. That is, the intrinsic rate of the hydrolysis may depend on the DP and/or there may be interaction between subsites for binding the glucose residues of a substrate.     

Comparative studies on the effect of the freeze–thawing process on the physicochemical properties and microstructures of black tiger shrimp (Penaeus monodon) and white shrimp (Penaeus vannamei) muscle

The effects of different freeze–thaw cycles (0, 1, 3 and 5) on the physicochemical properties and microstructures of black tiger shrimp (Penaeus monodon) and white shrimp (Penaeus vannamei) muscle were investigated. White shrimp had greater exudate loss and higher α-glucosidase (AG), as well as β-N-acetyl-glucosaminidase (NAG) activities, than did black tiger shrimp, especially when the number of freeze–thaw cycles increased (P < 0.05). The decreases in Ca²⁺-ATPase activity, sulfhydryl group content and protein solubility with concomitant increases in disulfide bond formation and surface hydrophobicity were more pronounced in white shrimp muscle, than in black tiger shrimp muscle, particularly after five cycles of freeze–thawing (P < 0.05). The shear force of both shrimps was decreased after five freeze–thaw cycles (P < 0.05). The microstructure study revealed that the muscle fibers were less attached, with the loss of Z-disks, after subjection to five freeze–thaw cycles. Therefore, the freeze–thawing process caused denaturation of proteins, cell disruption, as well as structural damage of muscle in both shrimps. White shrimp generally underwent physicochemical changes induced by the freeze–thawing process to a greater extent than did black tiger shrimp.     

ENZYMIC QUANTIFICATION OF (1→3) (1→4)-β-D-GLUCAN IN BARLEY AND MALT

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 maltsamples.α-Amylasedoes not interfere with the assay. The method issuitable for the routineanalysis 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.