STRUCTURAL FEATURES

1.6 STRUCTURAL FEATURES

The structural features of β-glucans are important determinants of their physical properties and functionality, including their physiological responses when they are considered as ingredients in cereal-based foods and other formulated products. These features include ratios of β-(1→4)/β-(1→3) linkages, presence and amount of long cellulose-like fragments, ratios of cellotriosyl/cellotetraosyl units, and molecular size. 96

Values of molecular weight for cereal β-glucans have been reported in the literature in the range of 31 to 2700 × 10 3 , 35 to 3100 × 10 3 , 209 to 416 × 10 3 , and

21 to 10 × 10 3 for barley, oat, wheat, and rye, respectively (Table 1.4). Other molecular characteristics of cereal β-glucans obtained by laser light-scattering detec- tors such as polydispersity index (M w /M n ) and radius of gyration (R g ) were found ranging from 1.2 to 3.1 154,156,158,159 and 30 to 75, 40,43,45,75,115,123,133,136,157–159 respectively. The apparent discrepancies in the molecular weight estimates of cereal β-glucans might originate from varietal and environmental (growth) factors, 62,71,86 aggregation phenomena (dependent on the structural features and solvent quality), 43,44,133,156,160,161 and the analytical methodology used for the determination of these values (detector, standards). 62,116

Depolymerization events (endogenous or microbial β-glucanases from contam- inating microorganisms) taking place during the extraction step, 110,116,137,140,151,162 as well as differences in extraction and isolation methods (solvent and temperature affect the solubilization) affect the molecular size of the isolated polysaccharide. Increasing temperature of extraction can lead to an increase in molecular size of the extracted cereal β-glucans. 4,38,83,99,114,137 However, an opposite trend has also been observed 3,68,98 that in some cases was attributed to small differences in fine structure. 3

22 Functional Food Carbohydrates

TABLE 1.4

Molecular Weights of Cereal β-Glucans

Molecular

Source Detection Method Weight (10 –3 ) References

Barley Sedimentation velocity

Woodward et al. 3,153 HPSEC with MALLS

Saulnier et al. 115 HPSEC with FD ( β-glucan standards)

Beer et al. 62 GPC with RI (pullulan standards)

Morgan and Ofman 137 HPSEC with MALLS

Bohm and Kulicke 40 HPSEC with MALLS and RI

Gomez et al. 133 HPSEC with LALLS, RI, and FD ( β-

Wood et al. 116 glucan standards) HPSEC with MALLS

Knuckles et al. 75 HPSEC with FD ( β-glucan standards)

Cui et al. 41 HPSEC with 90˚ laser LS, DP, and RI

Wang et al. 154 HPSEC with RI ( β-glucan standards)

Vaikousi et al. 21 HPSEC with RI ( β-glucan standards)

Lazaridou et al. 18 HPSEC with MALLS and RI

Irakli et al. 45 Oat

HPSEC with RI (dextran standards)

Zhang et al. 83 HPSEC with FD ( β-glucan standards)

Beer et al. 62 HPSEC with FD ( β-glucan standards)

Beer et al. 117 GPC with RI and FD ( β-glucan

Autio et al. 79 standards) GPC with MALLS

Malkki et al. 136 GPC and FD ( β-glucan standards)

Jaskari et al. 144 HPSEC with FD ( β-glucan standards)

Beer et al. 117 HPSEC with LALLS, RI, and FD ( β-

Wood et al. 116 glucan standards) HPSEC with FD

Johansson et al. 123 HPSEC with FD

Aman et al. 155 HPSEC FD ( β-glucan standards)

Cui et al. 41 HPSEC with 90˚ laser LS, DP, and RI

Wang et al. 156 HPSEC with 90˚ laser LS, DP, and RI

Wang et al. 154 HPSEC with RI and MALLS

Roubroeks et al. 157,158 HPSEC with RI ( β-glucan standards)

Lazaridou et al. 17,18 HPSEC with MALLS and RI

Skendi et al. 43 Wheat

Cui et al. 41 HPSEC with RI ( β-glucan standards)

HPSEC with FD ( β-glucan standards)

Lazaridou et al. 18 Rye

HPSEC with LALLS, RI, and FD ( β-

Wood et al. 116 glucan standards) HPSEC with LALLS

21 Roubroeks et al. 159

Note : HPSEC = high-performance size exclusion chromatography; MALLS = multiple-angle laser light-scattering detector; FD = fluorescence detector with calcofluor postcolumn; LALLS = low-

angle laser light-scattering detector; RI = refractive index detector; LS = light-scattering detector; DP = differential viscometer detector; GPC = gel permeation chromatography.

Cereal β -Glucans: Structures, Physical Properties, and Physiological Functions 23

Studies of the molecular weight of cereal β-glucans have often dealt with isolated fractions of significantly lower molecular weight than the native cell wall polysac-

charides. Several studies also showed that the molecular weight of cereal β-glucans decreases during the isolation and purification procedures. 43,112,116,134,136,144 Usually,

harsher extraction conditions, such as high or low pH or prolonged extraction times, especially at high temperatures, can lead to recovery of low molecular size β- glucans. 62,75,116,117,119,137 Moreover, high-speed homogenization, sonication, 109,134 and high shear rates 112,134 were shown to reduce viscosity and molecular weight. Also, the purification step with ammonium sulfate seems to reduce molecular sizes of the extracted cereal β-glucans. 38,116,124

Many researchers have used several techniques to fractionate β-glucan popula- tions differing in molecular size to study the influence of molecular size on functional

properties of β-glucans or to achieve β-glucans with distinct physical and molecular characteristics. Such methods were partial degradation by acid hydrolysis 14,21 or controlled depolymerization by lichenase 157 for different periods of time, as well as ultrasonication. 4,15,40 As aforementioned, Morgan and coworkers 137,151,162 proposed a process for controlling the molecular size of extracted β-glucans by altering the extraction time if no deactivation of endogenous enzymes is applied to the flour. Furthermore, fractionation of oat and barley β-glucans into populations with different molecular sizes has been achieved with ammonium sulfate; with increasing concen- tration of salt, there was an increasing molecular size of the precipitated β-glucan fractions. 38,39,154 Wang et al. 154 found that the starting β-glucan concentration and temperature also seem to affect the fractionation efficiency. A clear separation of the fractions was possible at values of the overlapping parameter, c[ η], lower than ~3.5. Moreover, the higher the temperature, the lower the amount of ammonium sulfate that was necessary to precipitate a fraction of similar M w .

The fine structure of cereal β-glucans consists predominantly of β-(1→3)-linked cellotriosyl and cellotetraosyl units. 1,3–6,163 Longer cellulosic oligosaccharides in

smaller amounts (~5 to 10%), with a degree of polymerization between 5 and 20, have been also identified. 2–3,5–7 Blocks of two or more adjacent (1 →3) linkages are absent or present in a very low frequency. 1,2,4–6,41 Moreover, there were some indi-

cations for the presence of alternating (1 →3) and (1→4) sequences in the cereal β- glucan chain. 157,158 Although the distribution of β-(1→3) linkages in the polysaccha- ride is not random, statistical analysis of the sequence of cellulose-like oligomers showed a rather random distribution in the polymer chain for cellotriosyl and cel- lotetraosyl segments. 163,164 From biochemical experiments in vitro with active syn- thases in isolated Golgi membranes, the biochemical features and topology of cereal β-glucan biosynthesis are found to be closely parallel to those of cellulose. 165 Accord- ing to a current model for biosynthesis of mixed-linkage (1 →3),(1→4)-β-glucans proposed by Buckeridge et al., 165 the (1 →3),(1→4)-β-glucan synthase is that of a cellulose core-like synthase that makes cellobiosyl and even-numbered cellodextrin units, and a distinct glycosyl tranferase adds a third glycosyl residue to complete the cellotriosyl and higher odd-numbered units. Further investigation of the synthase activity in vitro showed that the cellodextrin unit distribution is altered drastically depending on the uridine diphospate (UDP)–Glc concentration. The suboptimal UDP–Glc concentrations favor the synthesis of longer cellodextrin units in β-glucan,

24 Functional Food Carbohydrates

particularly the cellotetraosyl unit, whereas at the highest UDP–Glc concentrations tested, the cellotriose units were predominant of the total polymer synthesized. 165

Despite the structural similarity of β-glucans from different genera of cereals, as suggested from methylation analysis and their almost identical nuclear magnetic resonance (NMR) spectra, oat, barley, and wheat β-glucans are, in fact, structurally distinct, as shown by quantitative high-performance liquid chromatography (HPLC) analysis of lichenase-released oligosaccharides. 1,5,17,41 The enzyme lichenase, a (1 →3),(1→4)-β-D-glucan-4-glucanohydrolase (EC 3.2.1.73), specifically cleaves the (1 →4)-glycosidic bond of the 3-substituted glucose residues in β-glucans, yield- ing oligomers with different degrees of polymerization (DPs). The major products for the cereal β-glucans are 3-O-β-cellobiosyl-D-glucose (DP3) and 3-O-β-cellotri- osyl-D-glucose (DP4), but cellodextrin-like oligosaccharides are also released from the polymer regions containing more than three consecutive 4-linked glucose resi- dues. The DP of the long cellulose-like fragments has been found to vary between

5 and 20, with DPs 5, 6, and 9 being the most abundant. 6,7,14,17,18,21,38,43–45,93 The literature data on oligosaccharide distribution of cereal β-glucans from their respec- tive lichenase digests are given in Table 1.5. Generally, the oligosaccharide distri- bution within the same genera of cereals was found to be similar and showed major differences only between β-glucans of different origins. 5,16,18 The amount of the trisaccharide (DP3) for the β-glucans follows the decreasing order of wheat (67 to 72%), barley (52 to 69%), and oat (53 to 61%), whereas the relative amount of the tetrasaccharide (DP4) follows the increasing order of wheat (21 to 24%), barley (25 to 33%), and oat (34 to 41%). However, the total of tri- and tetrasaccharide of β- glucans is similar among the cereal genera, resulting in a similar total amount (5 to 11%) of cellulose-like oligomers with DP 5 among the cereal β-glucans. The dif- ferences in tri- and tetrasaccharide amounts observed among β-glucans from differ- ent cereal sources are also reflected in the molar ratio of cellotriose to cellotetraose units (DP3/DP4) following the order of wheat (3.0 to 4.5), barley (1.8 to 3.5), rye (1.9 to 3.0), and oat (1.5 to 2.3); this ratio is considered a fingerprint of the structure of cereal β-glucans.

Literature data also indicate that there are differences within the same genera as well, which could be attributed to genotypic and environmental factors. A narrower range of the DP3/DP4 ratio in domestic cultivars of Avena sativa (2.05 to 2.11) than

in other cultivars of Avena (1.81 to 2.33) has been noticed by Miller et al. 82 Jiang and Vasanthan 69 found the molar ratio of tri- to tetraose units in the β-glucans from waxy barley varieties to be somewhat higher (2.6 to 2.8) than from normal and high- amylose varieties (2.3 to 2.6). Wood et al. 93 also reported a higher DP3/DP4 molar ratio for β-glucans from waxy barleys (3.0) than from nonwaxy cultivars (2.7 to 2.8). Storsley et al. 44 found that the water-extractable (at 45 and 95˚C) β-glucans from normal amylose varieties exhibit lower DP3/DP4 molar ratios (2.5 to 2.9) than high-amylose (2.9 to 3.1) and waxy varieties (3.1 to 3.2). In the latter study, a higher amount of long cellulose-like fragments was found in water-extractable β-glucans at 95˚C for varieties of barley with the anomalous amylose–amylopectin ratio (12.0 to 17.5%) than for varieties with normal starch (8.2 to 10.7%); however, no significant structural differences were observed among the different barley varieties for water-extractable β-glucans obtained at lower extraction temperatures (45˚C).

Cereal β -Glucans: Structures, Physical Properties, and Physiological Functions 25

TABLE 1.5

Structural Features of Cereal β-Glucans

Molar Ratio

Source DP3 DP4

DP ≥5

DP3/DP4

2.4 c Wood et al. 5 —

— Miller et al. 82

— Doublier and Wood, 14 Wood et al. 78 —

55.0–58.1 a 34.2–36.0 a 7.7–8.9 a 2.1–2.2

— Miller and Fulcher 166 57.6 b 34.1 b 8.2 b 1.7 —

Izydorczyk et al. 39

53.4–53.8 a 40.4–41.4 a —

— Johansson et al. 123 58.3 a 33.5 a 8.1 a 2.2 —

Cui et al. 41

55.6–55.9 b 33.6–34.4 b 7.1–7.5 b 1.6–1.7

2.4 d,e Colleoni-Sirghie et al. 85

54.6–56.8 a 35.3–36.3 a 7.7–9.2 a 2.0–2.1

2.3–2.6 e Skendi et al. 43 56.7 a 34.6 a 8.7 a 2.2 —

Wang et al. 154

2.4–2.8 e Lazaridou et al. 17,18 —

54.2–60.9 a 33.8–36.7 a 3.6–9.7 a 2.0–2.3

2.3–2.6 e Dais and Perlin 1 —

2.5 d Westerlund et al. 130 —

2.4 d,e Roubroeks et al. 157 Barley

2.2–2.6 c Woodward et al. 2,3 —

56–61 a 28–32 a 6–13 a 2.3–2.9

2.4 c Wood et al., 5 Wood 167 62.1 a 29.4 a 8.4 a 2.8 —

Wood et al. 78

59.2–64.9 a 25.3–30.4 a 9.4–10.2 a 2.6–3.4

2.4 c Saulnier et al. 115

56.8–61.6 b 26.1–32.3 b 10.6–11.2 b 1.8–2.4

— Izydorczyk et al. 7,38 63.7 a 28.5 a 7.8 a 3.3 —

Cui et al. 41

51.8–61.9 a 28.1–32.1 a 6.3–12.5 a 2.3–2.8

— Jiang and Vasanthan 69

61.5–64.3 a 27.9–30.1 a 7.8–8.6 a 2.7–3.0

— Wood et al. 93

59.4–64.3 a 24.8–31.0 a 8.2–17.5 a 2.5–3.2

1.9–2.2 d Storsley et al. 44 66.0 a 25.7 a 8.2 a 3.4 —

Wang et al. 154

62.0–69.3 a 26.2–29.1 a 4.5–8.9 a 2.8–3.5

2.1–2.8 e Vaikousi et al. 21

62.0–63.3 a 27.5–29.2 a 8.8–9.1 a 2.8–3.0

— Lazaridou et al. 18

2.2–2.7 e Irakli et al. 45 —

57.7–62.4 a 29.4–32.9 a 7.7–9.5 a 2.3–2.8

1.9–2.3 c Balance and Manners 98 —

2.3–2.6 e Dais and Perlin 1 —

2.4 d Henriksson et al. 168 Rye

— Wood et al. 5,78 —

2.3 d Roubroeks et al. 159 Wheat

— Wood et al. 5 72.3 a 21.0 a 6.7 a 4.5 —

Cui et al. 41 67.1 a 24.2 a 8.7 a 3.7 —

Lazaridou et al. 18 a Weight percent from the chromatograms of the lichenase digests.

b Mole percent from the chromatograms of the lichenase digests. c Calculated from methylation analysis.

d Calculated from 1 H-NMR data. e Calculated from 13 C-NMR data.

26 Functional Food Carbohydrates

Furthermore, analysis of oat aleurone β-glucan showed a smaller proportion of the cellotetraosyl units than that of the endospermic β-glucan, 6 whereas whole groats and oat bran β-glucans seemed to be having similar trisaccharide/tetrasaccharide ratios. 5 Similarly, Izydorczyk et al. 71 found that among the products obtained from pearling and roller milling of various barley cultivars, the ratio of tri- to tetrasaccharides in β-glucans from pearling by-products was higher than that from flour and a fiber-rich fraction. The β-glucans from the former fraction probably originated from the aleurone tissue, whereas the β-glucans from the latter fraction originated from the endosperm cell walls. Despite the genetic and environmental variations, some discrepancies in the calculated values of the DP3/DP4 ratio for cereal β-glucans might arise from variations in the sensitivity of techniques employed, as well as from the uncertainties in the response factors for the oligosaccharides in the analytical system used because of the lack of appropriate pure oligosaccharide standards.

The calculated ratios of the two types of linkages (1 →4) to (1→3) in the native cereal β-glucan structures, based on NMR data and methylation analysis, were found within the range of 1.9 to 2.8 (Table 1.5). Izydorczyk and coworkers 7,38,44 fractionated cereal β-glucan using different aqueous or alkali conditions and different ammonium sulfate concentrations, and obtained fractions exhibiting differences in the (1 →4)/(1→3) ratio from 1.9 to 5.3, as well as other distinct molecular/structural characteristics, as described in the following section. Even small differences in the proportions of the two linkages seem to be enough to influence significantly the physical properties of cereal β-glucans, such as the solubility, conformation, and aggregation tendency of the polymeric chains. 3,163