Composition and molecular structure of polysaccharides released from barley endosperm cell walls by sequential extraction with water, malt enzymes, and alkali (original) (raw)

Abstract

Isolated and purified endosperm cell walls (CW), used in this study, were derived from a Canadian malting barley variety, AC Metcalfe, grown in three different environments in Canada in 2003, and varying in grain protein and b-glucan contents, as well as in grain hardness. The CW were initially extracted with water at 45 C and subsequently digested with barley malt crude enzyme extract resulting in two fractions designated CW-WE45 and CW-MD, respectively. The remaining non-digested cell wall material (CW ND ) was further fractionated by sequential extraction with water at 95 C (CW ND -WE95), saturated barium hydroxide (CW ND -BaE), and 1 N sodium hydroxide (CW ND -NaE) at 25 C. Composition and molecular structure analyses were carried out for all fractions including the remaining cell wall residue (CW RES ). Extraction of CW with water followed by digestion with malt crude enzyme extract solubilized the majority of b-glucans (w55e70%) and glucomannans (w60e80%) but only a small portion of arabinoxylans (w20e30%) present in the intact CW. The CW-WE45 and CW ND -WE95 fractions consisted mostly of b-glucans exhibiting high average molecular weights (M w ) (2e3 Â 10 6 ), whereas the CW ND -BaE consisted mainly of arabinoxylans with M w about 1e1.5 Â 10 6 . The CW ND -NaE contained almost equal amounts of b-glucans and arabinoxylans and a small amount of glucomannans, whereas the CW RES contained approximately equal proportions of b-glucans, arabinoxylans and glucomannans. b-Glucans in CW ND -WE95, CW ND -NaE, and CW RES exhibited a higher ratio of 3-O-b-D-cellobiosyl-D-glucose to 3-O-b-D-cellotriosyl-D-glucose (DP3/DP4) compared to b-glucans in CW-WE45 and CW-MD. b-Glucans in CW ND -NaE showed the highest level of long cellulosic oligosaccharides with DP ! 5, whereas those in the CW RES had the highest DP3/DP4 ratio. The CW-MD was fractionated by ultrafiltration into high (CW-MD HMW ) and low-molecular weight (CW-MD LMW ) sub-fractions, with weight-average M w of w150e350 Â 10 3 and <10 Â 10 3 , respectively, as confirmed by size-exclusion chromatography. The monosaccharide composition of the sub-fractions indicated a more extended enzymic degradation of b-glucans and glucomannans than arabinoxylans. Some differences in composition and molecular structure of the cell wall constituents among the three barley samples were related to their solubility and enzymic digestibility.

Figures (9)

Fig. 1. Sequential fractionation scheme of barley endosperm cell walls (CW) with water, malt extract and alkali.

Fig. 1. Sequential fractionation scheme of barley endosperm cell walls (CW) with water, malt extract and alkali.

* Results of duplicate analyses, CV < 5%. Yield and monosaccharide composition of CW fractions obtained upon sequential extraction of intact CW with water, malt extract, and alkali Table |

* Results of duplicate analyses, CV < 5%. Yield and monosaccharide composition of CW fractions obtained upon sequential extraction of intact CW with water, malt extract, and alkali Table |

Fig. 2. Relative amounts of carbohydrates released sequentially from the CW with each solvent/enzymes treatments, expressed as % of individual polysaccharides originally present in the intact CW.

Fig. 2. Relative amounts of carbohydrates released sequentially from the CW with each solvent/enzymes treatments, expressed as % of individual polysaccharides originally present in the intact CW.

Fig. 3. High-performance size exclusion profiles of water- and alkali-extracted CW fractions of barley sample B. Further to the fine structure of cell wall polysaccharides, their molecular size has also been identified as an important determinant of t properties. The heir solubility, extractability and aggregation weight-average molecular weight (M,,) and molecular weight distribution of polymers in the CW fractions were examined chromatography using high-performance — size-exclusion (HPSEC). Representative elution profiles of all fractions except for the CW-MD are shown in Fig. 3. Generally, broad and asymmetric peaks were observed for all fractions, but large differences in the shape and retention time of the eluting species were evident among different frac- tions. The CWyp-NaE fractions showed the broadest elution profiles in agreement with their polymeric heterogeneity (ara- binoxylans, B-glucans and mannose containing polymers). The M,, values for the CW fractions are presented in Table 3. For

Fig. 3. High-performance size exclusion profiles of water- and alkali-extracted CW fractions of barley sample B. Further to the fine structure of cell wall polysaccharides, their molecular size has also been identified as an important determinant of t properties. The heir solubility, extractability and aggregation weight-average molecular weight (M,,) and molecular weight distribution of polymers in the CW fractions were examined chromatography using high-performance — size-exclusion (HPSEC). Representative elution profiles of all fractions except for the CW-MD are shown in Fig. 3. Generally, broad and asymmetric peaks were observed for all fractions, but large differences in the shape and retention time of the eluting species were evident among different frac- tions. The CWyp-NaE fractions showed the broadest elution profiles in agreement with their polymeric heterogeneity (ara- binoxylans, B-glucans and mannose containing polymers). The M,, values for the CW fractions are presented in Table 3. For

Weight-average molecular weight and polydispersity index of water- and al- kali-extractable CW fractions

Weight-average molecular weight and polydispersity index of water- and al- kali-extractable CW fractions

Fig. 4. High-performance size exclusion profiles of: (a) the malt enzymes ex- tracted CW fractions (CW-MD) for three barley samples (grey line indicates the profile of the crude malt extract by itself) and (b) the sub-fractions of CW-MD obtained by ultrafiltration for barley C.

Fig. 4. High-performance size exclusion profiles of: (a) the malt enzymes ex- tracted CW fractions (CW-MD) for three barley samples (grey line indicates the profile of the crude malt extract by itself) and (b) the sub-fractions of CW-MD obtained by ultrafiltration for barley C.

Yield and monosaccharide composition (mol%) of the high- and low-molecular weight sub-fractions of CW-MD * Numbers in parentheses represent mol% of monosaccharides detected in the monomeric form. > Results of duplicate analyses, CV < 5%. Table 4

Yield and monosaccharide composition (mol%) of the high- and low-molecular weight sub-fractions of CW-MD * Numbers in parentheses represent mol% of monosaccharides detected in the monomeric form. > Results of duplicate analyses, CV < 5%. Table 4

* Results of duplicate analyses, CV < 3.5%. Relative distribution (peak area %)* of free glucose monomers and oligomers in the low-molecular weight CW-MD,; mw sub-fraction

* Results of duplicate analyses, CV < 3.5%. Relative distribution (peak area %)* of free glucose monomers and oligomers in the low-molecular weight CW-MD,; mw sub-fraction

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