Influence of Non-Starch Polysaccharides Structure on the Metabolisable Energy of U.K. Wheat Fed to Poultry

Journal of Cereal Science 29 (1999) 77–88 Article No. jcrs.0213, available online at http://www.idealibrary.com on

Influence of Non-Starch Polysaccharides Structure on the Metabolisable Energy of U.K. Wheat Fed to Poultry S. C. Austin∗, J. Wiseman† and A. Chesson∗ ∗Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, U.K.; †University of Nottingham, Department of Agriculture and Horticulture, Sutton Bonington Campus, Loughborough LE12 5RD, U.K. Received 8 September 1997

ABSTRACT With the inclusion of wheat in European poultry diets at 600 g/kg, or more, there is increasing concern that its apparent metabolisable energy (AME) is more variable than would be predicted by conventional analysis. Twelve samples of wheat with a range of AME values (8·34–13·74 MJ/kg dry matter when fed to broiler chicks aged 11–14 d at 750 g/kg diet) were used to investigate the causes of this variability. AME was not correlated with the amount of total water-soluble non-starch polysaccharide (sNSP), soluble arabinoxylan (the major polysaccharide contributing to NSP) or (1→3, 1→4)-b-glucan released from the grain or with the viscosity of aqueous extracts. Surprisingly, in vitro viscosity was negatively related to soluble (r2=0·61) and total (r2=0·82) arabinoxylan. This was thought to be due to the slow, but cumulative, action of endogenous hydrolases in the stored grain. Soluble NSP from each wheat was characterised by measurement of molecular weight distribution and the structural features of arabinoxylan determined from the amount and nature of the oligosaccharides released following treatment with an endo-xylanase. Oligomer molecular weight was determined by matrix-assisted laser desorption/ionisation time of flight mass spectrometry and structure by NMR. Multivariate analysis of the 32 variables measured provided a three-term model able to explain approximately 0·80 of the variation between wheat samples: AME=8·07+11·16(XRAX)+30·67(AX-6)−0·355(sNSP) Two terms (XRAX, the proportion of arabinoxylan resistant to hydrolysis by xylanase and AX-6, the properties of branched six-sugar present in hydrolysates) reflected the degree of branching of arabinoxylan and were positively associated with AME while the third term, the amount of sNSP present, was negatively related.  1999 Academic Press

Keywords: wheat grain, poultry feed, apparent metabolisable energy, non-starch polysaccharide, arabinoxylan structure, xylo-oligosaccharides.

 : AME=apparent metabolisable energy; GC=gas chromatography; HPAEC=high performance anion-exchange chromatography; HPLC= high performance liquid chromatography; MALDITOF-MS=matrix assisted laser desorption/ionisation time of flight mass spectrometry; NMR=nuclear magnetic resonance; NSP=non-starch polysaccharides. Corresponding author: Dr S. C. Austin, Laboratory for Molecular Structure, NIBSC, South Mimms, Potters Bar, Herts, EN6 3QG, U.K. Tel: 01707 654753; Fax: 01707 646730; E-mail: [email protected] 0733–5210/99/010077+12 $30.00/0

INTRODUCTION Wheat is the preferred cereal for use in many European broiler diets because its nutritional value, measured as apparent metabolisable energy (AME) is higher than that of other locally available cereals such as barley and rye. However, as the amount of wheat included in feeds has increased to the level where it typically comprises 600 g/kg or more of a European poultry diet, some wheat samples have been found to exhibit lower than  1999 Academic Press

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expected AME values. The consequences of this low AME wheat phenomenon are serious for poultry producers, since a reduction of as little as 0·5 MJ/kg from the expected AME value can make the difference between profit and loss1. Depression of AME in cereals has been attributed to a fraction of the non-starch polysaccharides (NSP) present in grain cell walls leaching out and increasing the in vivo viscosity of the digesta. This results in an increase in the size of the unstirred layer associated with the wall of the digestive tract, which is thought to reduce the absorption of nutrients through the gut wall and impede the passage of host digestive enzymes into the lumen of the gut2. The problem has been recognised for many years in barley and oats, ascribed to (1→3, 1→4)-b-glucans solubilised from the endosperm wall3–6, and in rye where soluble arabinoxylans were considered the primary agent6,7. Wheat was generally considered free from such problems until a clear inverse relationship was also demonstrated between AME and the amount of soluble NSP (or soluble arabinoxylan) extracted from some Australian wheat samples exhibiting the low AME phenomenon8–10. Further, it was shown that low ME effects could be created by the addition of isolated arabinoxylan to otherwise problem-free diets8 and that low AME wheat diets could be improved by the addition of appropriate polysaccharide hydrolases9,10. However, the same relationship has not been so evident in wheat grown in Europe and North America. This is due, in part at least, to the greater uniformity of wheat samples within any one growing year, storage of much of the wheat before use11 and levels of inclusion in the diet. Only under experimental conditions in which inclusion levels of wheat in broiler diets exceeded present commercial practice has the more extreme depression of AME been seen in U.K. wheat12. However, even in this case, the relationship between concentration of soluble NSP and AME was too weak to provide the measure of prediction required by the industry who have the option of treating problem feeds with exogenous enzymes. These observations suggest that the amount of soluble NSP leached from the grain may not be the dominant factor in the low AME wheat phenomenon, particularly when amounts solubilised are near to the threshold where effects become evident. Structural characteristics of the arabinoxylan and interactions of soluble NSP with other macromolecules which promote solution viscosity and, in consequence,

depress wheat AME may be equally important. The aim of this work was to establish which attributes of the NSP had the greatest effects on wheat fed to broiler chicks and whether the measurements of such factors held sufficient predictive capacity to allow potentially problem wheat samples to be identified prior to use. EXPERIMENTAL Plant material Twelve samples of wheat were selected from a range of U.K. wheat previously tested at the University of Nottingham for their AME value when fed at an inclusion of 750 g/kg to broiler chicks12. Selection was made to cover as wide a range of AME values as possible while maintaining an even spread of values (Table I). Most of the samples were harvested in 1992, but two, obtained from the National Institute of Agricultural Botany, were from the 1991 harvest. Wheat was ground to pass a 3 mm screen before incorporation into diets which also contained soya protein isolate (150 g/kg), vegetable oil (50 g/kg) included to minimise textural and intake problems associated with the diet, and a vitamin/mineral premix (50 g/kg). Diets were fed for a period of 6 d to six replicates of a cage containing two male broiler chicks of initial age 9 d. For the last 3 d a total excreta collection was made. Diets and excreta were analysed for dry matter and gross energy allowing calculation of wheat AME (assuming the AME values for the other ingredients). Extraction of NSP Soluble NSP was extracted from the samples in small quantities for quantitative analysis and in larger quantities for molecular weight determination and for hydrolysis to oligosaccharides and subsequent structural characterisation. Milled wheat (150 mg—small scale; 5 g—larger scale) was treated with boiling ethanol (80% v/v) for 20 min, cooled, centrifuged (20 000 g, 30 min) and the residue dried under vacuum. For small scale isolations the residue was treated with 1 mL dimethyl sulphoxide at 80 °C for 30 min, then 4 mL of amylase (Termamyl 300L, Novo Nordisk) solution (3% v/ v, pH 7·0), which had been heated to 100 °C for 10 min to destroy any enzymes present other than amylase, was added and the mixture incubated at 80 °C for 2 h. The samples were centrifuged (600 g,

Arabinoxylan structure and AME of wheat

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Table I The apparent metabolisable energy (AME) for broiler chicks and the total non-starch polysaccharide (NSP) content of samples of feed wheat. NSP was estimated by summing values for the individual monosaccharides measured after starch extraction and hydrolysis of the starch-free residue AME (MJ/kg DM)

Ara (g/kg)

Xyl (g/kg)

Man (g/kg)

Gal (g/kg)

Glc (g/kg)

UA (g/kg)

b-Glu (g/kg)

A

8·43

B

8·43

C

9·29

D

9·43

E

9·89

F

10·95

G

11·22

H

11·41

I

12·00

J

12·63

K

13·67

L

13·74

16·9 (0·7) 24·0 (0·7) 26·3 (0·1) 18·4 (0·4) 12·4 (1·3) 24·9 (2·4) 12·7 (0·9) 27·1 (3·1) 18·8 (1·0) 17·4 (1·0) 23·0 (0·7) 26·5 (0·3)

29·0 (0·4) 48·1 (1·0) 47·4 (0·7) 25·9 (4·3) 26·9 (2·0) 45·1 (4·2) 24·1 (1·9) 53·9 (4·4) 33·6 (2·0) 28·4 (1·7) 43·5 (1·4) 47·7 (0·9)

2·6 (0·1) 2·3 (0·1) 1·8 (0·1) 2·0 (<0·1) 1·7 (0·2) 1·9 (0·2) 2·0 (0·2) 2·0 (0·3) 2·2 (0·1) 2·1 (<0·1) 2·4 (0·1) 2·2 (<0·1)

4·09 (<0·1) 4·8 (1·5) 3·5 (0·1) 3·7 (0·1) 3·5 (0·2) 4·1 (0·3) 3·1 (0·3) 3·3 (0·7) 3·9 (0·1) 3·3 (0·1) 3·7 (0·4) 4·1 (0·2)

31·7 (0·7) 34·4 (0·8) 40·1 (3·1) 32·0 (0·9) 39·3 (1·9) 37·3 (2·5) 36·9 (3·0) 39·0 (3·8) 36·2 (2·7) 33·0 (1·7) 34·7 (1·1) 39·1 (1·4)

3·8 (0·1) 4·1 (0·5) 4·0 (0·4) 3·8 (0·1) 4·4 (0·1) 3·6 (0·5) 3·8 (0·2) 4·0 (0·3) 3·9 (0·1) 3·5 (0·2) 3·6 (0·1) 4·1 (0·1)

5·9 (0·5) 6·7 (0·1) 6·7 (0·1) 5·8 (0·2) 6·2 (0·2) 7·1 (0·2) 6·0 (0·2) 7·2 (0·0) 5·8 (0·1) 5·6 (0·3) 6·5 (0·1) 7·2 (0·4)

Sample

Total NSP (g/kg) 88·1 (1·3) 117·5 (3·8) 123·1 (2·8) 85·5 (3·5) 88·0 (3·1) 117·0 (9·7) 82·6 (5·7) 129·2 (12·1) 98·6 (5·6) 87·6 (3·2) 110·8 (3·4) 123·5 (2·2)

Ara=arabinose, Xyl=xylose, Man=mannose, Gal=galactose, Glc=glucose, UA=uronic acids, b-Glu=(1→3, 1→4)-bglucan (all values quoted as anhydrous sugars). Standard deviations shown in parentheses.

5 min) and the supernatant recovered. Supernatants were equilibrated in a water bath (50 °C) for 3 min and 0·25 mL of an aqueous solution of porcine pancreatin (10% v/v), and Promozyme 200L (pullulanase, EC 3.2.1.41 Novo Nordisk, 25% v/v) was added to complete the breakdown of starch and protein. Both enzyme preparations were examined for potentially interfering enzymes which were confirmed to be absent. The samples were incubated at 50 °C for 30 min then cooled to room temperature. For the larger scale extraction of soluble NSP, 50 mL of the Termamyl solution was added and the samples were incubated at 80 °C for 1 h with constant shaking. The mixture was then centrifuged (20 000 g, 30 min), the supernatant collected and the residue re-extracted under the same conditions with a fresh batch of enzyme. The combined supernatants were dialysed against distilled water for 3 d at 4 °C. In each case, after extraction, sufficient ethanol was added to the NSP solutions until the concentration reached 80% v/v. Samples were then cooled in ice for 30 min (small scale) or in a cold room at

4 °C overnight (large scale) to precipitate NSP which was collected by centrifugation (small scale—600 g, 5 min; larger scale—20 000 g, 30 min) and dried.

Analysis The total carbohydrate content of samples was determined by the phenol-sulphuric acid method13, total starch by an enzymatic method14 and total NSP as described by Theander and Westerlund15. Neutral sugars were measured by GC as their alditol acetate derivatives16 after hydrolysis of NSP. Acidic sugars were determined colorimetrically17. The polymer (1→3, 1→4)-b-glucan was measured directly following the enzymatic procedure of McCleary and Codd18 using (1→3, 1→4)-b-glucan and glucose test kits (Megazyme, Australia). Esterbound phenolic acids were released from grain or grain extracts with 1  NaOH at room temperature, removed from solution by binding to C18 solid phase extraction cartridges, eluted with

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methanol and determined by HPLC. Soluble protein in wheat extracts was measured by dye-binding with Coomassie Brilliant Blue G-250 (Bio-Rad Laboratories, Hercules, CA, U.S.A.) using the method detailed by the supplier. The viscosity of NSP solutions was determined by weighing wheat samples (1 g) into screw cap tubes containing magnetic stirring bars and extracting with 10 mL boiling ethanol (80% v/v) as described. Termamyl solution (5 mL, 3% v/v, pH 7) was added to the dried residue and the tubes incubated at 80 °C for 2 h with constant stirring. After cooling, the tubes were centrifuged (350 g, 5 min) and the supernatant filtered through Whatman GF/A filter paper. The viscosity of this liquid was measured at 25 °C with a Brookfield DVIII rheometer working in LV mode fitted with a CP-40 cone and plate revolving at 30 rev/min.

Preparation of oligosaccharides Arabinoxylan oligosaccharides were prepared by adding 25 mL of a cloned xylanase solution (2% v/v, SP628, Novo Nordisk) to 1 g commercially available wheat arabinoxylan (Megazyme, Australia). The absence of enzymes other than endoxylanase was previously confirmed by extended incubation of the concentrated enzyme with other potential substrates. The arabinoxylan suspension was incubated at 60°C for 84 h with constant stirring. After cooling in an ice-bath, any resistant polysaccharide was precipitated with 80% v/v aqueous ethanol, collected by centrifugation (25 000 g, 30 min), and then dried and weighed. The supernatant was transferred to a round bottom flask, the ethanol removed by rotary film evaporation and the residue re-dissolved in water and freezedried. A sample of the oligosaccharide mixture (>500 mg) was dissolved in water (3 mL) and applied to a column (2·5×75 cm) of Biogel P2 (Bio-Rad Laboratories, Hercules, CA, U.S.A.) and eluted with water at 60 °C with a flow rate of 20 mL/h. Fractions (7 mL) were collected and those containing carbohydrate were freeze-dried and weighed. Fractions were monitored by matrixassisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS) to check for purity and to provide an indication of molecular weight. Those found to contain a mixture of oligosaccharides were re-applied to the column and separated under the same conditions. Fractions (5 mL) were collected, analysed for total car-

bohydrate and positive fractions examined using MALDI-TOF-MS. Those containing oligosaccharides of the same molecular weight were pooled. The purified oligosaccharides were freezedried and stored under vacuum over P2O5. Structural fingerprints of each of the wheat samples were produced by digesting the NSP isolated from each of the wheat samples (100 mg) with endo-xylanase (0·15 mL) in water (5 mL) for 84 h. After ethanol precipitation to remove enzyme protein and any residual polysaccharide, the oligosaccharide mixture was dried by rotary evaporation, re-dissolved in water to a concentration of 200 lg/mL and an aliquot (25 lL) analysed by high performance anion-exchange chromatography (HPAEC) using a Carbopac PA1 column (4×250 mm, Dionex) at 20 °C with a flow rate of 1 mL/min and gradient elution (0–0·1 min: 5% A, 75% B, 20% C; 0·1–30 min: 29% A, 51% B, 20% C; 30–35 min: 0% A, 50% B, 50% C; 35–40 min: 5% A, 75% B, 20% C; where A=1  sodium acetate in 0·1  NaOH, B= water and C=0·5  NaOH). A pulsed amperometric detector was used with E1=+0·05 V, E2=+0·6 V and E3=−0·6 V. Individual oligosaccharides prepared from the commercially available arabinoxylan were also analysed under the same conditions and the observed retention times used to identify the peaks on the chromatograms from wheat samples.

Molecular weight determination The molecular mass of oligosaccharides was determined by MALDI-TOF-MS. A sample of oligosaccharide was dissolved in water (2 mg/mL) and an aliquot (1 lL) was placed on a sample plate and mixed with an equal volume of the matrix solution 2,5-dihydroxybenzoic acid (120 mg dissolved in 10 mL 70:30 v/v methanol:water). The mixture was allowed to dry before the sample plate was introduced into a Finnigan Mat Lasermat MALDI-TOF-MS. Each oligosaccharide sample was vapourised and ionised in positive ion mode using the lowest possible laser power consistent with a good mass spectrum. Polysaccharide molecular weight (Mr) was determined by size exclusion chromatography. Isolated NSP (1·5 mL of a 2 mg/mL solution) was applied to a Sephacryl S-400 column (1·5×112 cm) and eluted with sodium azide solution (0·02% w/v) at a flow rate of 18 mL/h

Arabinoxylan structure and AME of wheat

at 25 °C. Fractions (5 mL) were collected and analysed for total carbohydrate and Mr was determined by comparison of retention times with those of dextrans of known molecular weight separated on the same column. NMR identification of oligosaccharides Isolated oligosaccharides were treated with D2O (99·9 atom%) to deuterium-exchange hydroxyl protons, freeze-dried, re-dissolved in D2O (100%) and 1H spectra recorded at 27 °C on a Jeol LA300 spectrometer operating at 300·4 MHz equipped with a TH-5 multinuclear probe. Chemical shifts were measured with reference to external 3-(trimethylsilyl)-1-propane-sulphonic acid. Total correlation spectroscopy (TOCSY) experiments were recorded using the pulse sequence 90°-t1-SLacq where SL stands for a multiple of the MLEV17 sequence19,20. The MLEV-17 sequence utilised 90° and 180° pulse widths of 36 ls and 72 ls, respectively. The total mixing time was 112 ms. The spectral width was 1000 Hz in both dimensions and 512 experiments of 512 data points were recorded. The data matrices were multiplied in each time domain with a phase shifted sine function (shifted p/3) prior to phase-sensitive Fourier transformation. Rotating-frame nuclear Overhauser enhancement spectroscopy (ROESY) were accumulated using the pulse sequence 90°t1-SL-acq, where SL stands for the DANTE pulse train21. The DANTE pulse train utilised a 45° pulse width of 59 ls with a 118 ls pulse interval and a total mixing time of 200 ms. The spectral width was 3200 Hz in both dimensions and 1024 experiments of 1024 data points were recorded. The data matrices were multiplied in each time domain with a phase shifted sine function (shifted p/2) prior to phase-sensitive Fourier transformation. Statistical analysis Genstat 5 for Windows (Version 3.2) was used to analyse data by multiple linear regression analysis. A model of the type shown in equation (1) was used to predict AME from the chemical analysis of the grain. Y=C+a1X1+a2X2+.........anXn

(1)

Although the final model could only support three variables because of the limited number of wheat

81

samples analysed, these were selected from a pool of 32. The variables considered were: the concentration of soluble arabinose (sara), xylose (sxyl), mannose (sman), galactose (sgal), (1→3, 1→4)b-glucan (smlg), uronic acid (sua), NSP (snsp), arabinoxylan (araxyl), the arabinoxylan to (1→3, 1→4)-b-glucan ratio (axmlg), arabinoxylan branching (ax), galactose+mannose (galman), protein (sprot), E-ferulic acid (efer) and Z-ferulic acid (zfer), the data for the Mr, distribution, Mr, >300 000 (mw300), Mr, 200 000–300 000 (mw200–300), Mr, 100 000–200 000 (mw100– 200), Mr, 20 000–100 000 (mw20–100) and Mr, <20 000 (mw20), the data from the chromatograms of the oligosaccharides produced by xylanase digestion which included X-1 (X1), X-8 (X8), X-9 (X9), AX-5 (AX5), AX-6 (AX6), AX-8 (AX8), AX-9 (AX9), AX-10 (AX10) and the unidentified oligomers eluting with retention times 7·1 min (t7.1), 13·7 min (t13.7), 15·8 min (t15.8) and 20·4 min (t20.4) and finally, the amount of arabinoxylan which was not hydrolysed by the xylanase (XRAX). Initially, the single variable showing the best correlation with AME was selected. Other variables were added singly and the additional variable which best explained AME was retained and the process repeated.

RESULTS The total NSP in the 12 grain samples ranged from 87·6–129·2 g/kg dry matter with a mean value of 104·3 g/kg (Table I). Arabinoxylan, estimated from the sum of the arabinose and xylose content, contributed on average slightly more than half the total NSP but was highly variable with concentrations which varied from 36·8 to 81·0 g/ kg. The concentration of (1→3, 1→4)-b-glucan was more consistent with a mean of 6·4 g/kg (range 5·6–7·2 g/kg). Approximately 18% of the total arabinoxylan and 29% of the (1→3, 1→4)-bglucan was solubilised at 80 °C under the extraction conditions described with a modest correlation between the total and soluble arabinoxylan content (r2=0·71) and a rather weaker relationship between total and soluble (1→3, 1→4)-b-glucan (r2=0·40). A small amount of glucose, in a form sufficiently large to be precipitated by ethanol, was found in hydrolysates of the soluble NSP fraction of wheat (Table II). Since (1→3, 1→4)-b-glucan could account for only a small fraction of this glucose, it

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Table II Soluble NSP content of wheat samples determined after extraction from grain at 80 °C in the presence of amylase. The total value excludes glucose detected in hydrolysates but includes the concentration of (1→3, 1→4)-b-glucan which was separately determined Wheat sample

Ara (g/kg)

Xyl (g/kg)

Man (g/kg)

Gal (g/kg)

Glc (g/kg)

UA (g/kg)

b-glu (g/kg)

A

3·5 (0·3) 5·9 (0·4) 4·4 (0·2) 3·2 (0·4) 3·5 (0·5) 4·6 (0·8) 3·4 (0·3) 5·7 (0·4) 3·2 (0·4) 3·1 (0·4) 3·6 (0·1) 3·8 (0·4)

4·6 (0·5) 10·3 (1·0) 7·7 (0·2) 4·3 (0·6) 4·8 (0·6) 8·6 (1·4) 4·3 (0·3) 10·2 (1·1) 4·4 (0·3) 4·0 (0·6) 6·1 (0·1) 6·8 (0·7)

0·4 (0·6) 1·1 (0·1) 0·5 (0·5) 0·3 (0·5) 0·3 (0·4) 0·9 (0·2) 0·4 (0·6) 0·4 (0·3) 0·0 (<0·1) 0·3 (0·4) 0·1 (0·2) 0·4 (0·3)

3·3 (0·4) 1·8 (0·1) 1·1 (0·9) 3·0 (0·2) 2·7 (<0·1) 2·0 (0·4) 2·5 (0·2) 1·6 (0·2) 2·4 (0·2) 2·7 (0·3) 1·8 (0·2) 1·7 (0·3)

28·6 (4·5) 41·1 (3·9) 26·9 (13·5) 29·4 (7·7) 28·6 (6·0) 39·5 (2·8) 27·6 (3·2) 38·9 (5·6) 26·7 (6·1) 27·8 (6·0) 31·7 (1·0) 35·1 (2·3)

4·2 (0·5) 2·3 (0·1) 2·2 (0·1) 4·0 (0·3) 3·8 (0·3) 2·2 (<0·1) 3·9 (0·5) 2·3 (0·2) 4·0 (0·1) 3·8 (0·3) 2·0 (0·1) 2·3 (0·3)

1·7 (0·2) 2·1 (0·1) 2·0 (0·3) 1·2 (<0·1) 1·5 (0·2) 2·5 (0·2) 1·6 (0·2) 1·6 (0·1) 1·5 (0·1) 1·3 (0·2) 2·8 (0·3) 2·5 (0·1)

B C D E F G H I J K L

Soluble NSP∗ (g/kg) 17·8 (1·2) 23·5 (1·4) 17·8 (1·2) 16·2 (1·1) 16·7 (1·3) 20·8 (2·6) 16·2 (0·1) 21·8 (1·2) 15·5 (1·1) 15·2 (1·9) 16·5 (0·3) 17·5 (1·4)

Ara=arabinose, Xyl=xylose, Man=mannose, Gal=galactose, Glc=glucose, UA=uronic acids, b-Glu=(1→3, 1→4)-bglucan (all values quoted as anhydrous sugars). Standard deviations shown in parentheses.

most probably derived from incomplete hydrolysis of starch and was excluded from the calculation of soluble NSP content. No relationships could be detected between AME and total or soluble NSP, soluble arabinoxylan or (1→3, 1→4)-b-glucan, or with any of the individual monosaccharide residues contributing to NSP. Similarly, AME was not associated with the starch content of the grain. A modest negative correlation, however, was found between AME and the concentration of soluble galactose and mannose residues (r2=0·58) but only when data from wheat C was excluded from the analysis. In most wheat samples, galactose and uronic acids residues were more readily solubilised than other components of NSP (Tables I and II). Wheat C was the exception and the solubility of these two sugar residues was half of that observed in other samples. Similarly a relationship between AME and the ratio of soluble arabinoxylan to (1→3, 1→4)-b-glucan (r2=0·54) was evident only when one sample (wheat H) was excluded. Extracts of a constant weight of grain gave in

vitro viscosity values which ranged between 2·0 and 8·5 mPa/s (Table III) but there was no evidence of the expected negative correlation between viscosity and AME. Both the soluble and total arabinoxylan content of the wheat correlated with extract viscosity, with the soluble arabinoxylan showing a weaker relationship (r2=0·61) than the total arabinoxylan (r2=0·82). Since arabinoxylan was the major component of NSP and expected to be the main contributor to viscosity this was not surprising. However, both relationships were negatively related. The Mr distribution of the soluble NSP also varied between the wheat samples (Table III) but no relationship with AME was found. Six branched oligosaccharides were isolated from the commercially available arabinoxylan following digestion with xylanase and separation by size exclusion chromatography. Their molecular mass was determined by MALDI-TOF-MS and structural details determined by NMR (Fig. 1). Structures AX-9 [Fig. 1(e)] and AX-10 [Fig. 1(f )] have not been previously described. It was not

Arabinoxylan structure and AME of wheat

Table III

The viscosity of soluble NSP extracted from a constant amount of wheat and the molecular weight distribution of the component polymers

Wheat sample A B C D E F G H I J K L

83

Proportion of soluble NSP (%) >300 k

200–300 k

100–200 k

20–100 k

<20 k

Extract viscosity (mPa/s)

13 0 1 4 2 1 8 1 1 4 5 7

32 0 18 0·5 7 4 7 11 6 7 25 15

6 30 15 0 17 18 16 22 21 12 34 26

7 41 18 2 29 46 18 17 21 25 25 32

42 29 48 93·5 44 30 52 49 51 51 11 20

5·61 2·07 2·09 5·21 8·39 2·24 7·40 2·66 7·15 5·29 3·36 2·85

possible to fully assign the NMR spectra of these oligosaccharides because of imperfect spectral resolution. However, their structures were derived using the anomeric signals (Table IV) as structure reporter groups and the mass data from the MALDI-TOF-MS. These six identified branched oligomers AX-5 to AX-10 and the linear xylose oligomers X-1 to X-12, were included in HPAEC separations and their retention times recorded (Table V). The oligosaccharide mixtures obtained by digesting the soluble NSP extracted from the grain samples with the same xylanase were then separated under identical conditions. Data from these separations was normalised to allow comparison between samples by defining the total area under the chromatogram as equal to unity. The contribution made by each peak to the total area was then calculated and expressed as a fraction (Table V). Evident structural differences in arabinoxylans from the different wheat samples were present but no correlation was found between any of the individual structural features and AME. Finally, the amount of undigested arabinoxylan remaining after enzyme digestion, designated xylanase resistant arabinoxylan (XRAX), was measured and analysed for sugar content. XRAX represented 4–6% of soluble arabinoxylan (0·36–0·72 g/kg wheat dry matter) and was found to be highly branched with arabinose:xylose ratios of 1·51– 2·98. Values above 2 would require side chains of more than a single monosaccharide residue. A weak, positive, linear correlation was found between AME and XRAX (r2=0·37).

Since no single feature of soluble NSP could explain the variation in AME when the grains were fed to poultry, the data was subjected to multiple regression analysis. Selection from 32 variables produced equation (2) which explained 76·9% of the variance in AME and was significantly related (p<0·05). AME=8·07+11·16(XRAX)+30·67(AX-6) −0·355(sNSP)

(2)

The terms XRAX and AX-6, the concentration of the branched hexasaccharide whose structure is shown in Figure 1(b), provided information on the extent of branching within the arabinoxylan and sNSP, the amount of soluble NSP, that could be extracted. DISCUSSION The properties of soluble NSP likely to affect digesta viscosity, other than concentration, are the structure and molecular weight of the contributing polysaccharides and any interactions between polymers. Any differences between (1→3, 1→4)b-glucans, which are unbranched and relatively regular in structure, are most likely to relate to molecular weight. In contrast, structural heterogeneity amongst arabinoxylans from different wheat samples is well established22–26 and has been confirmed in the current study. Partial degradation of arabinoxylan with an endoxylanase resulted in mixtures of up to 12 oligosaccharides, with each wheat sample examined differing in the presence

S. C. Austin et al.

84

(a) O

O O O

O

O

O O

O CH2OH

O

CH2OH

(b) O O

O O O

O

O

O O

O CH2OH

(c)

O O

CH2OH O

O O O

O O

O

O O

O

O CH2OH

(d)

O O

CH2OH O

O O O

O O

O

O O

O

O

O CH2OH O CH2OH

O

CH2OH

(e) O O

O O O O

O

O

O O O

O

O

O

O CH2OH

O CH2OH

O

CH2OH

(f) O O

O O O O

O CH2OH

O

O

O

O O O

O

O O

O

O CH2OH

Figure 1 Structure of the six branched oligosaccharides isolated from wheat arabinoxylan after hydrolysis with an endoxylanase. (a) AX-5; (b) AX-6; (c) AX-7; (d) AX-8; (e) AX-9; (f ) AX-10. Proton NMR data used for the identification of AX5 to AX-8 was the same as that previously published by Hoffmann and colleagues33.

Arabinoxylan structure and AME of wheat

Table IV

1

H-NMR assignments for the anomeric protons of oligosaccharides AX-9 and AX-10

AX-9 m/z=1231

arabinogalactans in the soluble NSP but suggested that their levels were too low to be of any significance. The galactose and mannose observed in this study were also present in very low amounts and it is possible that their correlation found with AME may be an associated phenomenon rather than causal in nature. The ratio of arabinoxylan to (1→3, 1→4)-b-glucan has not previously been reported as having an influence on AME but might suggest some form of interaction between the two polymers. However, both of these correlations were fairly weak and a larger pool of wheat samples would be required to confirm these observations. The lack of any correlation between extract viscosity and AME is contrary to the findings of others28,29, who have shown that intestinal viscosity is strongly negative related to nutrient digestibility. Since the conditions in the chick digestive tract are likely to be radically different from those in the in vitro measurement of extract viscosity, the two sets of data are not strictly comparable. Although no interactions, other than that between arabinoxylan and (1→3, 1→4)-b-glucan, could be demonstrated in vitro, the upper digestive tract offers far greater opportunities for interpolymer interactions, particularly with proteins. Possibly of far greater significance was the unexpected finding of a negative correlation between extract viscosity and arabinoxylan concentration. Most others who have examined this relationship

AX-10 m/z=1358

Residue

H-1

Residue

H-1

a-Xylp-1 b-Xylp-1 b-Xylp-2 b-Xylp-3 b-Xylp-4 b-Xylp-5 b-Xylp-6 a-Araf3×3 a-Araf2×5 a-Araf3×5

5·179 4·580 4·477 4·509 4·463 4·634 4·437 5·387 5·218 5·267

a-Xylp-1 b-Xylp-1 b-Xylp-2 b-Xylp-3 b-Xylp-4 b-Xylp-5 b-Xylp-6 b-Xylp-7 a-Araf2×4 a-Araf3×4 a-Araf3×6

5·176 4·577 4·482 4·469 4·631 4·459 4·506 4·432 5·215 5·264 5·385

Xylp-1 denotes xylopyranose at the reducing end, Xylp-2 denotes the next xylose in the chain, etc. Araf3×3 denotes arabinofuranose side chain linked to position 3 of Xylp 3, Araf2×5 denotes arabinofuranose side chain linked to position 2 of Xylp 5, etc. Chemical shifts are expressed in ppm relative to external DSS. The m/z values were measured as [M+Na]+ by MALDI-TOF-MS.

and relative proportions of these oligomers (Table V). Interactions between different polysaccharides have not, apparently, been considered as an explanation for differences in AME. Choct and Annison27 reported the presence of glucomannans and

Table V t(R)

HPAEC separation of the oligosaccharides obtained after hydrolysis of wheat soluble NSP with an endo-xylanase

ID

Wheat sample A

2·7 7·1 10·0 10·8 11·3 13·0 13·7 15·8 19·3 20·4 21·5 22·4

85

X-I 0·279 UI 0·156 X-8 0·073 X-9 0·050 AX-5∗ 0·022 AX-6 0·036 U2 0·249 U3 0·075 AX-8 0·022 U4 0·014 AX-10 0·012 AX-9 0·010

B

C

D

E

F

G

H

I

J

K

L

0·427 0·030 0·059 0·152 0·000 0·126 0·032 0·000 0·076 0·033 0·043 0·021

0·631 0·000 0·025 0·088 0·000 0·105 0·059 0·000 0·024 0·017 0·036 0·016

0·401 0·098 0·027 0·074 0·000 0·119 0·101 0·000 0·035 0·035 0·066 0·044

0·597 0·126 0·038 0·043 0·014 0·030 0·098 0·014 0·018 0·008 0·007 0·006

0·656 0·000 0·023 0·072 0·000 0·098 0·037 0·000 0·027 0·015 0·045 0·027

0·514 0·107 0·061 0·060 0·017 0·038 0·119 0·042 0·017 0·011 0·008 0·007

0·494 0·000 0·031 0·116 0·000 0·152 0·028 0·000 0·063 0·035 0·049 0·032

0·259 0·191 0·064 0·084 0·040 0·075 0·170 0·047 0·028 0·014 0·016 0·013

0·592 0·078 0·021 0·066 0·000 0·085 0·073 0·000 0·018 0·018 0·029 0·020

0·561 0·000 0·031 0·044 0·013 0·151 0·049 0·000 0·047 0·028 0·063 0·012

0·558 0·000 0·030 0·099 0·000 0·127 0·079 0·000 0·032 0·017 0·032 0·025

X-1 to X-9 denotes linear xylo-oligosaccharides; AX-5 to AX-10, branched xylooligosaccharides carrying arabinose side chain(s) and U1 to U4, xylo-oligosaccharides of unknown structure. Results are expressed as the fraction of the total area contributed by each peak. t(R)=retention time in min and ID=identity of the peak.

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have found the expected positive correlation2,8,28,30,31. The most likely explanation for this difference relates to the age of the sample. All of the wheat samples used here were harvested in 1991/92 and stored for approximately 6 months before the AME trials. There was then a second period of storage before the analytical work was started. Endogenous xylanases, in the stored grain32, may slowly degrade NSP resulting in an increased solubility of arabinoxylans with decreased molecular weight. Any reduction in the molecular weight of a polysaccharide would make it less likely to form the large intermolecular networks which cause increases in solution viscosity. Practical support for this view is provided by low quality wheat whose AME tends to improve with time, with the greatest part of any improvement occurring within the first 6 months of storage11. A similar situation has been observed with exogenous enzyme supplementation. While the intestinal viscosity of birds fed enzyme supplements is reduced, the concentration of soluble NSP may increase10. This is due to the action of the exogenous enzymes solubilising otherwise insoluble NSP from the cell walls of the grain while simultaneously reducing the molecular weight of the released polysaccharide below the size where they contribute significantly to intestinal viscosity. Little attention has been paid to endogenous enzyme activity as a possible confounding factor in studies of grain polysaccharides. As a result, the extent of any modification to carbohydrate solubility, chain length and fine structure which occurs during normal conditions of storage remains to be determined. Since relatively few bonds need to be cleaved to affect viscosity, it is likely that this measure is a particularly sensitive index of change. However, the present results suggest that small changes to the fine structure of arabinoxylan may not be as apparent or significant. Nonetheless, this work highlights the need to take account of changes on storage as a possible variable in all studies of grain NSP and that for nutritional studies, animal and laboratory work ideally should be synchronised. This was not possible in the present case because of the need to select from a large number of trials, samples of wheat which showed the widest spread of AME values. Multivariate analysis of the data pointed to the importance of branching within arabinoxylan, the predominant polysaccharide found in wheat NSP. Two of the three factors able to explain >80% of the variation in AME between wheat samples

related to this facet of structure. It seems unlikely that this relationship could be sustained if there was extensive structural modification of AX in the stored grain. What is likely to be important is the distribution of the branches within a molecule. If branching is concentrated in small blocks, then large stretches of unbranched xylose residues would be available for the formation of junction zones. A more even distribution of branching along the backbone of the molecule would allow only small blocks of linear xylan between branches and reduce the opportunity for the formation of junctions between polymers. Additionally, any unbranched sections that are available for the formation of junction zones will tend to be short, reducing their stability. Increased branching or a more even spread of branching would be expected to be negatively related to the capacity to enhance viscosity and, thus, positively related to AME. The two variables preferentially selected by the model which offer a measure of arabinoxylan branching (the concentration of AX-6 and XRAX) were both positively related to AME implying a reduced capacity for inter-chain interactions. This is supported by the absence of any detected relationship between AME and the concentration of xylose or linear xylo-oligomers in enzyme digests. The third component of the model, the concentration of soluble NSP, was negatively related to AME which is consistent with the expectation that an increase in soluble NSP concentration leads to an increase in digesta viscosity and, in turn, to a reduction in AME. However, selection of this component was surprising in the light of the lack of any relationship with soluble NSP or arabinoxylan concentration with viscosity and its selection may have been driven by the (1→3, 1→4)-b-glucan content of NSP. If this is the case, then this component of the model may be more dominant in freshly harvested grain where a clearer relationship between soluble arabinoxylan and viscosity would be expected. The model in its present form predicts AME to a reasonable extent, although not to within the 0·5 MJ/kg required by the industry. It should also be noted that the error terms associated with the AME determination were of this order of magnitude. Increasing the number of wheat samples would allow a greater number of variables to be included in the model and would be expected to improve accuracy and reduce errors. However, the model has demonstrated that araboxylan structure is at least of equal importance to NSP con-

Arabinoxylan structure and AME of wheat

centration as a factor determining the AME of wheat. This study has also shown that it is possible to identify low AME wheat from a combination of chemical and statistical analyses.

Acknowledgements The authors would like to thank S. McCrae (RRI), G. Duncan (RRI), M. Franklin (SASS) and N. Nichol (Nottingham) for their help and advice. The work described here was funded by the Home Grown Cereals Authority and forms part of a report to the Authority (Project Report 133).

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