Hemicelluloses: Science and Technology - American Chemical Society

Chapter 4

Isolation and Characterization of Arabinoxylan from Oat Spelts Downloaded by UNIV OF ARIZONA on November 19, 2015 | http://pubs.acs.org Publication Date: October 7, 2003 | doi: 10.1021/bk-2004-0864.ch004

B. Saake, N. Erasmy, Th. Kruse, E. Schmekal, and J. Puls Institute of Wood Chemistry and Chemical Technology of Wood, Federal Research Centre of Forestry and Forest Products, 21031 Hamburg, Germany

Oat spelts contain high amounts of xylan whereas the lignin content is comparatively low. Accordingly this raw material is an interesting candidate for the isolation of xylan. The variation in chemical composition of oat spelts from different sources was investigated. Xylans were isolated by alkaline extraction under various conditions. A NaOH concentration of 5% (w/v) at a temperature of 90 °C enabled the isolation of xylan in high yield and purity. The total alkali charge, based on the raw material, could be reduced by lowering the liquid to solid ratio. With a simple H O bleaching procedure high brightness values of 90 % ISO could be reached. Bleached xylan powders excelled by a high brightness stability. 2

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2

© 2004 American Chemical Society

In Hemicelluloses: Science and Technology; Gatenholm, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

Downloaded by UNIV OF ARIZONA on November 19, 2015 | http://pubs.acs.org Publication Date: October 7, 2003 | doi: 10.1021/bk-2004-0864.ch004

53 Oat spelts are a raw material rich in xylan. They are collected at the factory sides of oat mills in large quantities since they can make up to almost 40% of the total weight of grain processed in a mill. In the seventies oat spelts were a major source for the production of furfural accounting for about 22% of the total production (/). However, the importance of oat spelts for this application has declined due to lower production costs in countries of Afrika and Asia, where bagasse or rice residues are the raw materials of choice. In addition the utilisation of furfural is in general declining in industrialized countries. Today oat spelts are mainly used as an additive for animal feed, in most cases after grinding and pelleting. However, the price of this product is rather low, especially considering the energy input for production. Lehrfeld reported in 1996 that 70 US $ per ton can be obtained and prices today should be even lower (2). During the last years there exists an emerging interest for the application of xylan as a polymer. Xylans were tested as gel forming or thermoplastic material (3,4,5), as filler for polypropylene (6), and as a component for paint formulation (7) or for the coating of cellulosic fibres (8). For higher value products the application for pharmaceuticals might be of interest for instance as tabletting material (9), for the treatment of wounds (10) or for preventing blood coagulation (11). Some biological and physiological effects of xylans are summarized by Ebringerova and Hromadkova (12). Further studies have focused on the derivatisation of xylans to ethers and esters (13). Besides of the large number of possible applications xylan is not yet available in large quantities on the market. Xylans are components of many plant groups including monocotyls, dicotyls and even algae. However, for a technical isolation procedure not all raw materials are favourable. In recent years several groups tried to pursue the development of isolation procedures of xylan based on raw materials, like wood (14), corn cobs (75), corn hulls (16), or straw from various agricultural plants (17, 18). In generell xylans from different raw materials show a wide variation in their structure, which are summarized in several reviews for wood hemicelluloses (19, 20), grass hemicelluloses (21) and cereals (22). Arabinoxylans from cereals cell walls are of importance for cereal utilisation or flour properties. These arabinoxylans often have a high amount of side groups or side chains, and molar masses can reach several hundred thousands or even millions (23). For xylan isolated from oat spelts molar masses of 22,000 g/mol have been reported (24). The ratio of xylose, arabinose and 4-O-methyl glucuronic acid for those sample was 7.6:1:0.03. Accordingly, these xylans resemble xylans isolated from different straw types, which is in accordance with the fact that both, straw and spelts, are lignified plant tissues. For lignocellulosic materials there are some arguments in favour of grasses compared to wood as raw material. The first mentioned materials have higher xylan and lower lignin contents, while the lignin structure is easier degradable. For the isolation procedure several techniques have been applied which normally include an alkaline extraction as the main step. Sodium hydroxide or


54


aqueous ammonia are the most common extraction media. Mild steaming treatment (25), refining (14,26) or milling (17,18) of the raw material have been applied to increase accessibility or reduce the necessary reactor volume. For lignocellulosic materials extraction yields can be further improved by delignification treatments which reduce the hindrance of extraction by lignin carbohydrate bonds. Treatments with sodium chlorite (18) and hydrogen peroxide (17) have been applied prior to extraction while hydrogen peroxide, peracetic acid and performic acid have been used as well during the extraction stage (27). Extraction yields can be improved as well by the application of ultrasound during the extraction (75), while twin screw extrusion might be a technical approach to design a high throughput process (28).

Experimental Raw Material All oat spelts were provided by Peter Kolln K G a A (Elmshorn/Germany). Starch residues were reduced by removing material smaller than 1 mm by sieving. Prior to xylan extraction oat spelts were treated in a Sprout Waldron refiner.

Xylan Extraction and Precipitation Climatized oat spelts, corresponding to a dry weigh of 50 g were mixed with the NaOH solution. The NaOH concentration was varied from 2-19% (w/v) and the consistency from 10-30%o. Extraction treatments were performed in autoclaves, rotating in an oil bath for 60 min including the heating up period. The autoclaves were equipped with valves for the application of oxygen pressure, if needed. After the reaction, autoclaves were cooled down. The reaction mixture was transferred into a hollow steel cylinder, containing a filter cloth on a perforated steel plate on the bottom of the cylinder. A plunger fitting into the cylinder was used to apply a pressure of 10 MPa using a hydraulic press. The alkaline extraction liquor passed the perforated plate on the bottom of the cylinder and was collected in a tray. The liquor was neutralised using acetic acid and slowly precipitated in the threefold volume of ethanol. The pH was adjusted to 5.5 and the precipitate was recovered by filtration. The xylan was re-dispersed in water, the volume equivalent to the volume of the starting liquid. The solution was re-precipitated in the threefold volume of ethanol, the pH adjusted to 4.0 and the precipitate recovered by filtration. The xylan was washed first with ethanol and then with ether. Samples were dried in the fume hood from ether. During this procedure the particles size was reduced every now and then to insure the formation of a fine powder. The extracted oat spelts residue was mixed with 100 ml acetic acid (20%) in a glass beaker. After 10 min the residue was transferred on a buchner funnel and


55 the acetic acid was washed out with water. The samples were climatised for yield determination and further analysis.


Bleaching of Xylans Peroxide bleaching of extracted xylans was performed analogously to the procedures used for pulp bleaching in polyethylen bags in a thermostated water bath at 80 °C for 2 hours at a consistency of 10%. The NaOH, water glass, and peroxide charges were calculated, based on dry xylan. After the treatment xylans were recovered by one precipitation step into the threefold volume of ethanol. The pH of the liquid was adjusted to 4.0. The xylan was recovered by filtration and drying from ether, analogously to the procedure for extracted xylan. Monosaccharide Analysis Prior to carbohydrate analysis oat spelts and extraction residues were ground in a vibration grinding mill (HSM 100, Herzog, Osnabriick/Germany). A two step hydrolysis was used for spelts and extraction residues applying 72% sulphuric acid in the first and 2.5% sulphuric acid in the second step. Extracted xylans were analysed in a one step procedure applying 4% of sulphuric acid. Monosaccharides were then determined by borate-complex anion exchange chromatography. Details of the hydrolysis and chromatography procedures were reported previously (29, 30). Starch was analysed by aqueous extraction, enzymatic hydrolysis and chromatographic determination of the glucose (37) Size Exclusion Chromatography SEC was performed on the following column set: G R A M 30, 100, 3000 (8x300 mm) and guard column (8x50 mm, Polymer Standard Service, Mainz/ Germany). 0.05 M LiBr in DMSO:water (90:10) was chosen as mobile phase with a flow rate of 0.4 ml/min at 60 °C. The amount of the soluble material passing the column was determined by concentration calibration with xylopentaose as a standard. Molar mass data were calculated from viscositydetection and universal calibration as published previously (24). Brightness and Brightness Stability of Xylans 3 g of xylan powder was pressed into a pellet of 45 mm diameter and 5 mm heights using a small press for the production of B a S 0 brightness standards. The measurements were then performed with an Datacolor 2000 instrument. Brightness stability was tested placing xylan powder in a large crystallizing dish to maximise the surface exposed to the light source. A daylight source was used for irradiation. Every day the powder was transformed into a pellet for 4


56 brightness measurement exposure.

and afterwards dispersed in the dish for further

Results and Discussion


Analysis of Different Oat Spelts Oat spelts from different sources were analysed regarding their chemical composition. The samples were not collected according to botanical species, since the grain wholesaler are collecting and mixing products from several farmers. Nevertheless, between spelts from oats purchased from Finland, Australia and two German sources major differences occurred. The hydrolysis residues listed in Table I are an indication for the lignin content of the spelts. The sample with the highest lignin content were spelts from Finnish oats while the lowest content was determined for quality B from Germany and the material produced from Australian oats. The maximum difference accounted to 5% which is a substantial advantage for low lignin content material. In view of the fact that oat spelts contain an arabinoxylan, the possible product yield can be compared after adding up the xylose and arabinose content for each sample. Here the lowest amount was found for quality B from Germany (21.9%) while the highest content was determined for the spelts from Australian oats (33%). Again this difference of more than 10% clearly demonstrates that a comparison of different oat varieties has to be considered for further studies. For all samples the glucose contents in Tabel I not only reflects the cellulose but as well the starch. Therefore the residual starch content was investigated separately. Here a variation between 0.8 and 10.2% was found. For the extraction work a

Table I. Hydrolysis residue, monosaccharide composition, and starch content of oat spelts from different sources

Origin

Hydrolysis residue

Xylose

23.6 18.8 22.9 18.5 21.3

29.7 30.1 27.0 19.7 29.5

Arabinose

Total glucose

Glucose from starch

% based on raw material Finland Australia Germany-A Germany-B Material for extraction

2.9 2.9 2.6 v 2.2 2.7

34.0 35.4 37.9 46.7 36.5

0.8 4.9 10.2 2.6 4.5



57 representative sample was taken from the oat mill, which was a mixture of different raw materials representing the production process. This material is listed in the last line of Table I and had a hydrolysis residue of about 21.3% and a arabinoxylan content of 32.7%, not adjusted for water addition. The acetyl, ferulic and p-coumaric acid content of xylans was not determined, since these side groups are removed in an alkaline extraction and cannot contribute to the product yield. 4-O-methyl glucuronic acid contents are low in these xylans. Only 0.2-0.3% survive the hydrolysis procedure used for the raw materials. Assuming a xylan yield of 30% this would amount to 0.8 to 1%, based on the xylan. A ' H N M R study of xylan extracted in this work indicated about 1-1,5% of 4-Omethyl glucuronic acid in the xylan. Accordingly a significant proportion of these substituents are destroyed under the hydrolysis conditions applied for the raw materials. Since the total amount is still low the analysis of 4-O-methyl glucuronic acid was not included in the study as a routine analysis. Galactose and rhamnose are present in small quantities adding up to less than 2 % and will not be discussed in detail. The ester groups (acetyl, feruloyl, p-coumaroyl) were not quantified. These groups are saponified under the alkaline extraction conditions applied and therefore not relevant for the characteristics and the yield of arabinoxylan in this study. Non-Pressurized Alkaline Extraction First the alkaline extraction at ambient temperature was investigated using NaOH concentrations from 5 to 12.5% (w/v) and consistencies of 10% for 1 hour extraction time. However, under those conditions xylan yields were very low and did not exceed 8%, based on raw material. Therefore the effect of reaction temperatures was investigated using different NaOH concentrations. The results for 5% (w/v) NaOH are summarized in Table II.

Table II. Yield, carbohydrate composition and molar mass of xylans extracted at different temperatures Temp. XylanSEC Carbohydrate analysis yield M Hydrolysis Xylose Arabinose Glucose residue °C g/mol % % based on total carbohydates % w

70 80 90

34.6 38.5 40.4

14.5 13.7 15.4

70.1 74.7 72.4

11.2 12.7 11.7

16.7 10.2 13.4

29,300 30,200 30,080

NOTE: Extraction at 10% consistency, 5% (w/v) NaOH, 1 h



58 At 70 °C a yield of 34.6% extracted powder could be achieved while a further increase of the reaction temperature up to 90 °C enabled a maximum yield of 40.4%. The hydrolysis residue of roughly 14-15% indicated that the xylans still have a high amount of lignin impurities. The carbohydrate fraction was made up to more than 80% by arabinoxylan, while between 10-17% of the carbohydrates were glucose, which resulted mainly from starch impurities and to a minor extent from low molar mass cellulose. Molar masses were in the magnitude of 30,000 g/mol, which is equivalent to 227 monosaccharide units per molecule. Considering the xylose:arabinose ratio of 5.9 to 6.4 for these samples the polymer backbone, calculated as weight average degree of polymerisation ( D P J , amounted to roughly 195 xylose units while every sixth unit was substituted by arabinose. In general the results indicate that an effective extraction of oat spelt xylan requires the application of elevated temperatures. It can be assumed that even at the high alkaline concentration applied, elevated temperatures are needed to effectively cleave lignin carbohydrate bonds in the limited time of the alkaline treatment. Oxygen Aided Alkaline Extraction Alternatively to non-pressurized extraction the application of 0.6 MPa 0 pressure was investigated. These experimental conditions should generate an alkaline oxygen bleaching effect and lower the lignin content in both, the extracted xylan powder and the extraction residue. 2

Effect of Consistency and Alkaline Concentration

on 0 -Aided 2

Extraction

The total alkaline charge required for xylan extraction was of major importance for the feasibility of the isolation process. These factors were dependant on the alkalinity of the extraction liquor and the consistency used in the process. In Figure 1 the effect of alkalinity on the yield of xylan and extraction residue is depicted for 10% and 25% consistency. The upper picture for 10% consistency demonstrates that an increase of NaOH concentration from 2.2 to 5% resulted in a continuous increase of xylan yield. Totalling the yield of xylan and residue it became apparent that up to 20% of the starting materials were not recovered. This material remained in the precipitation liquid and should consist of extractives, lignin fragmentation products, and low molar mass carbohydrates. A further analysis of those components was not pursued at this stage of the project. By comparison of the upper and lower x-axis it became apparent that 5% NaOH concentration at 10% consistency resulted in an enormous total alkali charge of more than 45%, based on raw material. Rising the consistency to 25% (Figure 1, lower graph), enabled far higher alkali concentrations up to 15%),


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NaOH-solution [% w/v] 2

3

4

5

50

•Sj 40

— - -o —

Xylan Residue

CD


E ^ 30

2 c o

TJ

Hemicelluloses: Science and Technology - American Chemical Society

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