WO2014170498A1

WO2014170498A1 - Enzymatic production of polysaccharides from gum tragacanth

Info

Publication number: WO2014170498A1
Application number: PCT/EP2014/058092
Authority: WO
Inventors: Jørn Dalgaard Mikkelsen; Hassan Ahmadi GAVLIGHI; Malwina MICHALAK; Anne S. Meyer; Marcel Tutor ALE; Dennis Sandris NIELSEN
Original assignee: Danmarks Tekniske Universitet
Priority date: 2013-04-19
Filing date: 2014-04-22
Publication date: 2014-10-23

Abstract

Plant polysaccharides, relating to the field of natural probiotic components, can comprise structures similar to human milk oligosaccharides. A method for enzymatic hydrolysis of gum tragacanth from the bush-like legumes of the genus Astragalus, using a combination of pectin hydrolases and a xylogalacturonan hydrolase, is described. Fractions with different oligo- and/or polysaccharide compositions and structure are separated according to molecular weight.

Description

Enzymatic production of polysaccharides from Gum Tragacanth

Field of the Invention

The present invention relates the field of natural probiotic components such as plant polysaccharides comprising structures similar to human milk oligosaccharides. More particular, the present invention relates to enzymatic hydrolysis of gum tragacanth from the bush-like legumes of the genus Astragalus.

Background of the invention

There is an increasing interest in the development and identification of new carbohydrates having prebiotic effects. Prebiotic carbohydrates are defined as selectively fermented compounds that cause specific probiotic changes in the gastrointestinal microbiota

(currently mainly understood as stimulating the growth of lactobacilli and bifidobacteria), which in turn confer benefits upon well-being and health of the host. The beneficial growth stimulation of specific probiotic colonic bacteria is generally explained by the capability of these bacteria to cleave the glycosidic linkages in the prebiotic carbohydrates. A primary example of natural prebiotics is the group of human milk oligosaccharides, which constitutes a structurally diverse family of galactose-, glucose, N-acetyl-glucosamine, sialic acid, and fucose-substituted lactose-based structures containing different types of glycosidic linkages, and which are almost unique to human breast milk. Human milk oligosaccharides were thus originally recognized as being responsible for the "bifidus factor" of human milk, and involved in the relationship between the intestinal bacteria and lower incidences of infectious diarrhea in breast-fed infants. Galacto-oligosaccharide (GOS) and fructo- oligosaccharide (FOS) are two main types of oligosaccharides that are recognized as prebiotics and which have been tested in human trials and which are already available in the market (Gopal, P. K et al., Int. Dairy J. 2001, 11, 19-25; Roberfroid, M. B. et al., J. Nutr. 1998, 128, 11-19). Foods containing these compounds are generally categorized as functional foods.

Gum tragacanth is a natural gum obtained from the dried sap from the stem, for example in the form of an exudate, of several species of the Middle Eastern bush-like legumes of the genus Astragalus, also known as "locoweed" and "goat's-horn". Astragalus species is one of the few natural plant sources of L-fucose-substituted polysaccharides. The gum has been used commercially for well over 2000 years, is approved for food use in both the US and Europe, and is widely used as an emulsifier and thickener in emulsion systems in different food, pharmaceutical and cosmetic applications. Gum tragacanth is made up of highly substituted, heterogeneous hydrophilic polysaccharides containing L-arabinose, D-galactose, D-glucose, D-xylose, L-fucose, L- rhamnose, and D-galacturonic acid, and confers high viscosity when in aqueous solution (Balaghi, S. et al., Food Biophys. 2010, 5, 59-71). When solubilized in water, the gum is usually categorized in a "soluble" fraction, tragacanthin, and an "insoluble" fraction, bassorin, respectively( Balaghi, S. et al., Food Hydrocolloids 2011, 25, 1775-1784). The water-soluble tragacanthin fraction appears to resemble pectin, and contains linear chains of galacturonic acid (probably 1,4-a-linked) and arabinogalactan structures as well as fuco- xylogalacturonans, whereas bassorin is believed to be mainly composed of a mixture of xylo- and fuco-xylo-substituted polysaccharides ( Balaghi, S. et al., Food Hydrocolloids 2011, 25, 1775-1784).

Terminal, fucosyl residues are also found in some human milk oligosaccharide structures (as a l-2, a l-3, and a l-4 substitutions), hence gum tragacanth saccharides could have prebiotic effects mainly because of the L-fucose-substituted polysaccharides. However, the high viscosity inducing effects of gum tragacanth partly prevent unfolding of this potential, because the viscosity means that only low levels are used, since inclusion of even very low levels of gum tragacanth in food products changes the physico-chemical properties of the products significantly.

Recently, enzyme technology has been used to produce various potentially prebiotic oligo- and polysaccharides from plant fiber polysaccharides for food applications. In particular, the combination of enzymatic depolymerization of various natural polysaccharides and membrane reactor separation has proven useful for lowering the viscosity of hydrocolloid polysaccharides and for obtaining prebiotic compounds of different molecular size and structural composition. In previous studies it has been shown that both the molecular size and the structural make-up of the resulting poly- and oligosaccharides have a significant influence on their potential prebiotic bacterial growth promoting properties (Al-Tamimi, M .et al, J. Appl. Microbiol. 2006, 100, 407-414; Thomassen, L. V. et al., Appl. Microbiol.

Biotechnol. 2011, 90, 873-884; Hoick, J. et al., Process Biochem. 2011, 46, 1039-1049).

In a previous work, the present inventors have shown that pectinase catalyzed

depolymerization of gum tragacanth could reduce the viscosity of the gum in solution, and the product was separation into different molecular size fractions via membrane filtration ( Gavlighi, H. et al, J. Agric. Food Chem., DOI: 10.1021/jf304795f, Publication Date (Web) : January 23, 2013)). The resulting fuco-xylooligosaccharides (FXO) fractions had different molecular sizes, structures and types of linkages and showed different growth-stimulating effects on selected gut-relevant bacteria. Plant cell walls (mainly primary cell walls) consist basically of three major polysaccharides: cellulose, hemicellulose and pectin. Cellulose is the most abundant plant polysaccharide and consists mainly of p-(l- 4)-linked D-glucan chains. Xyloglucan is a component in

hemicellulose in the primary cell wall and consists of a cellulose-like (l- 4)-linked β-glucan backbone with single unit of a-D-xylopyranosyl (a-D-xylp) substituents attached at 0-6.

Some Xylp residues are further substituted at 0-2 by β-D-galactopyranosyl (β-D-Galp) or a- L-fucose-p-l,2-galactose in position C-2. Pectin has a complex structure including homogalactronan, rhamnogalactronan I and II and xylogalactronan.

In order to degrade plant cell walls different enzymes may be used to hydrolysis or modify the different polysaccharides constituting the plant wall. Pectolytic enzymes are classified base on their mode of action on pectin structures. Pectin and pectate lyases split a-l,4-D galacturonan linkages in homogalactronan by β-elimination and introduce a double bond between C4 and C5 of the newly formed non-reducing end. While pectin lyases (E.C.

4.2.2.10) prefer to act towards highly methylated pectins, pectate lyases (endo acting (EC 4.2.2.2) and exo-acting (EC 4.2.2.9)) are most active towards pectate or low methoxyl pectins. Pectin methylesterase (PME, E.C. 3.1.1.11) removes methoxyl groups from pectin, and at the same time decreases the affinity of Pectin lyase for this substrate. This results in the formation of methanol and less highly methylated pectin. Pectinase or

polygalacturonase (PG, E.C.3.2.1.15) exists in two forms: endo-PG and exo-PG. Both types act only on pectin with a degree of esterification of less than 50 - 60 %. Endo-PG acts randomly on the a-l,4-polygalacturonic backbone and results in a pronounced decrease in viscosity. Exo-PG acts at the non-reducing end of the chain. Exo-PG releases small fragments from the chain and does not significantly reduce the viscosity.

Xylogalacturonan (XGA) exists in cell walls and extrudates from many plants, such as for example peas, soya beans, apples, onions, cotton seeds and watermelons. The degree of xylosylation of the homogalacturonan backbone is different in different sources of XGA. For instance, in apple XGA three out of four GalA residues carry a xylosyl side chain, whereas in watermelon XGA is found in one out of four GalA residues.

In fruit juice clarification industry, complete degradation of pectin structure is the crucial problem and the hairy regions of pectin remain resistant to degradation and XGA cause membrane fouling in the ultrafiltration process.

Recently, a xylogalacturonan hydrolase enzyme (XGH) belonging to the GH 28 family was discovered in Aspergillus tubingensis, and was reported to act on the galacturonic acid backbone by cleaving a-l,4-D galacturonan linkages in an endo-fashion and has a requirement for xylose side chains ( Van der Vlugt-Bergmans, C. et al., Applied and

Environmental Microbiology, 66(1), 36-41; WO 99/41386).

Bauer, S. et al., Proceedings of the National Academy of Sciences, 103(30), 11417-11422 disclosed the cloning of a xylogalacturonase from Aspergillus fumigatus (Afu8g06890). XGA is also found in gum tragacanth from the Astralagus species and consists of a linear chain of a-(l,4)-linked D-galacturonic acid residues in which β-D-xylose residues are β- (l,3)-linked to parts of the GalA residues. Zandleven, J. et al., Biochem J. (2005) 387, 719- 725 discloses digestion of XGA from saponified gum tragacanth preparations with the XGH (GH 28), resulting in oligosaccharides comprising GalA_mXyl_n units and the disaccharide GalAXyl.

In some plants, such as for example the Astralagus species, some of the xylose residues in XGA are further linked to fucose or galactose to form p-(l,3)-linked disaccharide units such as a-L-fucose-(l, 2)-D-xylose and p-D-galactose-(l, 2)-D-xylose (Figure 2). α-1-Fucosyl residues are frequently found at the nonreducing terminal end of various sources, including human milk oligosaccharides (many of which are fucosylated), fucoidan, xyloglucan and tragacanth gum (Figure 1). It has been reported that milk fucosyl- oligosaccharides and specifically fucosyl a l,2-linked molecules inhibit binding of the pathogenic Campylobacter to intestinal mucosa and may therefore represent a novel class of antimicrobial agents. In addition, from previous field studies it has been shown that the high ratio of 2-fucosylated oligosaccharides in human milk results in a lower risk of symptomatic infection with enterotoxigenic Escherichia coli in breast-fed infants and thus a significant and clinically relevant protection against diarrhea.

Summary of the invention

The present invention concerns a new method for depolymerizing gum tragacanth, which method comprises the treatment of a sample of gum tragacanth with a combination of two or more hydrolyzing enzymes. The depolymerization leads a mixture of oligo- and polysaccharides which can be separated into different fractions, for example according to molecular weight, where the different fractions possess different biological properties.

In one embodiment, there is provided a water-soluble fraction of gum tragacanth, which is treated with a first polysaccharide degrading enzyme, such as a pectinase. The resulting mixture is separated into sized oligo- and/or polysaccharide fractions, wherein at least one fraction comprises oligo- and/or polysaccharides which are enriched in arabinose and galactose and at least one fraction comprises polysaccharides being enriched in fucose. The latter fraction comprising polysaccharides being enriched in fucose is treating with a second polysaccharide degrading enzyme.

In a second embodiment gum tragacanth oligo- and/or polysaccharide fraction(s) enriched in fucose is obtained by a method wherein the gum tragacanth solubilized in an aqueous solution, such as water, is treated with a pectin degrading enzyme, the resulting mixture of oligo- and/or polysaccharides separated into different aqueous fractions, for example according to molecular weight, each fraction containing oligo- and/or polysaccharides containing different amounts of bound fucose, treating (one or more of) the aqueous fraction(s) containing polysaccharides with the highest amount(s) of bound fucose with a xylogalacturonan hydrolase enzyme, and separating said enzyme treated polysaccharide solution(s) into different aqueous fractions according to molecular weight, each fraction containing oligo- and/or polysaccharides with different amounts of bound fucose.

Additional hydrolytic enzymes may be used in the method in order to depolymerize the gum to a higher degree. Further, the two hydrolysis steps may be combined leaving out the intermediate separation step.

The gum tragacanth is isolated from an Astragalus sp., preferably an Astragalus specie which is rich in fucose, such as Astragalus gossypinus and powdered before use. The extruded and powdered gum tragacanth or any polysaccharide fraction thereof may be saponified before the enzymatic hydrolysis. Oligo- and/or polysaccharide compositions and fraction thereof may be used as prebiotics and/or antimicrobial agents in a functional food, such as in milk formula, as travelers food additives, etc., or may be used as a therapeutic agent, such as a prebiotic, antimicrobial agent or anti-cancer agent.

Figures Figure 1 Shows structures of natural fucose-rich glycans

Figure 2 shows a schematic representation of the xylogalacturonan hydrolase action

Figure 3 shows molecular size profiles of gum tragacanth samples of different molecular size: HAG1 < 2 kDa; 2 kDa < HAG2 < 10 kDa; and HAG3 > 10 kDa, obtained after enzymatic modification and membrane separation. Tra Control indicates the track of the original gum tragacanth starting material.

Figure 4 shows the structure of partial part of gum tragacanth structure and

xylogalacturonan hydrolyse attack. Figure 5 shows molecular size profiles of gum tragacanth samples of different molecular size: HAG3.1 < 2 kDa; 2 kDa < HAG3.2 < 10 kDa; and HAG3.3 > 10 kDa, obtained after enzymatic modification and membrane separation.

Detailed description of the invention The aim of the present invention is to develop a method to depolymerize the highly xylolated and fucosylated gum tragacanth by enzymatic hydrolysis in order to obtain oligo- and/or polysaccharides with different molecular size, structure and content of fucose and to exploit and utilize the functional and biological properties of these oligo- and

polysaccharides, for example as food additives in functional foods or as therapeutic agents. Gum Tragacanth

Gum tragacanth is obtained from Astragalus species having a high content of xylose and fucose. Extruded, dried gum may be purchased from commercial providers or obtained directly from the plants. For the present work and illustrative for the present invention, the gum tragacanth was selected from Iranian Astragalus gossypinus because of its particularly high fucose content (Balaghi, S. et al., Food Biophys. 2010, 5, 59-71; Gavlighi, H. et al., Food Hydrocolloids 2013, 31, 5-14), which was recently found to amount up to about 235 mg fucose/g dry matter, whereas the fucose content in other gum tragacanth samples obtained from five other species of Iranian Astragalus bush plants were found to range from 30-115 mg/g dry matter. Before enzymatic treatment, the dried extruded gum is powdered by mechanical forces and dissolved in a suitable aqueous solvent.

Total content of the carbohydrate content in gum tragacanth

Quantitation of the monosaccharaide composition of gum tragacanth can be done by means of acid hydrolysis of the gum, followed by quantitation of the liberated monosaccharides. The monosaccharide content of the hydrolysates can be determined in many ways, for example by use of high-performance size exclusion chromatography with pulsed

amperometric detection, HPAEC-PAD. The separation and the quantification method using HPAEC-PAD were accomplished in the present work in one single run without the need for pre-derivative treatment.

Table 1 shows the content of different sugars present in the galacto-polysaccharide structures (pectin) in gum tragacanth from different Astragalus species. A. gossypinus is the preferred source of gum tragacanth due to its high content of fucose and xylose. Table 1

Composition of Astin ofie gum tragacanth, soluble, insoluble, and full gum fractions *

Varieties A parrowiamis A fluetmus A. rahemis A gossypinus A microcephalia A compaam

Sol Insol Full Sol Insol Full Sol Insol Full Sol Insol Full Sol Insol Full Sol Insol Full

Ratio (I) 64 36 76 24 55 45 35 65 50 50 45 55

Methoxyl (mg/g) 25c 24c 26c 29b 33b 34b t 9d 16d I 7e 44a 54a 55a 30b 26c 32b 30b 32 b 36b

Acetyl (mg/g) 27a 26a 28a 27a 27a 29a 23b 22bc 24bc 24b 27a 27ab 24b 21c 24bc 25b 23b 25bc

Sugar composition (mg/g dry matter)

Fucose 46cd 55cd 49de 68c 76c 70cd 27d 26d 27e 210a 249a 236a 101b 13 lb 1 16bc 98b 1 15b 108b

Rhamnose 22a 22a 22a 20a 12ab 18abc 27a 14ab 21ab 23a 12ab 16c 25a l i b 18bc 15a !2ab 13abc

Ara inose 276b 310b 2SSb 206c 219c 209c 320a 410a 360a 49d 47e 48d 187c 174d 181 c 185c 160d 171c

Galactose 128a 127b 128b 121ab !06c 1 171K 129a 159a 143a 49c 33e 39d 112ab 73d 93c 104b 73d 87c

Glucose 15d 56ab 30d 18cd 39ab 23e 32a 151 ab 86c 4e 4b 4Γ 22bc 201a 1 12a 26ab 147ab 93b

Xylose 186b 204b 193c 216b 229b 21 Sb l 7J b 128c 152d 282a 268a 273a 180b 217b 199b 23 lab 227b 229b

Galacturonk .acid 300b 192bc 209bc 290bc 244ab 298ab 274bc 91c 109c 380a 337a 335a 370ab 129bc 218bc 187c 146bc 244ab

^Λ Values with different letters in each row are significantly different (p < 0,05).

The polysaccharide structure

The complete structural elucidation of polysaccharide and enzymatic hydrolysis of oligomers was done in the present work by methylation analysis to reveal essential structural information on structure and linkage. This method (an ingenious approach for determining linkage positions) was developed in the late 1960s and early 1970s. For glycosyl linkage analysis, the sample was permethylated (Ciucanu & Kerek, 1984), depolymerized, reduced, and acetylated; and the resulting partially methylated alditol acetates (PMAAs) analyzed by gas chromatography-mass spectrometry (GC-MS) as described by York et al (York, W. S. et al.„ In Methods Enzymol., Arthur Weissbach, H. W., Ed. Academic Press: 1986; Vol. 118, pp 3-40). The PMAAs were analyzed on a Hewlett Packard 5975C GC interfaced to a 7890A

MSD (mass selective detector, electron impact ionization mode); separation was performed on a 30m Supelco 2330 bonded phase fused silica capillary column. However, as the analyses of these linkage data are sophisticated, they are usually incomplete and difficult, at least because of the following reasons: (1) most polysaccharides are heterogeneous with high molecular weights, (2) there are many combinations of linkage types between any two monosaccharides, and (3) there is a lack of a complete set of standard materials of all linkage types that could be used as reference standards. Other approaches for the structural elucidation may also be applied.

Depolvmerization of gum traqacanth (the first enzymatic step) Pectin

Gum tragacanth polysaccharides mainly belong to the pectin group. Pectins are complex branched heteropolysaccharides primarily containing an a-(l-4) polygalacturonic acid backbone which can be randomly acetylated and methylated. Three different pectins have been isolated from plant cell walls: homogalacturonans, rhamnogalacturonan I and rhamnogalacturonan II.

Pectin ase and Pectinesterase Specificities

Homogal acturonan

P oly-a 1 -4 D-galacturonic acid backbone with random -partial m ethylation and a cetylation

P ectin ase P ecti nase

Rhamnogalaclironan I

Alternating a -(1 -2)-Ι_ -rha mnosyl- a -(1 -4)-D -galacturonosyl backbone

with two types of branching com posed of aribonfuranose or galactose oligomers

Oligo-fl- -3)-D -Ara bi nose branching

Homogalacturonans are composed of the simple a-(l-4) poiygaiacturonic acid backbone.

Substituted homogalacturonans are modifications of this backbone with β-D-xylose branching at C3, or apiofuranose substitutions in the backbone with p-D-Apiosyl-(l,3')-p-D- Apiose branching.

Rhamnogaiacturonan I contains alternating a-(l-4) galacturonosyl and a-(l-2) rhamnosyl residues, with primarily oligo a-(l-3) arabinose and oligo β-(1-4) galactose branching.

Rhamnogaiacturonan II is composed of the simple a-(l-4) poiygaiacturonic acid backbone with complex branching with composed of up to 11 different monosaccharide types.

Pectinase

Pectinase (polygalacturonase, EC# 3.2.1.15, catalyzes the random hydrolysis of 1,4-a-D- galactosiduronic linkages in pectin and other galacturonans. (Figure 1) Pectinases may be obtained from many sources and are commercially available from several companies. As non-limiting examples of commercially available pectinases useful in the method according to the present invention may be mentioned pectinases from Aspergillus aculeatus (Novozymes Pectinex Ultra SPL), from Aspergillus niger (Novozymes Pectinex 3XL), and from Rhizopus species.

For the present work and illustrative for the present invention, Pectinex® BE Colour, 3600 MOE units/mL, pH optimum 3.5-4.0, temperature optimum 45-55 °C, was obtained from Novozymes A/S (Bagsvaerd, Denmark). This enzyme preparation is derived from Aspergillus niger, and mainly contains pectin lyase and polygalactronase activity, and is approved as a processing aid for industrial food applications.

Additional enzymes, such as fungal pectin methyl esterases, could also be included in order to de-methylate the tragacanth backbone and the methylated galacturonic acid, and to avoid the base treatment step. Other pectolytic enzymes, such as Pectolase, Xymex, etc. from Novozymes, A&B enzymes, DSM or Dupont, could also be added in hydrolysis process. Alternatively, the different enzymes or combinations of enzymes could be applied in different treatment steps.

Separation of oligo- and polysaccharide fractions (the first separation step)

After treatment of the gum tragacanth solution with a suitable pectin hydrolase or combination of different hydrolases, the resulting depolymerized mixture of di-, oligo- and polysaccharides is fractionated in order to collect one or more fractions comprising the oligo- and/or polysaccharides having the highest content of bound fucose compared to the total content of these carbohydrates in gum tragacanth. The separation thus results in an enrichment of oligo-, and polysaccharides with a high content of fucose. According to a preferred embodiment of the present invention, the depolymerized mixture is separated according to molecular size.

The separation principle may for example be achieved by differential exclusion from the pores of the packing material, of the sample molecules as they pass through a bed of porous particles. The elution of polysaccharide is then achieved over time where larger molecules stay less time in the pores and thus elute first and the smaller molecules stay longer time in the pores and therefore elute later.

In one embodiment of the present invention, the mixture is separated into three fractions HAG1 <2 kDa; 2kDa < HAG2 < 10 kDa and HAG3 > 10 kDa. The fraction having a molecular weight above 10 kDa ("HAG3") is selected for further hydrolysis in the second enzymatic step. The monosaccharide compositions of the three fractions, HAGl, HAG2 and HAG3, illustrating the present invention vary significantly and also vary compared to the starting material which is dominated by galacturonic acid, xylose, and fucose. The high molecular weight fraction (HAG3) gets enriched in fucose during the enzymatic depolymerization, with the final product containing more than 15%, more than 20% and preferably more than 25% w/w fucose of the total amount of dry matter in the fraction. This fraction, HAG3, is also rich in galacturonic acid and xylose. This result agrees with the expected attack pattern of the pectinases, since the pectinases are not able to attack the substituted fuco-xylo- galacturonic acid structures. The enzymatic degradation depleted the gum tragacanth polysaccharides in HAG3 in rhamnose, arabinose, galactose, and glucose, which is in accord with previous findings that have indicated that, in addition to polygalacturonase and pectin lyase, the enzyme preparation contains side-activities that can catalyze the cleavage of several different kinds of bonds in complex plant cell wall structures. Analogously, the smaller fractions, HAGl and HAG2, became very rich in arabinose and galactose and had significantly lower fucose and xylose contents than HAG3. The level of galacturonic acid was high in all three enzymatically treated fractions, although lowest in the HAGl. The enzymatic process thus seemed to cleave the pectin-resembling, polygalacturonic acid parts of the gum tragacanth polysaccharide backbone(s), leaving the xylo-galacturonan, and especially the fuco-xylo-galacturonan stretches in the gum tragacanth intact, i.e. left in the HAG3 fraction. Hence, enzymatic depolymerization using pectinase in combination with membrane separation may be used to produce significantly different gum tragacanth polysaccharide fractions with respect to both molecular size and composition.

Membranes for use in separation according to molecular weight can be selected among any available membranes with the desired cut off value. Molecular Weight Cut Off or MWCO refers to the lowest molecular weight solute (in Daltons) in which 80%, preferably 90% of the analytes/molecules are retained by the membrane. Membrane materials may for example be selected from mineral materials (such as alumina, titanium, zirconium oxides or some glassy materials), cellulose nitrate (CN), mixed cellulose ester, cellulose acetate, nylon, polyethersulfone (PES), polytetrafluoroethylene (PTFE) and the like. High-performance size exclusion chromatography (HPSEC) was performed in order to determine the molecular size of oligomers/polymers, produced via enzymatic

depolymerization of gum tragacanth according to the present work. Alternatively, LC-MS is also feasible, however preferably for smaller and more defined structures. Treatment of high-molecular fraction(s) with xyloqalacturonan hydrolase (the second enzymatic step)

In the second enzymatic hydrolysis of the present invention, at least the fraction with the highest amount of xylose and fucose obtained from the prior separation step is mixed in a suitable aqueous solvent with a suitable xylogalacturonan hydrolase (XGH). Preferably, the XHG is an endo-enzyme which acts between two xylosidated GalA units or between a xylosidated GalA unit and a GalA unit in the oligo- or polysaccharide (Figure 4).

In one embodiment of the present invention, fraction HAG3 is subjected to hydrolysis by a suitable XGH. The oligo-/polysaccharide fractions obtained in the separation step may advantageously be saponified before treatment with the XGH.

In a further embodiment of the present invention, a xylogalacturonan hydrolase from Aspergillus fumigatus is use in the second hydrolysis step. The enzymes was produced from a Pichia pastoris clone transformed with the xylogalacturonase gene Afu8g06890, obtained from the Fungal Genetic Stock Center, as described by Bauer et al (Bauer, Vasu, Persson, Mort, & Somerville, 2006).

Optimum condition for enzyme activity was determined and used for reaction on the HAG3 product which was saponified and then applied for enzymatic degradation. The reaction was done in batch reactor and different molecular size fractions were achieved by membrane separation at 2 and 10 kDa based on HPSEC.

In another embodiment, xylogalacturonan hydrolase from Aspergillus tubingensis (GH 28) may be used in the second hydrolysis step. Xylogalacturonan hydrolase from other sourses may be used in the same way.

In an alternative method, the two enzymatic hydrolysis steps are performed at the same time by adding both enzymes (enzyme compositions) at the same time.

Separation of oligo- and polysaccharide fractions (the second separation step)

The second separation may be performed in the same way as the first separation step. In one embodiment, the mixture of di-, oligo- and polysaccharides obtained after treatment of HAG3 with a xylogalacturonan hydrolase is separated into three fractions HAG3.1 <2 kDa; 2kDa < HAG3.2 < 10 kDa and HAG3.3 > 10 kDa. Other cut-off values may be selected if desired. Additional steps involving enzyme treatment and/or separation and fractionation technique such as Supercritical fluid extraction (SFC) may be applied in order to obtain a desirable product. Also, purification steps may be used to provide sub-fractions comprising specified oligo- and/or polysaccharides with beneficial properties. In accordance with the above discussion, in a first aspect the present invention concerns a method for depolymerizing gum tragacanth, comprising a) providing a water-soluble fraction of gum tragacanth, b) treating said fraction with a first polysaccharide, e.g. pectin degrading enzyme, c) separating said treated polysaccharide fraction into sized poly- and oligosaccharide fractions, wherein at least one fraction comprises poly- and/or oligosaccharides being enriched in arabinose and galactose and at least one fraction comprises polysaccharides being enriched in fucose, and d) treating said fraction comprising polysaccharides being enriched in fucose with a second polysaccharide degrading enzyme. In a second aspect, the present invention concerns a method for obtaining a gum

tragacanth oligo- and/or polysaccharide fraction enriched in fucose, comprising the steps of a) solubilizing gum tragacanth in an aqueous solution, b) treating said solubilized gum tragacanth with a pectin degrading enzyme, c) separating said enzyme-treated tragacanth solution into different aqueous fractions (according to molecular weight), each fraction containing oligo- and/or polysaccharides containing different amounts of bound fucose and xylose, d) treating (one or more of) the aqueous fraction(s) containing the polysaccharides with the highest amount(s) of bound fucose with a xylogalacturonan hydrolase enzyme, and e) separating said enzyme treated polysaccharide solution(s) into different aqueous fractions according to molecular weight, each fraction containing oligo- and/or polysaccharides with different amounts of bound fucose.

In one embodiment of the present invention the pectin degrading enzyme is a pectinase (EC# 3.2.1.15), such as a A. niger pectinase, e.g. Pectinex®.

In another embodiment of the present invention the separation in step c) is according to molecular weight, and the aqueous fraction treated in step d) is the fraction with the highest molecular weight. Alternatively, more than one fraction may be subjected to hydrolysis with a xylogalacturonan hydrolase enzyme, either separately or pooled in one sample. This may be needed or desired if in the separation step cut-off values for the molecular weight or chosen such that more fractions contain desirable fucose-containing polysaccharides. In a preferred embodiment, the fraction(s) enriched in fucose after the enzyme treatment in step b) and separation in step c) has a molecular weight larger than about 10 kDa. In another embodiment, the polysaccharides in said fraction enriched in fucose have an average molecular size of approximately 110 kDa and comprise intact fuco- xylo-galacturonan stretches. In a further embodiment of the present invention the enzyme used in step d) is a

xylogalacturonan hydrolase enzyme, e.g. a xylogalacturonanase, such as a

xylogalacturonanase from A. tubingenis or from A. fumigatus, preferably from A. fumigatus.

In another embodiment of the present invention the gum tragacanth is from an Astragalus sp. , preferably an Astragalus specie rich in fucose, such as Astragalus gossypinus.

Saponification of tragacanth gum or polysaccharide fractions thereof

Methyl and acetyl groups of the tragacanth gum solution from A. gossypinus (1% w/v) or polysaccharide fractions thereof can be removed by adding a base, such as NaOH, followed by dialysis against water using a low molecular cut off membrane, such as cross flow membrane filtration. After dialysis, the solution may be freeze dried before the enzymatic treatment

In a third aspect, the present invention concerns an oligo- and/or polysaccharide

composition comprising intact fuco-xylo-galacturonan stretches obtainable in accordance with the first and/or second aspect of the present invention as discussed above or any oligo- and/or polysaccharide fragment thereof. Oligo- and/or polysaccharide fragments of the isolated fractions may be obtained by purification techniques known in the art.

Biological properties and therapeutic use

Many woman do not breast feed their infants due to problems or cultural differencies. Infant formulas contain sublements with sugar fibers as galactooligosaccharides (GOS) or Fructooligosaccharides (FOS), which has only partly replaced the superior effects of the Human Milk Oligosaccharides.

Solving the problem of food for infants - the formula containing the very potent Fucosylated oligosaccharides, which could be a potent component to combat human pathogens

(Campylobactor)

Fucosylated oligosaccharides could also be used together with antibiotics to combat multiresistant bacteria in humans, e.g. urinary infectionsMany woman do not breast feed their infants due to problems or cultural differencies. Infant formulas contain sublements with sugar fibers as galactooligosaccharides (GOS) or Fructooligosaccharides (FOS), which has only partly replaced the superior effects of the Human Milk Oligosaccharides.

(Campylobactor)

Fucosylated oligosaccharides could also be used together with antibiotics to combat multiresistant bacteria in humans, e.g. urinary infections

In a further aspect, the present invention relates to an oligo- and/or polysaccharide composition or oligo- and/or polysaccharide fraction thereof as discussed above for use as a prebiotic and/or an antimicrobial agent in a functional food, such as a milk formula, travelers food additives and also natural antimicrobials for control of pathogen such as Clostridia.

In yet another aspect, the present invention related to an oligo- and/or polysaccharide composition or oligo- and/or polysaccharide fraction thereof according to claim 10 for use as a therapeutic agent, such as a prebiotic, antimicrobial agent or anti-cancer agent.

EXAMPLES Example 1

Preparation of HAG3

Dextran standards with molecular weight of 10, 40, and 110 kDa were from Pharmacia (Uppsala, Sweden). Pullulan standard with molecular weight of 1.3 kDa and 400 kDa, and L- fucose, L-rhamnose, D-arabinose, D-galactose, D-glucose, D-xylose, and D-galacturonic acid were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Trifluoroacetic acid (TFA) (99%) was from Merck (Darmstadt, Germany). Sodium hydroxide (NaOH) standard solution HPLC grade was from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Dried raw gum tragacanth from Astragalus gossypinus was ground using an OBH Nordica coffee mill (OBH Nordica A/S, Copenhagen, Denmark) to pass through a 500 μιη sieve. The grounded tragacanth gum (10 g/L) was dissolved in deionized water (4 L, milliQ water) during stirring for 2 h at room temperature (22 °C). After heating the tragacanth gum solution to 50 °C, using a Sartorius Biostat heating cap set-up, 10 ml_ Pectinex® BE Colour enzyme (equivalent to addition of 9000 MOE units/L) was added, and after 2 h of reaction, when the viscosity of the solution had dropped from 1.2 Pa-s to 0.57 Pa-s, continuous filtration of the enzymatically treated gum tragacanth solution was initiated by using a cross-flow Hydrosart 10 kDa membrane (Millipore, Sartorius). This filtration was continued overnight (18 h). The initial viscosity drop to about 0.6 Pa-s was needed for allowing initiation of membrane fractionation to obtain differently sized molecules. After the first overnight filtration, the retentate was heated to 100 °C for 10 min to stop the enzymatic reaction. This retentate (~ 1 L) was defined as "HAG3" > 10 kDa. The permeate (~3 L), i.e. the enzymatically degraded product, was then separated further using another cross-flow Hydrosart membrane set-up with a 2 kDa membrane; this second filtration was stopped after collection of ~ 1.5 L of permeate, and this permeate was defined as "HAG1", and the retentate remaining was defined as "HAG2". Finally, the three collected fractions: HAG1 < 2 kDa; 2 kDa < HAG2 < 10 kDa; and HAG3 > 10 kDa, were freeze dried and stored frozen at -20 °C until further analysis.

In an alternative procedure, methyl and acetyl groups of the tragacanth gum solution from A. gossypinus (1% w/v) were removed by slowly adding 5 M NaOH in water (cold) until pH 13 and stirring for 24 hr at 4 °C. After that, the saponified solution was dialysed against deionized water at 4 °C using a 5kDa molecular weight cut off polyethersulfone membrane (Sartorius Stedim Biotech) and cross flow membrane filtration. After the dialysis was completed, the solution was freeze dried, and re-dissolved prior to use.

HPSEC analyses confirmed the depolymerization of the gum into different molecular weight fractions, and revealed that the average size of the high molecular weight fraction population, HAG3, was approximately 110 kDa (Fig. 3). By a rough estimate, the HAG3 fraction thus contained branched polysaccharides having degrees of polymerization (DP) ~60650 monosaccharides; the HAG2 polysaccharides were DP ~ 12-60 ; whereas the HAG1 fraction contained oligomers of maximum 10-12 DP. The majority of polysaccharides in the HAG3 fraction has a DP of above 110 (~ 110-650) determined by gelpermeation/size exclusion. The monosaccharide composition of the A. gossypinus gum tragacanth starting material and of the membrane separated products from the enzymatic reaction were determined after TFA hydrolysis (2 M, 2 h, 121 °C) (20) by use an ICS3000 ion chromatography system equipped with a CarboPacTM PA20 (3 mm x 150 mm) analytical column (Dionex Corp., Sunnyvale, CA), using an elution programme described previously (12).

The lyophilized fractions from the membrane filtration and the gum tragacanth starting material were dissolved in 0.1 M sodium acetate buffer (NaOAc) (pH = 6), filtered through a 0.20 μιη syringe-tip nylon membrane filter (VWR International, USA), separated for high performance size exclusion chromatography on a Shodex OHpak SB-806 HQ (8.0 mm x 300 mm) column (Showa Denko KK, Kawasaki, Japan), and eluted with 0.1 M NaOAc (pH = 6) at a flow rate of 0.5 mL/min at 30 °C on a system consisting of a P680 HPLC pump, an ASI- 100 automated sample injector using a refractive index detector Shodex RI-101 (Showa Denko KK, Kawasaki, Japan). The injection volume was 25μΙ. The column was calibrated with Pharmacia Dextran (T10, T40, and T110 kDa) and pullulan (1.3kDa and 400 kDa) as standards (21).

The monosaccharide composition of the three fractions turned out to vary significantly, and to differ from the starting material (Table 2). The data confirmed the dominance of galacturonic acid, xylose, and fucose in the starting material (Table 2). The high molecular weight fraction (HAG3) in essence got enriched in fucose during the enzymatic

depolymerization, with the final product containing almost 300 mg fucose/g dry matter

(Table 2). This fraction, HAG3, was also rich in galacturonic acid and xylose, ~380 and 320 mg/g, respectively (Table 2).

The smaller fractions, HAG1 and HAG2, became very rich in arabinose and galactose;

hence, the arabinose content was 293 mg/g in HAG1, 130 mg/g in HAG2, and ~30 mg/g in HAG3, but HAG1 and HAG2 had significantly lower fucose and xylose contents than HAG3. The level of galacturonic acid was high in all three enzymatically treated fractions, ranging from 196-380 mg/g, although lowest in the HAG1 (196 mg/g). Sugar Composition A. gossypinus* HAG1 HAG2 HAG3 (mg/g dry matter)

Fucose 236±5^b 25.5±2.7^d 102±6^C 296±3^a

Rhamnose 16.0±4.2^C 31.5±4.3^b 65.3±1.4^a 2.8±0.6^d

Arabinose 48.1±2.4^C 293±2^a 130±l^b 32.2±0.6^d

Galactose 38.9±1.7^C 123±10^a 83.7±0.9^b 8.9±0.0^d

Glucose 3.5±0.2^C 49.7±3.3^a 23.3±0.1^b 0.7±0.1^c

Xylose 273±3^b 56.0±3.5^d 207±14^c 322±7^a

Galacturonic Acid 335±16^ab 196±22^c 310±12^b 378±12^a

Table 2. Tragacanth gum, A. gossypinus, monosaccharide composition of different molecular size fractions: HAG1 < 2 kDa; 2 kDa < HAG2 < 10 kDa; and HAG3 > 10 kDa, obtained after enzymatic modification and membrane separation. Different superscript letters a, b, c, d indicate significantly different levels of the monosaccharides row-wise in the raw material and the fractions (p < 0.05). *Starting material

Example 2

Preparation of HAG3 fractions

The highly fucosylated gum tragacanth fraction (HAG3) was enzymatic hydrolysed to obtain oligo- and polysaccharide with different molecular sizes. The Pichia pastoris clone transformed with the xylogalacturonase gene Afu8g06890, was obtained from the Fungal Genetic Stock Center as described by Bauer et al (Bauer, Vasu, Persson, Mort, & Somerville, 2006). XGH was producedby high cell density fermentation : Cryocultures were prepared from the most productive P. pastoris transformants. Inoculation of P. pastoris cells was prepared with MGY Minimal Glycerol Medium in shaking flaks as detailed by Higgins and Cregg (14). The genetically engineered strain was grown in a 5 I

Sartorius Biostat Aplus fermentor (Sartorius, Company) in a basal salt media. The fermentor was equipped with monitors and controls for pH, dissolved oxygen, agitator speed, temperature, air flow and oxygen level. The fermentation process took approximately 95 h and consisted of three phases: glycerol batch, glycerol fed-batch and methanol fed-batch. The pH was adjusted and maintained automatically at 5.0 using ammonia. The cultivation temperature for the glycerol batch and glycerol fed-batch phases was typically controlled at 30 °C, whereas the temperature in the methanol fed-batch phase was kept at 28 °C. After approximately 21 h of fermentation the glycerol carbon source was exhausted, and the glycerol fed-batch phase was initiated automatically by feeding approx. 300 ml of glycerol (50%, W/V). The glycerol fed-batch phase continued for 5 h. After exhaustion of the glycerol carbon source, the methanol fed-batch phase was initiated. The methanol concentration was gradually increased over 16 h to 0.5% in order to adapt the P. pastoris cells to the toxic organic inducer. In total 2 I of methanol were added over the next 70 h. The growth of the P. pastoris cells was followed by measuring the optical density at 600 nm (OD600) in samples collected at specified time intervals throughout the fermentation process. The same samples were used to determine the enzyme and protein levels after methanol induction. After the end of the fermentation process, the P. pastoris cells were collected by centrifugation at 5300 xg at 5 °C. The cell paste was discarded and the supernatant was recovered. The supernatant was subjected to sterile filtration using a crossbow reactor equipped with a 0.4 ^J m filter to remove any residual P. pastoris cells. The permeate was concentrated by ultrafiltration using the cross-flow filter reactor equipped with a 10 kDa cutoff membrane (Millipore, Sartorius). Further purification was carried out on a Cu2+ affinity column (CIM® IDA-8f ml Tube Monolithic Column, Bioseparations). The affinity column chromatography was carried according to the procedure outlined by

Bioseparations. The column was equilibrated with binding buffer: 20 mM epps buffer (4-(2- hydroxyethyl)-l-piperazinepropanesulfonic acid) adjusted to pH 7.4, containing 2 mM of CaCI2 and 0.5 M of NaCI. After loading of the enzyme sample and washing with the binding buffer, the bound enzyme was eluted with a linear gradient from 0 mM to 500 mM of imidazole. The protein peak containing the RGI Lyase was collected and the fractions were desalted using PD-10 columns (GE Healthcare) to remove the imidazole.

The reaction was done in optimized condition for Xylogalactronase enzyme in the same batch reactor as in the first enzymatic step (example 1).

Methyl and acetyl groups of the tragacanth gum fragment solution from A. gossypinus (1% w/v) were removed by slowly adding 5 M NaOH in water (cold) until pH 13 and stirring for 24 hr at 4 °C. After that, the saponified solution was dialysed against deionized water at 4 °C using a 5kDa molecular weight cut off polyethersulfone membrane (Sartorius Stedim Biotech) and cross flow membrane filtration. After the dialysis was completed, the solution was freeze dried.

Saponified HAG3 was prepared in dionized water and adjust pH to optimize condition via HCI as substrate with 10 g/l concenteration in this reaction. Optimum conditions for the enzymatic activity was determined and used for hydrolysis of saponified HAG3. The results showed that optimum condition for enzyme activity was: Temperature 42°C, pH 3.5. The reaction was run in a batch reactor from the previous hydrolysis step.

Different molecular size fractions were obtained by using the same membrane separation 2 and 10 kDa as in the separation of HAG1, HAG2 and HAG3.

Different molecular size of oligo- and polysaccharide was produced in this process based on HPSEC as shown in Figure 5.

Claims

1. A method for depolymerizing gum tragacanth, comprising a) providing a water-soluble fraction of gum tragacanth, b) treating said fraction with a first polysaccharide degrading enzyme, c) separating said treated polysaccharide fraction into sized oligo- and/or

polysaccharide fractions, wherein at least one fraction comprises oligo- and/or polysaccharides being enriched in arabinose and galactose and at least one fraction comprises polysaccharides being enriched in fucose, and d) treating said fraction comprising polysaccharides being enriched in fucose with a second polysaccharide degrading enzyme.

2. A method for obtaining a gum tragacanth oligo- and/or polysaccharide fraction enriched in fucose, comprising the steps of a) solubilizing gum tragacanth in an aqueous solution, b) treating said solubilized gum tragacanth with a pectin degrading enzyme, c) separating said enzyme-treated tragacanth solution into different aqueous fractions (according to molecular weight), each fraction containing oligo- and/or polysaccharides containing different amounts of bound fucose, d) treating (one or more of) the aqueous fraction(s) containing the polysaccharides with the highest amount(s) of bound fucose with a xylogalacturonan hydrolase enzyme, and e) separating said enzyme treated polysaccharide solution(s) into different aqueous fractions according to molecular weight, each fraction containing oligo- and/or polysaccharides with different amounts of bound fucose.

3. A method according to claim 1 or claim 2, wherein the pectin degrading enzyme is a pectinase (EC# 3.2.1.15), such as a A. niger pectinase, e.g. Pectinex®.

4. A method according to any one of claims 1 to 3, wherein the separation in step c) is according to molecular weight, and the aqueous fraction(s) treated in step d) is (are) the fraction(s) with the highest molecular weight(s).

5. A method according to claim 4, wherein the fraction(s) enriched in fucose after the enzyme treatment in step b) and separation is step c) has a molecular weight larger than about 10 kDa.

6. A method according to claim 5, wherein the polysaccharides in said fraction enriched in fucose has an average molecular size of approximately 110 kDa.

7. A method according to any one of claims 1 to 6 wherein the enzyme used in step d) is a xylogalacturonan hydrolase enzyme, e.g. a xylogalacturonanase, such as a

xylogalacturonanase from A. tubingenis or from A. fumigatus, preferably from A.

fumigatus.

8. A method according to any one of claims 1 to 7, wherein the gum tragacanth is from an Astragalus sp., preferably an Astragalus specie rich in fucose, such as Astragalus gossypinus.

9 A method according to any one of claim 1 to 8, wherein the oligo- and/or

polysaccharides enriched in fucose comprise intact fuco-xylo-galacturonan stretches.

10. A method according to any one of claims 1-9, wherein step b and/or step d comprise addition of further hydrolytic enzymes.

11. A method for depolymerizing gum tragacanth to obtain one or more oligo- and/or

polysaccharide fraction enriched in fucose, as defined in any one of claims 1-10, wherein step b and step d are combined and step c is deleted.

12. An oligo- and/or polysaccharide composition obtainable in accordance with a method according to any one of claims 2 to 11 or any oligo- and/or polysaccharide fraction thereof.

13. An oligo- and/or polysaccharide composition or oligo- and/or polysaccharide fraction thereof according to claim 12 for use as a prebiotic and/or an antimicrobial agent in a functional food, such as a milk formula, travelers food additives.

14. An oligo- and/or polysaccharide composition or oligo- and/or polysaccharide fraction thereof according to claim 12 for use as a therapeutic agent, such as a prebiotic, antimicrobial agent or anti-cancer agent.