GB2464769A - Nutriment containing an arabinoxylo-oligosaccharide and a water soluble arabinoxylan - Google Patents

Abstract

A feed or food product enriched with two distinct types of water extractable arabinoxylan molecules comprising (i) an arabinoxylo-oligosaccharide (AXOS) and (ii) a water soluble arabinoxylan (WS-AX), wherein such nutritional prebiotic supplements are shown to have a positive effect on bifidogenic bacteria and butyric acid production with an inhibitory effect on intestinal protein fermentation, for improving gastro-intestinal health. Preferably the food product comprises on a dry weight basis between 0.75 to 15% (w/w) of water soluble arabinoxylan (AX-WS) and between 1.0 and 15% arabinoxylo-oligosaccharide. The food product may further comprise a water unextractable arabinoxylan. The food products are typically cereal based foods.

Description

NUTRIMENT CONTAINING ARABINOXYLAN AND OLIGOSACCHARIDES

FIELD OF THE INVENTION

The present invention relates to feed and food products enriched with a given amount of two distinct types of water extractable arabinoxylan molecules. Said food products may further comprise a suitable amount of a water unextractable arabinoxylan.

BACKGROUND OF THE INVENTION

The invention relates to the positive effect on gastro-intestinal health, and more particularly on the gut microbiota, of food, food ingredients or nutritional supplements with particular compositions of arabinoxylans. Arabinoxylan (AX), also referred to as pentosan, is a major constituent in the cell wall of many plant species. For instance in cereal grains, AX occurs at 5-10% of dry weight of the grains. In general, AX from cereals consists of a backbone of beta-(1-4)-Iinked D-xylopyranosyl residues (xylose), some of which are mono-or disubstituted with alpha-L-arabinofu ranosyl residues (arabinose) (Izydorczyk and Biliaderis,1995). The ratio of arabinose to xylose (A/X ratio or average degree of arabinose substitution) in cereal AX ranges from 0.10 to over 1.0, depending on tissue and plant species. In addition, more minor substituents can be attached to the xylose residues such as acetyl, alfa-glucu ronyl, alfa-4-O-methylglucu ronyl, galactu ronyl, xylosyl, rhamnosyl, galactosyl, or glucosyl side chains, or short oligosaccharide side chains (Izydorczyk and Biliaderis, 1995; Andersson and Aman, 2001). Hydroxycinnamic acids, mainly ferulic acid, and to a lesser extent dehydrodiferulic acid, p-coumaric acid, or sinapic acid, are present as substituents as well, and they are generally linked to the C-(O)-5 position of terminal arabinose units (Izydorczyk and Biliaderis, 1995; Andersson and Aman, 2001). AX in cereals occurs in two forms, a water extractable form, also referred to as WE-AX, and a form that is water-unextractable (WU-AX) most likely due to covalent or non-covalent interactions with neighbouring AX molecules and other cell wall components such as proteins, cellulose or lignin (Andersson and Aman, 2001; Courtin and Delcour, 2002). In wheat grains, the AX present in aleurone and seed coat tissues are mainly water-unextractable AX (WU-AX) and have a low AIX ratio (about 0.1 to 0.4), while AX from the pericarp tissues are WU-AX with a high A/X ratio (about 1.0 to 1.3) (Andersson and Aman, 2001; Barron et al. 2007). The AX in the endospermic tissues of wheat are either WU-AX or -WE-AX with an intermediate AIX ratio (about 0,5 to 0,7) (lzydorczyk and Biliaderis, 1995; Andersson and Aman, 2001).

Part of the WU-AX in cereal grains can be solubilised by low dose endoxylanase treatment.

The enzyme-solubilized AX (ES-AX) has similar physicochemical properties as WE-AX (Courtin and Delcour, 2002). We will here refer to the group of WE-AX and ES-AX as water-soluble AX (WS-AX).

Prebiotics are compounds, usually oligosaccharides, that can not be digested by enzymes of the upper gastro-intestinal tract but are fermented selectively by some types of intestinal bacteria in the large intestine (Gibson and Roberfroid, 1995; Roberfroid, 1988; Van Loo, 2004). Ingestion of prebiotics causes a shift in the composition of the intestinal bacterial population, typically characterised by a relative increase in Lactobacillus and Bifidobacterium species. This shift in the intestinal microbiota is associated with improved overall health, reduced gut infections, better absorption of minerals, and suppression of colon cancer initiation (Van Loo, 2004; Macfarlane et al. 2006).

Fermentation of prebiotics by colonic bacteria gives rise to production of short chain fatty acids (SOFA) such as acetate, propionate, butyrate and lactate, which act as electron sinks of respiration in the anaerobic environment of the gut. The presence of SOFA in the intestines contributes to a lower pH, a better bio-availability of calcium and magnesium, and inhibition of potentially harmful bacteria (Teitelbaum and Walker, 2002; Wong et al. 2006).

Among the SOFA, butyrate appears to be of greatest interest as butyrate is a preferred energy source for colonocytes (Roediger, 1982), stimulates colon epithelial cells, thereby increasing the absorptive capacity of the epithelium (Topping and Olifton, 2001), and inhibits the growth of colonic carcinoma cells, both in vitro and in vivo (Scheppach et al 1995). The cancer-suppressing properties of dietary fibres appear to correlate with their ability to generate butyrate upon colonic fermentation (Perrin et al. 2001).

The selective stimulation by prebiotics of certain colonic bacteria, such as Lactobacilli and Bifidobacteria, which typically use saccharolytic pathways to fuel their energy needs, is in some cases paralleled by suppression of protein fermentation in the colon (van Nuenen et al. 2003; De Preter et al. 2004; Geboes et al. 2005). Reduced protein fermentation in the colon is a desired outcome, as the amino acid degradation pathways in bacteria result in the production of potentially toxic catabolites such as ammonia, amines, phenols, indoles, and thiols, some of which have been implicated in bowel cancer (Bone et al 1976; Johnson, 1977; Visek 1978) and in exacerbation of diseases such as ulcerative colitis (Ramakrishna et a11991).

Preparations of xylo-oligosaccharides (XOS, oligosaccharides consisting of 3-1,4-linked D-xylopyranosyl units) with predominance of oligosaccharides with a degree of polymerisation (OF) of 2-3 (xylobiose and xylotriose), have been shown to cause a significant increase in the level of Bifidobacteria and SOFA in the faeces and caecum of rats (EP 0265970B1; Campbell et al., 1997; Hsu et al., 2004), and the colon of humans (Okazaki et al., 1990).

Such xylobiose-rich XOS preparations also suppress early symptoms of chemical-induced colon carcinogenesis in rats (Hsu et al., 2004) and enhance the absorption of calcium in the colon (Toyoda et al., 1993). A preparation consisting predominantly of arabinoxylo-oligosaccharides (AXOS) with a DP of 3-5 (arabinosylxylobiose, arabinosylxylotriose, arabinosylxylotetraose, and diarabinosylxylotetraose) has also been shown to increase the levels of Bifidobacteria in the intestines of rats and mice (Yamada et al., 1993). Experiments described in W02006/002495 have provided evidence that AXOS with an intermediate average DP (avDP) ranging from 5 to 50 have better prebiotic properties than AXOS with higher avDP, and are less sweet than AXOS preparations with a lower avDP. Addition of such AXOS preparations to the diet causes a significant increase in the number of Bifidobacteria present in the caecum of chickens, caecum of rats, and faeces of humans (W02006/002495). Next to the degree of polymerisation of AXOS fragments, also the ratio of arabinose to xylose may influence the prebiotic potential.

SUMMARY OF THE INVENTION

The present invention provides feed and food products enriched with a given amount of two distinct types of water extractable arabinoxylan molecules. Said food products may further comprise a suitable amount of a water unextractable arabinoxylan.

DETAILED DESCRIPTION

List of figures Figure 1: Effect of different oligosaccharides on the concentrations of acetate (A), propionate (B), butyrate (C) and branched short-chain fatty acids (the sum of isovalerate and isobutyrate concentrations) (D) in the cecum of rats after 14 days of feeding. Data are expressed per g dry weight intestinal sample. The box represents the 0.25 and 0.75 quartiles; the median is the square in the box; the whiskers are at the minimum and maximum values. Medians (n = 10) without a common letter differ, p < 0.05.

Figure 2: Effect of different oligosaccharides on the concentrations of acetate (A), propionate (B), butyrate (C) and branched short-chain fatty acids (the sum of isovalerate and isobutyrate concentrations) (0) in the colon of rats after 14 days of feeding. Data are expressed per g dry weight intestinal sample. The box represents the 0.25 and 0.75 quartiles; the median is the square in the box; the whiskers are at the minimum and maximum values. Medians (n = 10) without a common letter differ, p < 0.05.

Figure 3: Effect of different oligosaccharides on the concentration of ammonium ions in the cecum of rats after 14 days of feeding. Data are expressed per g dry weight intestinal sample. The box represents the 0.25 and 0.75 quartiles; the median is the square in the box; the whiskers are at the minimum and maximum values. Medians (n = 10) without a common letter differ, p < 0.05.

Figure 4: Effect of different oligosaccharides on the concentration of bifidobacteria in the cecum of rats after 14 days of feeding. Data are expressed per g dry weight intestinal sample. The box represents the 0.25 and 0.75 quartiles; the median is the square in the box; the whiskers are at the minimum and maximum values. Medians (n = 10) without a common letter differ, p < 0.05.

Figure 5: Schematic representation of the influence of the avDP on the prebiotic and fermentation properties of AXOS. Acetate and butyrate are based on measurements in the colon, branched SOFA, ammonium ions and bifidobacteria are based on measurements in the cecum.

Figure 6: Effect of different types of arabinoxylans and their combinations on the concentration of acetate (A), propionate (B) and butyrate (0) in the colon of rats after 14 days of feeding. Concentrations are expressed in mmol per kg on fresh weight basis of colon content. Error bars indicate the standard deviation. Different letters above the bars indicate significant difference at p < 0.05.

Figure 7: Effect of different types of arabinoxylans and their combinations on the summed concentrations of isovalerate and isobutyrate in the colon of rats after 14 days of feeding.

Concentrations are expressed in mmol per kg on fresh weight basis of colon content. Error bars indicate the standard deviation. Different letters above the bars indicate significant difference at p < 0.05.

Description

As used herein arabinoxylo-oligosaccharides or "AXOS" refers to oligosaccharides derived from arabinoxylans comprising a main chain of 3-1,4-linked D-xylopyranosyl units to which 0-2 and/or 0-3 ct-L-arabino-furanosyl units are preferably linked. For the purpose of the present invention it is preferred that the average degree of substitution of the arabinoxylo-oligosaccharides varies between 0.15 and 0.35. Preferably, the average degree of polymerisation of the arabinoxylo-oligosaccharides varies between 3 and 50, more preferably between 3 and 20, for instance between 3 and 10 or between 3 and 8. Typically the arabinoxylo-oligosaccharides or AXOS can be solubilised in a sufficient amount of a 65/35 (v/v) ethanol-water mixture at 70°C. Arabinoxylo-oligosaccharides suitable for use in the method according to the present invention can be obtained by partial hydrolysis of arabinoxylans extracted from cereals or cereal derived material. More preferably, the arabinoxylo-oligosaccharides are obtained by hydrolysis of arabinoxylans extracted from bran, for instance wheat or rye bran.

As used herein "water soluble arabinoxylans" or "WS-AX" refers to arabinoxylan molecules, which can be solubilised in a sufficient amount of water at 70°C, but not in a 65/35 (v/v) ethanol-water mixture at the same temperature. These water soluble arabinoxylans preferably have an average degree of substitution between 0.15 and 0.6, more preferably between 0.15 and 0.35. The degree of polymerisation of these water soluble arabinoxylans typically exceeds 50 and can go upto 15000 corresponding to a molecular weight of about 2 million. Given the very high viscosity of the high molecular weight WS-AX it is preferred that the WS-AX for use in the present invention have an average degree of polymerization between 50 and 1000, more preferably between 50 and 500, for instance between 100 and 400. WS-AX are naturally present in many cereals and cereal flours and in particular in brans. Particularly high amounts of WS-AX are found in rye and in Yumai-34 as well as in meal, flour and bran derived thereof. Moreover, the WS-AX content of cereal flour, meal or bran can be increased by mixing an appropriate amount of an enzyme preparation comprising endoxylanase activity in said flour, meal or bran and subsequently incubating said moistened mix during an appropriate period of time. During the incubation period a fraction of the water-unextractable arabinoxylans comprised in said flour, meal or bran is solubilised. Preferably, said enzyme preparation further comprises at least one endoxylanase which is highly selective for WU-AX.

As used herein "water-unextractable arabinoxylans" or "WU-AX" refers to arabinoxylan molecules, which can not be solubilised in water at 70°C. These water-unextractable arabinoxylans may have preferably have an average degree of substitution between 0.1 and 1.3, more preferably between 0. 35 and 1. The degree of polymerisation of these water-unextractable arabinoxylans typically exceeds 200. WU-AX are present in relatively high amounts in most cereals and the flour, meal and brans derived thereof. Particularly, bran is a good source of WU-AX.

The present invention is based on the findings of a comparative study of the effects of different types of arabinoxylan molecules and combinations thereof on parameters related to gastrointestinal health. In an animal model the prebiotic and intestinal health related effects of the administration of xylan or arabinoxylan polysaccharides vary according to the physico-chemical properties and molecular weight of these molecules. Administration of AXOS and xylo-oligosaccharides through the diet increased acetate and butyrate concentrations in the colon, reduced intestinal protein fermentation and stimulated the presence of bifidogenic bacteria. Supplementing the diet with WS-AX had no significant effect on butyrate effect and a variable effect on the presence of bifidogenic bacteria, but clearly reduced the intestinal protein fermentation. Administration of WU-AX through the diet had no clear effect on bifidogenic bacteria or intestinal protein fermentation but had a positive effect on the butyric acid production in the colon. Interestingly, the combined administration of AXOS and WS-AX through the feed had a surprisingly potent inhibitory effect on the intestinal protein fermentation combined with a positive effect on the presence of the bifidogenic bacteria and the butyric acid production. So in a first objet the present invention provides feed and food products enriched with both AXOS and WS-AX. Said food products comprise on a dry weight basis between 0.75 and 15% (wlw) of WS-AX and between 1.0 and 15% AXOS. Preferably, said food products comprise more than 1 %, more preferably more than 1.25 %, such as for instance more than 1.5, 2 or 3% of WS-AX on dry weight basis. Preferably, said food products comprise more than 1.25%, more preferably more than 1.5%, such as for instance more than 2 or 3% of AXOS on dry weight basis.

Optionally, said food products may further comprise between 1 and 15% (wlw) of WU-AX on dry weight basis. Preferably, such food products comprise more than 1.25%, more preferably more than 1.5%, for instance more than 2 or 3% of WU-AX on dry weight basis.

It is preferred that the food products according to the present invention comprise between 0.25 and 10 g of AXOS per serving and between 0.3 and 10 g of WS-AX per serving.

Preferably such food products comprise more than 0.5, more preferably more than 1 g, for instance more than 2, 3 or 5 g of AXOS per serving and preferably not more than 10 g, for instance less than 8 g AXOS per serving. Preferably such food products comprise more than 0.5, more preferably more than 1 g, for instance more than 2, 3 or 5 g of WS-AX per serving and preferably not more than 10 g, for instance less than 8 g WS-AX per serving. Optionally, such food products further comprise between 0.3 and 20 g of WU-AX per serving. Preferably such food products comprise more than 0.5, more preferably more than 1 g, for instance more than 2, 3 or 5 g of WU-AX per serving and preferably not more than 10 g, for instance less than 8 g WU-AX per serving.

Given that cereals are a good source of appropriate arabinoxylan molecules the food products of the present invention are typically cereal based food products, comprising at least 10% (wlw), more preferably at least 25% (wlw), for instance more than 40% or 60% (wlw) of cereals or arabinoxylan-containing cereal derived material. Said arabinoxylan-containing cereal derived material comprises at least 0.1% (w/w), more preferably at least 0.5% (w/w), for instance more than 1% or 2% (wlw) of arabinoxylan material on a dry weight basis. Such cereal based products may comprise fillings or toppings, however, when calculating the concentration of AXOS, WS-AX and WU-AX on a dry weight basis for such food products, the dry weight of these toppings and/or fillings has to be subtracted from the total weight of the food product. Examples of cereal based food products are bread, cookies, crackers, breakfast cereals, pop tarts and the like.

In a preferred embodiment food products of the present invention comprise an amount of AXOS as indicated above next to between 25 and 99% (w/w) of flour, meal or other milling fraction of a rye variety, wherein said rye variety comprises on a dry weight basis between 1.5 and 8% (w/w) of WS-AX.

In a second preferred embodiment the food products of the present invention comprise an amount of AXOS as indicated above next to between 25 and 99% (w/w) of flour, meal or other milling fraction of a wheat variety, wherein said rye variety comprises on a dry weight basis between 1.5 and 8% (w/w) of WS-AX. Preferably, said wheat variety is Yumai-34.

In a third preferred embodiment the food product of the present invention comprises an amount of AXOS as indicated above next to between 50 and 99% (w/w) of flour, meal or other milling fraction of a cereal, wherein said cereal material is processed in order to release at least part of the WU-AX as WS-AX. Preferably, said cereal material is treated using an exogenous endoxylanase preparation at a dose which allows to double the WS-AX naturally present in said cereal fraction. More preferably, the enzymatic treatment of the said cereal material occurs in the production of the food product.

The term "cereal", in the context of the present invention, refers to plants of the botanical family of the Poaceae, including but not limited to species such a wheat, barley, oat, rye, sorghum, maize, and rice.

The term "bran' in the context of the present invention, means a cereal grain-derived milled fraction enriched in any or all of the tissues to be selected from aleurone, pericarp, seed coat, sepals, and petals, as compared to the corresponding intact cereal grain.

The invention is further illustrated by way of the illustrative embodiments described below.

Illustrative embodiment

EXAMPLES

EXAMPLE 1: Structurally different wheat-derived arabinoxylooligosaccharides have different prebiotic and fermentation properties in rats

Introduction

Arabinoxylan (AX)1, also referred to as pentosan, is a major constituent of the cell wall of cereals. AX typically makes up 5-10% of the dry weight of cereal grains. Cereal AX consists of a backbone of 13-(1 -4)-linked D-xylopyranosyl residues (xylose), some of which are mono-or disubstituted with a-L-arabinofuranosyl residues (arabinose) (1). The ratio of arabinose to xylose (average degree of arabinose substitution, avDAS) in wheat water-extractable AX varies with the cultivar (2) and, within a given wheat cultivar, with the fractions obtained from it by conventional roller milling (3). In addition, less abundant substituents such as acetic acid or (methyl)glucuronic acid are coupled to some of the xylose residues in some AX, while hydroxycinnamic acids such as ferulic acid and p-coumaric acid can be ester linked to the arabinose residues of cereal AX. Covalent cross-linking of AX chains occurs through dehydrodiferulic acid bridges (1). AX has raised interest as some types of oligosaccharides, derived by enzymatic or chemical cleavage of AX, exert prebiotic properties (4-7).

Prebiotics are compounds that cannot be utilized by enzymes of the upper gastrointestinal tract of healthy individuals but that are fermented selectively by some types of intestinal bacteria in the large intestine, thereby exerting a beneficial health effect on their host (4).

Ingestion of prebiotics causes a shift in the composition of the intestinal bacterial population, typically characterized by a relative increase in Lactobacillus and Bifidobacterium species.

This shift in the intestinal microbiota is associated with improved overall health (5, 6), reduced gut infections (5, 7), better absorption of minerals (5-8), and suppression of colon cancer initiation (6, 7, 9). The 13-(2-1)-fructans inulin and fructooligosaccharides (FOS) are frequently used and well-studied prebiotics.

Fermentation of prebiotics by colonic bacteria gives rise to production of unbranched short-chain fatty acids (SCFA) such as acetate, propionate, butyrate and lactate. The presence of SOFA in the intestine lowers pH (10-12), increases bioavailability of calcium and magnesium (6, 11), and inhibits the growth of potentially harmful bacteria (11, 12). Among the SOFA, butyrate appears to be of greatest interest as it is a preferred energy source for colonocytes (12-14), stimulates colon epithelial cells, thereby increasing their absorptive capacity (9), and inhibits the growth of colonic carcinoma cells, both in vitro (14) and in vivo (12, 14, 15). The cancer-suppressing properties of dietary fiber appear to correlate with their ability to generate butyric acid upon colonic fermentation (12, 14, 16).

The selective stimulation by prebiotics of certain colonic bacteria proceeds, in some cases, together with the suppression of protein fermentation in the colon (17-19). Reduced protein fermentation in the colon is desired, as the amino acid degradation pathways in bacteria result in the production of potentially toxic catabolites such as ammonia, other amines, phenols, indoles, and thiols, some of which have been implicated in bowel cancer (18-21) and exacerbation of ulcerative colitis (22). In this context, reduced concentrations of the branched SOFA isobutyrate and isovalerate, formed during the catabolism of branched chain amino acids (20, 21, 23), are desired, as they are an indicator of microbial protein fermentation in the gut.

Preparations of xylooligosaccharides (XOS, i.e. oligosaccharides consisting of 13-(1-4)-linked D-xylopyranosyl units) with predominance of oligosaccharides with an average degree of polymerization (avDP) of 2 to 3 (xylobiose and xylotriose), caused a significant increase in the concentrations of bifidobacteria and SOFA in the cecum and feces of rats (10, 24) and mice (25), and also in human feces (26-28). Such xylobiose-rich XOS preparations also suppressed early symptoms of chemically induced colon carcinogenesis in rats (24). A preparation consisting predominantly of arabinoxylooligosaccharides (AXOS) with an avDP of 3 to 6 (arabinosylxylobiose, arabinosylxylotriose, arabinosylxylotetraose, and diarabinosylxylotetraose) increased the concentrations of bifidobacteria in the intestines of rats and mice (29). In addition, an AXOS preparation with an avDP of 15 increased bifidobacterial counts in the cecum of chickens to a much higher extent than FOS (30).

However, so far, the influence of the avDP and the avDAS on the prebiotic potential of AXOS has not been studied systematically. Against this background, the aim of the present study is to investigate the structure-activity relation of AXOS through evaluation of changes in microbiota and fermentation metabolites in the cecum and colon of rats following administration of structurally different AXOS.

Materials and Methods Materials and chemicals Wheat pentosan concentrate (WPC, described by Oourtin and Delcour (31)) was from Pfeifer & Langen (Dormagen, Germany) and wheat bran was from Dossche Mills & Bakeries (Deinze, Belgium). XOS was the oligosaccharide preparation Xylooligo-95P (Suntory Ltd., Tokyo, Japan). This product consists predominantly of xylobiose, xylotriose, and xylotetraose (32). The FOS preparation was Orafti�P95 (avDP = 4) (Orafti, Tienen, Belgium). The inulin preparation was Orafti�HP (avDP = 25) (Orafti).

Enzyme preparations used were heat-stable a-amylase (Termamyl 1 20L, Novozymes, Bagsvaerd, Denmark), bacterial protease (Neutrase 0.8L, Novozymes), a glycosyl hydrolase (GH) family 11 Bacillus subtilis endoxylanase (Grindamyl H640, Danisco, Copenhagen, Denmark) and a GH family 10 endoxylanase from Aspergillus aculeatus (Shearzyme 500L, Novozymes). Endoxylanase activity was determined as described by Swennen et al. (33).

One unit of enzyme activity (EU) was the amount of enzyme required to yield an absorbance of 1.0 at 590 nm under the assay conditions (40 °C, pH 4.7, 10 mm). Units of amylase and protease activity (EU) were as defined by the suppliers.

All chemicals, bovine serum albumin and reagents were of at least analytical grade and supplied by Sigma-Aldrich (Bornem, Belgium).

Preparation of AXOS compounds Preparation of AXOS with avDP 0161 and avDAS of 0.58 (AXOS-61-0.58) WPC was solubilized in deionized water (1:10 w/v) and silica was added as an aqueous suspension (20% w/v) until a silica/protein ratio of 7:1 was achieved. The pH of the mixture was adjusted to 4.8 using 0.1 mol/L HCI. After 30 mm of stirring, the suspension was Büchner filtered and the residue discarded. Ethanol (95% v/v) was added to the filtrate under continuous stirring to a final concentration of 65% (v/v), and, after additional stirring (30 mm), settling (24 h, 4 °C) and centrifugation (10,000 x g, 30 mm, 4 °C), the obtained residue was dissolved in deionized water and lyophilized. The obtained material was sieved through a 250 iim sieve.

Preparation of AXOS with avDP of 12 and avDAS of 0.69 (AXOS-12-0.69) WPC was treated with silica as described for the preparation of AXOS-61-0.58. The recovered filtrate was further incubated at 30 °C during 24 h with the A. aculeatus endoxylanase (29 EU per g WPC). After inactivation of the enzyme by boiling (30 mm), the obtained solution was cooled. Ethanol (95% v/v) was added under continuous stirring to a final concentration of 65% (v/v), and after additional stirring (30 mm), settling (24 h, 4 °C) and centrifugation (10,000 x g, 30 mm, 4 °C), the precipitated material was removed. Ethanol (95% v/v) was added to the supernatant under continuous stirring to a final concentration of 80% (v/v), and, after additional stirring (30 mm), settling (24 h, 4 °C) and centrifugation (10,000 x g, 30 mm, 4 °C), the obtained residue was dissolved in deionized water and lyophilized. The obtained material was sieved through a 250.Lm sieve.

Preparation of AXOS with avDP of 15 and avDAS of 0.27 (AXOS-15-0.2 7) The production of this AXOS preparation was based on a procedure described by Swennen et al. (33). Briefly, wheat bran in water (1:7 w/v) was treated with Termamyl 1 20L (120 EU/kg wheat bran, 90 mm, 90 °C) and Neutrase 0.8L (32 EU/kg wheat bran, 4 h, 50 °C, pH 6.0).

After boiling (20 mm) and filtering the suspension, the destarched and deproteinized wheat bran (DDWB) was washed with water, and resuspended in deionized water (1:14 wlv). The suspension was incubated under continuous stirring with the B. subtilis endoxylanase at 1.4 EU per g DDWB (10 h, 50 °C), and for another 10 h at 50 °C after addition of a second dose of the endoxylanase (1.1 EU per g DDWB). After inactivation of the enzyme by boiling (30 mm), the solution was concentrated until ca. 20% dry matter in a falling film evaporator and finally spray-dried.

Preparation of AXOS with avDP 015 and avDAS of 0.27 (AXOS-5-0.27) AXOS-5-0.27 was prepared by incubating a solution (1:10 wlv) of AXOS-1 5-0.27 at 30 °C during 60 mm with the A. aculeatus endoxylanase at 75 EU per g AXOS-15-0.27. After inactivation of the enzyme by boiling (30 mm), the solution was lyophilized and the obtained material was sieved through a 250 l.im sieve.

Preparation of AXOS with avDP 013 and avDAS of 0.26 (AXOS-3-O.26) Wheat bran was destarched and deproteinized as described for the preparation of AXOS-15- 0.27. The resulting material was incubated under continuous stirring for 10 h at 50 °C with the B. subtilis endoxylanase at 1.2 EU per g DDWB, and for another 10 h at 50 °C after addition of the A. aculeatus endoxylanase (21 EU per g DDWB). After inactivation of the enzymes by boiling (30 mm), the solution was concentrated to 20% dry matter in a falling film evaporator and finally spray-dried.

Characterization of the isolated preparations Moisture and ash contents were analyzed according to AACCI methods 44-19 and 08-01, respectively (34).

Protein contents were determined according to a Dumas combustion method, using an automated Dumas protein analysis system (EAS varioMax N/ON, Elt, Gouda, The Netherlands), an adaptation of the AOAC Official Method for protein determination (35) and using 5.7 as the nitrogen protein conversion factor.

Total and reducing end sugar contents were determined by gas chromatographic analysis as described earlier (33). The avDP and avDAS of AXOS were calculated using formulae [1] and [2], respectively. The total AXOS content was calculated using formula [3].

[1] avDP = (% arabinose -0.7 x % galactose + % xylose) / % reducing end xylose [2] avDAS (% arabinose -0.7 x % galactose) / % xylose [3] AXOS = (% arabinose -0.7 x % galactose) * 132/150 + ((132 x (avDP-1) + 150))! (150 x avDP) x % xylose The correction for the % galactose in formulae [1], [2] and [3] is only done for WPC derived material as WPC contains soluble arabinogalactan peptides (0.7 is the fixed arabinose to galactose ratio in arabinogalactan peptides) (36). Destarched and deproteinized bran no longer contains such material as a result of the purification.

The factors 132 and 150 in the formulae above reflect the molecular mass of anhydropentose sugars and pentose sugars, respectively. As the anhydroxylose and anhydroarabinose units in AXOS are hydrated upon hydrolysis, a correction for this molecular mass shift has to be incorporated in the calculations.

Rat trial design The changes in microbiota and fermentation metabolites in the cecum and colon of rats following administration of structurally different AXOS were evaluated in a completely randomized controlled trial. Ninety 6-week-old male rats (Wistar, Elevage Janvier, Le Genest-St-lsle, France) were housed in stainless steel wire-bottom cages (2 rats per cage) in an environmentally controlled room (22 °C) with a 14!10 h lightldark cycle. For 6 days, rats were given free access to water and pellets (10 mm) of a basic diet (Table 1). The basic diet, mimicking the average Western human diet composition, was prepared and analyzed by Ssniff Spezialdiäten GmbH (Soest, Germany). The diet was designed and produced following the general feed standards for laboratory animals, and met all nutrient requirements of rats (37). Furthermore, the diet was formulated to allow a margin of safety for strain and individual differences. After 6 days of adaptation, the rats were randomly assigned to one of 9 different treatment groups (10 rats!group). All groups were given free access to pellets (10 mm) of basic diet to which the dosages of AXOS, XOS, FOS or inulin, mentioned in Table 2, were added. For the oligosaccharide-containing diets, the starch in the basic diet was replaced with the appropriate amount of oligosaccharide preparation.

Rats were weighed and feed intake was measured 3 times per week. After 14 days of treatment, all rats were weighed and killed with an overdose of 5-ethyl-5-(1-methylbutyl)- 2,4,6(1 H,3H,5H)-pyrimidinetrione (NembutalTM). Thereafter, the rats were dissected to collect the cecum and colon contents.

Before experimentation, the above experimental protocol was approved by the Ethical Committee on Animal Experiments of the K.U. Leuven.

Short-chain fatty acid analysis For measurement of non-branched and branched SCFA, the following were added to vials containing individual intestinal (cecal or colonic) samples (2.0 g, fresh weight): 0.5 mL 9.2 mol!L sulfuric acid, 0.4 mL of 0.75% (v!v) 2-methylhexanoic acid (internal standard), 0.4 g NaCI and 2.0 mL diethyl ether. After shaking the vials for 2 mm, they were centrifuged (3,000 x g, 3 mm) and the diethyl ether phases containing the organic acids (1.0 1iL) analyzed as described earlier (38).

Ammonium analysis Ammonium ions in cecal samples were liberated as ammonia by addition of magnesium oxide (0.4 g per g fresh weight of sample). Released ammonia was distilled from the sample into a boric acid solution (20.0 giL) using a 1062 Kjeltec Auto Distillation apparatus (FOSS Benelux, Amersfoort, The Netherlands). Ammonia was determined by titration using a 665 Dosimat (Metrohm, Herisau, Switzerland) and 686 Titroprocessor (Metrohm). Colonic ammonium ion concentrations could not be measured due to lack of sample.

Microbiological analyses by quantitative PCR The concentrations of total bacteria, lactobacilli and bifidobacteria in the cecum were measured by quantitative PCR. Extraction of metagenomic DNA from cecal samples was performed using the QlAamp DNA Stool Mini kit (Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions and starting from 0.2 g (fresh weight) sample. DNA amplification was performed in triplicate in 25 il reaction mixtures of the qPCR Core Kit for SYBR� Green I as described by the supplier (Eurogentec, Liege, Belgium) in MicroAmp Optical 96-well reaction plates with optical caps (PE Applied Biosystems, Nieuwerkerk aid ljssel, The Netherlands) using an ABI Prism SDS7000 instrument (PE Applied Biosystems).

For the detection of the number of copies of the 16S ribosomal RNA genes from total bacteria, the following PCR program, using 0.3 iimol/L of both 338f (39) and 518r (40) as the forward and reverse primer respectively, was performed: 50 °C for 2 mm, 95 °C for 10 mm, followed by 40 cycles of 94 °C for 1 mm, 53 °C for 1 mm, and 60 °C for 2 mm. By analogy, the number of copies of 16S ribosomal RNA genes from Bifidobacterium and lactobacilli were detected using 0.3 iimol/L of the primers 243f1243r and LactoF/LactoR (41) respectively, and the following thermal protocol: 50 °C for 2 mm, 95 °C for 10 mm, followed by 40 cycles of 94 °C for 20 s, 58 °C for 30 s, and 60 °C for 1 mm. Standard curves for quantification of bifidobacteria were constructed based on real-time PCR amplification using six different dilutions of DNA extracted from a culture of Bifidobacterium breve (strain LMG11O42), and from Lactobacillus brevis (strain LMG12023) for quantification of total bacteria and lactobacilli. Real-time PCR data obtained were plotted against the standard curve and corrected for efficiency of DNA extraction using a factor consisting of the mean DNA concentration of all samples divided by the DNA concentration of the individual experimental sample. Colonic bacterial concentrations could not be measured due to lack of sample.

Statistical analysis The rat trial was performed according to a completely randomized controlled design. Since the response variables were found to be not normally distributed based on a Kolmogorov- Smirnov test, the effect of the diets on different characteristics was analyzed by a non-parametric one way ANOVA at the 95% confidence level with the Statistical Analysis System software 8.1 (SAS Institute, Cary, NC). Hereto, the original response variables were transformed into their ranks and a classical one way ANOVA was performed on those ranks.

The model for the statistical analysis was Y=/J+a1+e with,uthe overall mean, a1 the main effect of diet i, Y,3the response (rank) for the jth rat following diet i, and e,3 the error term. A Tukey multiple comparisons procedure was used to find significant differences (p < 0.05) among the different diets.

Results Properties of AXOS Table 2 lists the properties of the different preparations in terms of total AXOS content, avDP and avDAS. All preparations were at least 70% pure in terms of AXOS content. Their avDP ranged from 3 to 61, and their avDAS from 0.26 to 0.69.

Feed intakes and body weights Feed intakes during the first and second weeks of the treatment period were 21.0 � 1.2 and 21.2 � 1.4 g/(ratday), respectively (overall means � SD of all groups). Body weights of the rats at the start of the treatment were 252 � 8.7 g, and reached 311 � 13.4 g and 360 � 19.0 g after 1 and 2 weeks of treatment, respectively (data not shown). Body weights and feed intakes did not differ among the groups.

Fermentation products Cecal concentrations of acetate, propionate and butyrate did not differ significantly from the control group for any of the treatment groups, although some of the treatment groups differed from one another (Figure 1). The AXOS-61-0.58 group showed higher cecal acetate and propionate concentrations compared to the AXOS-3-0.26, inulin and FOS groups and the AXOS-1 5-0.27, AXOS-5-0.27 and FOS groups, respectively. Colon acetate concentrations were significantly higher in rats fed with diets containing either AXOS-5-0.27, AXOS-3-0.26, XOS or inulin than in rats fed the control diet (Figure 2A), whereas propionate concentrations did not differ among the groups (Figure 2B). Colonic butyrate concentrations were significantly increased in the AXOS-5-0.27, AXOS-3-0.26, and XOS groups by more than 100% compared to concentrations in the control group (Figure 2C).

Cecal branched SOFA concentrations were significantly reduced versus control upon addition of AXOS-61-0.58, AXOS-12-0.69, AXOS-15-0.27, and AXOS-5-0.27, but not upon addition of AXOS-3-0.26, XOS, inulin and FOS (Figure 1 D). The reduction was the strongest in rats fed the AXOS-61 -0.58 supplemented diet and amounted to 72% compared to control.

Significant differences also showed between the highest avDP AXOS (AXOS-61 -0.58 and AXOS-15-0.27) and the lowest avDP AXOS (AXOS-3-0.26 and XOS). In the colon, concentrations of branched SOFA were significantly lower relative to the control for rats fed AXOS-61-0.58, but not for the other treatment groups. However, branched SOFA concentrations tended to be lower than in the control group in the AXOS-5-0.27 group (p 0.0681) (Figure 20).

Ammonium ion concentrations in the cecum were significantly reduced versus control for all treatment groups fed oligosaccharide preparations, except for the AXOS-12-0.69 group (Figure 3). Decreased ammonium ion concentrations can result from either reduced protein fermentation or increased assimilation of ammonium ions by carbohydrate fermenting bacteria (23).

Bacteria measurements The concentrations of total bacteria and lactobacilli did not differ among the groups (results not shown). However, the bifidobacterial content of the cecum was significantly increased in rats fed with any of the oligosaccharide preparations versus the control treatment, except for the AXOS-61-0.58, AXOS-12-0.69 and AXOS-15-0.27 groups (Figure 4). The increase compared to the control amounted to about 1 to 1.5 log units. The cecal bifidobacteria content was also significantly higher in the AXOS-5-0.27, AXOS-3-0.26, XOS, FOS and inulin groups than in the AXOS-61 -0.58 group.

Discussion Since the structure of non-digestible oligosaccharides influences the rate and extent of intestinal fermentation, one can assume that their relative bifidogenic effect and the fermentation products they generate strongly depend on their structure. In general, easily fermentable compounds are more exhaustively fermented in the proximal colon (42), where they may increase SOFA production and bifidobacteria concentrations. Fermentation of more slowly fermentable compounds proceeds beyond the proximal colon (38, 42), where they may suppress protein fermentation.

The present study clearly demonstrates that mainly the avDP, but not, or to a limited extent, the avDAS of AXOS preparations determine the effects they bring about in the intestines of rats (Figure 5). The avDP clearly influenced the bifidogenic potency, as addition of AXOS and XOS preparations with a rather low avDP (�= 5) increased bifidobacteria concentrations, whereas addition of AXOS with a higher avDP (�= 12) did not stimulate bifidobacteria development. The difference in bifidogenic effect between larger and smaller AX(OS) compounds is in line with some earlier observations. Wheat flour AX had no effect on bifidobacteria, bacteroides and clostridia population levels in an in vitro continuous fermentation system mimicking the human gastrointestinal tract, whereas xylanase pre-treated wheat flour AX clearly increased the bifidobacteria concentration and decreased the concentrations of bacteroides and clostridia (43). No remarkable changes in the fecal microbiota occurred at the bacterial group level after addition of maize AX to healthy human volunteers (44). In contrast, several studies reported wheat bran AXOS to exert bifidogenic effects in vitro (29, 45) and in vivo (29, 30). These studies collectively suggest that AX is not or only poorly bifidogenic, while its hydrolysis products XOS and AXOS stimulate bifidobacterial growth. In contrast to the above mentioned studies and the present results, Hughes et al. (46) in in vitro batch fermentation experiments observed that high molecular mass AX fractions (with molecular masses between 354 and 66 kDa) significantly increased bifidobacteria counts. Also in these experiments, the bifidogenic effect clearly increased with decreasing molecular mass (46).

The present study also shows a strong influence of the avDP on the formed fermentation products. Colonic acetate and butyrate production was increased upon addition of AXOS and XOS preparations with a low avDP (�= 5), but not with AXOS preparations with a higher avDP (�= 12). On the other hand, AXOS preparations with an avDP �= 5 lowered cecal branched SCFA, and thus suppressed protein fermentation, while the AXOS preparation with the highest avDP (AXOS-61 -0.58) also lowered the branched SCFA concentrations in the colon.

The main SOFA after fermentation of AXOS-61-O.58 were acetate and propionate. This is consistent with literature data, as in vitro fermentation of wheat endosperm AX results in the production of mainly acetate and propionate (47), while fermentation of AXOS (avDP 2-11) leads to not only acetate and propionate, but also butyrate (48). In contrast, Hughes et al. in in vitro batch fermentation experiments observed significantly increased concentrations of acetic, propionic as well as butyric acid upon addition of high molecular mass AX fractions (46). However, in vitro fermentation experiments differ in many respects from in vivo feeding trials. One notable difference is that, in vitro, the products of fermentation, like SOFA, accumulate in the fermentation vessel, while, in vivo, these fermentation products are not only produced in the lumen of the gut but also removed by uptake in the blood through the gut mucosa. Hence, unlike in in vitro experiments, increased SCFA production in vivo through fermentation of non-digestible oligosaccharides is not always reflected in increased concentrations in the gut content, and, therefore, in vivo trials only reveal changes in fermentation patterns that surpass a certain threshold.

The influence of the avDAS on the bifidogenic potency was not clear from the present results. Two couples of AXOS preparations with a similar avDP but different avDAS could be compared. AXOS-12-O.69 and AXOS-15-O.27 did not significantly increase the cecal bifidobacteria concentration, in spite of a positive trend for the AXOS-15-0.27 group. XOS and AXOS-3-0.26 both increased the cecal bifidobacteria concentration by one log unit. The fermentation products formed were similar in nature and abundance when comparing preparations with similar avDP. The AXOS-12-0.69 preparation had no significant effect on any of the measured characteristics except that it reduced the concentrations of branched SCFA in the cecum, while AXOS-15-0.27 reduced cecal ammonium ion as well as branched SCFA concentrations. As outlined above, regretfully, colonic ammonium ion concentrations could not be measured. Based on the similarity between cecal and colonic branched SOFA concentrations (Figures 1 D and 2D), however, results would be expected to be similar to cecal ammonium ion concentrations. The fact that the observed effects of AXOS preparations with a similar avDP but different avDAS were similar, suggested that the influence of the avDAS was small in the range studied.

Although the present results suggest only a weak influence of the avDAS on the bifidogenic potential and the fermentation products, several other studies reported an influence of the avDAS on AX and AXOS fermentability. After in vitro fermentation of rye AX fractions with different avDAS (ranging from 0.52 to 0.74), unfermentable residues with an avDAS of 1.1 were obtained, regardless the substrate (49). This suggests that the unfermented AX with high avDAS have a structure that is highly resistant to hydrolysis and fermentation and so remains unutilized by colonic microbiota (49). Similar results were obtained in an in vivo study with pigs. Oomparison of different rye milling fractions showed the fecal digestibility to be highest for endosperm AX (avDAS = 0.76) and aleurone AX (avDAS = 0.42), while pericarp/testa AX (avDAS = 1.04) is virtually not degraded through colonic fermentation (42).

Taken together with the data of the present study, the literature data suggest that the avDAS only has a marked negative impact on intestinal fermentability at relatively high values of 1.0 or above.

Among the AXOS preparations tested, AXOS-5-0.27 exhibited the best combination of desirable effects on gut health characteristics, in terms of increased acetate and butyrate concentrations in the colon, reduced intestinal protein fermentation (as deduced from reduced branched SOFA and ammonium ion concentrations in the cecum), and increased concentrations of bifidobacteria in the cecum. In comparison to the most optimal AXOS-based oligosaccharide preparation (AXOS-5-O.27) the fructan-based oligosaccharides FOS and inulin resulted in similar bifidogenic effects, yet they only caused an increase in production of colonic acetate (inulin) but not of butyrate. Inulin was not effective at reducing the cecal concentrations of branched SOFA.

In conclusion, in vivo evaluation of structurally different AXOS shows that the AXOS structure has a strong influence on the prebiotic potential and the formed fermentation products. In general, smaller AXOS result in stronger increases in intestinal butyrate concentrations and a significant bifidogenic effect, while larger compounds mainly lead to decreased branched SOFA concentrations. The influence of the avDAS seemed to be limited in the range studied.

These new insights into the structure-activity relation of AXOS open up new perspectives for the production and application of AXOS preparations with optimized prebiotic and fermentation properties.

EXAMPLE 2: Effect of WU-AX, WS-AX and AXOS preparations and combinations thereod on intestinal parameters Materials and methods Preparation of AXOS.

AXOS was prepared by FUGEIA NV (Leuven, Belgium) from wheat bran by endoxylanase treatment (see Swennen et al., 2006). The composition and characterisation of the AXOS preparation is shown in Table 3.

Preparation of WS-AX.

WS-AX was prepared from commercial wheat bran by treating wheat bran suspended in demineralised water (8 liter water per kg dry matter) with a commercial amylase (BAN 480 L, Novozymes, Bagsvaerd, Denmark; 1 ml enzyme preparation per kg dry matter) at 70°C under constant stirring for 90 minutes, followed by filtering and extensive rinsing of the residue with demineralised water. The destarched bran was then suspended in demineralised water (10 liter water per kg dry matter) and treated with a commercial xylanase (Multifect OX 12 L, Danisco, Copenhagen, Denmark; 0.25 ml enzyme preparation per kg dry matter) at 50°C under constant stirring for 8 h. The liquid phase was recovered after filtration. After inactivation of the enzyme by treatment of the filtrate for 10 minutes at 90°C, the solution was concentrated in a falling film evaporator and finally dried in a spray-drier. The composition and characterisation of the WS-AX preparation is shown in Table 3.

Preparation of WU-AX.

WU-AX was prepared from commercial wheat bran by treating wheat bran suspended in demineralised water (10 liter water per kg dry matter) with a commercial amylase (Termamyl 120 L, Novozymes, Bagsvaerd, Denmark; 1 ml enzyme preparation per kg dry matter) at 90°C for 90 minutes. After cooling of the mash to 50°C, the pH of the mash was adapted to pH 6.0 by addition of HCI and treated with a commercial protease (Neutrase 0.8 L Novozymes, Bagsvaerd, Denmark; 40 ml enzyme preparation per kg dry matter) at 50°C under constant stirring for 4 h. The mash was heated to 100°C and kept at this temperature for 10 minutes. After cooling to 60°C, the mash was filtered, the residue washed extensively with demineralised water, and finally dried in a lyophilisator. The composition and characterisation of the WU-AX preparation is shown in Table 3.

Characterisation of saccharides.

The content of total saccharides, reducing end saccharides, and free monosaccharides was determined by gas-liquid chromatographic analysis as described by Courtin et al. (2000). For determination of total saccharide content, 40 mg dry samples suspended in 2.5 ml distilled water or 2.5 ml saccharide-containing water extracts of samples were hydrolyzed by mixing with 2.5 ml 4.0 M trifluoroacetic acid and incubating at 110°C for 60 minutes. After the hydrolysis, the mixture was filtered and 3.0 ml of the filtrate was further treated by adding 1.0 ml of an internal standard solution (100 mg beta-D-allose in 100 ml of a 50% saturated benzoic acid solution), 1.0 ml of ammonia solution (25% v/v) and 3 drops of 2-octanol. The monosaccharides were reduced to alditols by addition of 200 t1 of sodium borohydride solution (200 mg sodium borohydride in 1.0 ml 2 M ammonia) and the sample was incubated for 30 minutes at 40°C. The reaction was stopped by addition of 400 l.tl of glacial acetic acid.

For the acetylation reaction, 500 l.tl of the sample containing the alditols was added to 5.0 ml of acetic anhydride and 500 il of 1-methyl-imidazole. After 10 minutes, the excess of acetic anhydride was removed by addition of 900 il ethanol to the sample. Alditol acetates were then concentrated in the organic phase by addition of water (10 ml) and potassium hydroxide solution (2 times 5.0 ml of 7.5 M solution, with an intermediate rest of a few minutes).

Bromophenol blue solution (500 p1, 0.04% w/v) was added as indicator for the aqueous phase. Aliquots of liil of the organic phase containing the formed alditol acetates were separated by gas chromatography on a Supelco SP-2380 polar column (30 m X 0.32 mm l.D.; 0.2 pm film thickness) (Supelco, Bellefonte, PA, USA) in an Agilent chromatograph (Agilent 6890 series, Wilmington, DE, USA) equipped with autosampler, splitter injection port (split ratio 1:20) and flame ionisation detector. The purified monosaccharides D-glucose, D-mannose, D-galactose, D-xylose, and L-arabinose were treated in parallel with each set of samples for calibration purposes, and calibration took into account partial degradation of the monosaccharide standards during the hydrolysis step (6% for 0-glucose, 8% for 0-mannose, 6% for D-galactose, 11% for D-xylose, and 5% for L-arabinose).

For determination of the reducing end saccharide content, 40 mg dry samples suspended in water or 2.5 ml saccharide-containing water extracts of samples, were mixed with 500 il of an internal standard (100 mg beta-D-allose in 100 ml of a 50% saturated benzoic acid solution) and 50 il ammonia solution (25% vlv) and 9 drops of 2-octanol. The saccharides were reduced to alditols by addition of 200 p1 of sodium borohydride solution (200 mg sodium borohydride in 1.0 ml 2 M ammonia) and the sample was incubated for 30 minutes at 40°C.

The reaction was stopped by the addition of 400 p1 glacial acetic acid. An aliquot of 2.5 ml of the sample containing reduced saccharides was hydrolyzed by addition of 500 p1 trifluoroacetic acid (99 %) and the sample was incubated at 110°C for 60 minutes. After hydrolysis, acetylation and gas chromatography analysis was performed as described above.

The purified monosaccharides 0-glucose, D-mannose, D-galactose, D-xylose, and L-arabinose were treated in parallel with each set of samples for calibration purposes.

For determination of the free monosaccharide content, 40 mg dry samples suspended in water or 2.5 ml saccharide-containing water extracts of samples, were mixed with 500 p1 of an internal standard (100 mg beta-D-allose in 100 ml of a 50% saturated benzoic acid solution) and 50 p1 ammonia solution (25% v/v) and 9 drops of 2-octanol. The saccharides were reduced to alditols by addition of 200 p1 of sodium borohydride solution (200 mg sodium borohydride in 1.0 ml 2 M ammonia) and the sample was incubated for 30 minutes at 40°C.

The reaction was stopped by the addition of 400 p1 glacial acetic acid. An aliquot of 2.5 ml of the sample containing reduced saccharides was acetylated and analysed by gas chromatography as described above. The purified monosaccharides 0-glucose, 0-mannose, D-galactose, D-xylose, and L-arabinose were treated in parallel with each set of samples for calibration purposes.

From the above described analyses, the following values were obtained: * %totxyl, %totara, %totgal, %totman, %totglu are the concentrations of total (polymeric and free) xylose, arabinose, galactose, mannose, and glucose, respectively, as determined by the total saccharide analysis procedure.

* %redxyl, %redara, %redgal, %redman, %redglu are the concentrations of reducing end xylose, arabinose, galactose, mannose, and glucose, respectively, as determined by the reducing end saccharide analysis procedure.

* %freexyl, %freeara, %freegal, %freeman, %freeglu are the concentrations of free xylose, arabinose, galactose, mannose, and glucose, respectively, as determined by the free monosaccharide analysis procedure.

The content of non-cellulosic bound glucose, bound galactose, bound mannose, bound xylose and bound arabinose was calculated by formulae (1), (2), (3), (4), and (5), respectively.

(1) (%totglu %freegIu)*162/180 (2) (%totgal %freegaI)*162/180 (3) (%totman %freeman)*162/180 (4) (%totxyl %freexyl)*1 32/150 (5) (%totara %freeara)*1 32/150 The content of arabinoxylan or AXOS (%AX/AXOS) in a sample was calculated by formula (6).

(6) (%totxyl%redxyI)*1 32/150 + (%totara -%freeara)*1 32/150 + (%redxyl -% freexyl) The average degree of polymerisation (avDP) of the arabinoxyan or AXOS was calculated using formula (7).

(7) (%totxyl -% freexyl + %totara -%freeara) / (%redxyl -% freexyl) The arabinose to xylose ratio (AIX ratio) of the arabinoxylan or AXOS was calculated using formula (8).

(8) (%totara-%freeara) I (%totxyl-%freexyl) Determination of WU-AX, WS-AX and AXOS content The content of WU-AX, WS-AX and AXOS content in a sample, for instance a food sample, can be determined by the following set of parallel analyses.

The total amount of AX or AXOS (TOT-AX/AXOS) in a sample was determined as follows: * Weigh accurately three 40 mg aliquots of the dry sample with known dry matter content.

* Determine the total saccharide, reducing end saccharide and free monosaccharide content on each of the 40 mg samples, respectively.

* Calculate the % TOT-AX/AXOS as the % AX/AXOS using the above formula (6) on the basis of 40 mg (multiplied by % dry matter) of the original sample The total amount of water soluble AX (= WS-AX + AXOS) in a sample was determined by the following procedure: * Weigh accurately about 1 g of the dry sample with known dry matter content, transfer to a capped centrifuge tube and add 40 ml of distilled water * Incubate for 20 minutes with constant shaking in a water bath at 70°C * Centrifuge the tubes at 10,000 x g for 15 minutes * Collect 30 ml of the supernatant * Determine the total saccharide, reducing end saccharide and free monosaccharide content on the 2.5 ml aliquots of the cleared supernatant samples.

* Calculate the % WS-AX/AXOS as the % AX/AXOS using the above formula (6), assuming a sample dry weight of 1000*2.5/40 mg (multiplied by % dry matter) of the original sample.

The total amount of ethanol soluble AX or AXOS (ETS-AX/AXOS) in a sample was determined by the following procedure: * Weigh accurately about 1 g of the dry sample with known dry matter content, transfer to a capped centrifuge tube and add 40 ml of 65% (v/v) ethanol.

* Incubate for 20 minutes with constant shaking in a water bath at 70°C * Centrifuge the tubes at 10,000 x g for 15 minutes * Collect 30 ml of the supernatant * Evaporate the solvent in a rotavap instrument until about 3 ml is left, add 20 ml water at 70°C, swirl well, transfer in a flask and adjust the volume to 30 ml * Determine the total saccharide, reducing end saccharide and free monosaccharide content on the2.5 ml aliquots of the cleared supernatants.

* Calculate the % ETS-AX/AXOS as the % AX/AXOS using the above formula (6), assuming a sample dry weight of 1000*2.5/40 mg (multiplied by % dry matter) of the original sample.

Calculate the % WU-AX using formula (9) (9) % WU-AX = % TOT-AX/AXOS -% WS-AX/AXOS Calculate the % WS-AX using formula (10) (10) % WS-AX = % WS-AX/AXOS -% ETS-AX/AXOS Calculate the % AXOS using formula (11) (11) % AXOS = % ETS-AX/AXOS Determination of moisture and ash content Moisture and ash contents were analysed according to AACC methods 44-19 and 08-01, respectively (Approved Methods of the American Association of Cereal Chemist, 10th edition. 2000. The Association, St. Paul, MN, USA).

Determination of protein content Nitrogen content and deduced protein contents were determined according to the Dumas combustion method, using an automated Dumas protein analysis system (EAS varioMax N/ON, Elt, Gouda, The Netherlands) that follows an adaptation of the AOAC Official Method for protein determination (Association of Official Analytical Chemists. Official Methods of Analysis, 16th edition. 1995. Method 990.03. AOAC Washington DC, USA). The protein content was deduced by multiplying the nitrogen content with the factor 6.25.

Animal trial conditions.

6-week-old male rats (Wistar) were purchased from Elevage Janvier (Le Genest-St-IsIe, France) and randomly assigned to 6 groups of 10 rats each. The rats were housed in stainless steel wire-bottom cages (2 rats per cage) in an environmentally controlled room (22°C) with a 14-10 h light-dark cycle. Rats were given free access to water and to pellets (10 mm) of the control' diet (Table 4) during 6 days. After 6 days of adaptation on the control diet, the rats were randomly assigned to one of 6 different treatment groups (10 rats/group), and the groups were each given free access during 14 days to pellets (10 mm) of one of the 6 diets described in Table 4.

Animals were weighed and feed intake was measured 3 times per week. After 14 days of treatment, all animals were weighed and euthanized by carbon dioxide asphyxiation.

Thereafter, the animals were dissected to collect the colon content.

Short chain fatty acid analysis. To vials containing intestinal samples (2 g) the following was added: 0.5 ml 9.2 M sulfuric acid, 0.4 ml of 0.75 % (vlv) 2-methylhexanoic acid (internal standard), 0.4 g NaCl and 2 ml diethyl ether. After shaking the vials for 2 minutes, the vials were centrifuged (3 mm at 3000 x g) and the diethyl ether phase transferred to glass vials.

The diethyl ether phase containing the organic acids was analysed on a gas-liquid chromatograph equipped with a EC-1000 Econo-Cap column (Alltech, Laarne, Belgium; dimensions: 25 m x 0.53 mm, film thickness 1.2 Ilm; acid-modified polyethylene glycol as liquid phase) and a flame ionization detector. Nitrogen was used as a carrier gas at a flow rate of 20 mL per minute and the column temperature and injector temperature were set at 130 and 195°C, respectively. Concentrations of SCFAs were calculated based on standards with known concentrations of the different acids. 2-Methyl hexanoic acid was used as an internal standard.

Statistical analyses. The effect of diets on different parameters was analysed by the non-parametric Kruskal-Wallis test at the 95% confidence level using the Analyse-it software, version 2.07. In case a statistically significant effect was observed for the factor diet, differences among each of the diets were analysed with Bonferroni error protection at the 95% confidence level.

Results Rats were used as an in vivo model to study the effect of dietary inclusion of different types of arabinoxylans in mammalians. To this end, preparations of low molecular weight arabinoxylo-oligosaccharides (AXOS), high molecular weight water soluble arabinoxylan (WS-AX) and high molecular weight water-unextractable arabinoxylan (WU-AX) were made and characterised (Table 3). The different preparations were added either alone or in combinations to different diets (Table 4), and a range of gut health related parameters were assessed after a 14 days feeding period.

No significant differences in body weight or daily feed intake were be observed between the different treatments.

As increased intestinal SOFA levels are a hallmark of shifts in the intestinal microflora induced by intake of prebiotic compounds (Macfarlane et al 2006), the concentration of the main SOFA, acetate, propionate and butyrate, were measured in the colon for the different treatment groups. No significant changes in colonic concentrations of acetate were observed in any of the AXOS or AX containing diets relative to the control diet (Figure 6A). Propionate concentrations were significantly elevated in the WS-AX supplemented diet, but not in any of the other diets (Figure 6B). The most marked changes were observed for the butyrate levels (Figure 6 0). WS-AX by itself did not affect butyrate concentrations, yet the combination of WS-AX and AXOS caused a significant increase by more than 2.5 fold relative to the control level. WU-AX, with or without AXOS addition, also significantly raised butyrate concentrations. Highest butyrate levels were found in rats fed the diet supplemented with the combination of WU-AX, WS-AX and AXOS (more than 4-fold higher than control levels).

The branched SOFA isobutyrate and isovalerate are formed during the catabolism of branched chain amino acids valine, leucine, isoleucine (Mortensen et al. 1992; Macfarlane and Macfarlane, 1995), and are an indicator of protein fermentation in the gut. The colonic contents of the rats were therefore assessed for the branched SOFA isobutyrate and isovalerate. Significant reductions in colonic branched SOFA levels were observed in all groups fed diets containing WS-AX, either alone or in combination with AXOS or with AXOS and WU-AX (Figure 7). The strongest decrease was observed for the combination of WS-AX and AXOS, which led to a decrease of branched SOFA by more than 90%, whereas WS-AX alone reduced branched WS-AX levels by 75%.

The most desired effects on gut health related parameters were observed with either the diet containing a combination of WS-AX and AXOS, or with the diet containing a combination of WS-AX, AXOS and WU-AX. With these diets the concentrations in the colon of butyrate, the most beneficial SOFA, is significantly boosted, whereas at the same time the level of branched SOFA, a marker of undesired protein fermentation, is decreased.

EXAMPLE 3: Effect of AXOS preparations in different cereal matrices on intestinal parameters Materials and methods Preparation of AXOS and maltodextrin.

AXOS was prepared by FUGEIA NV (Leuven, Belgium) from wheat bran by endoxylanase treatment. The composition and characterisation of the AXOS preparation is shown in Table 5. Maltodextrin (Maldex 150) was purchased from Syral Belgium NV (Aalst, Belgium).

Preparation of extruded cereals Extruded wheat flour was made by extruding wheat endosperm flour (Surbi, Dossche Mills & Bakery, Deinze, Belgium) in a Brabender twin screw extruder at a product exit temperature of 115°C. Extruded rye meal was made by extruding rye wholegrain meal (1740, Plange Mühle, Düsseldorf, Germany) in a Brabender twin screw extruder at a product exit temperature of 115°C. Extruded xylanase-treated wheat flour was prepared by mixing and kneading wheat endosperm flour (62 %), endoxylanase preparation (about 0.7%; Grindamyl H640, Danisco, Copenhagen, Denmark) and water (37%) followed by incubation at 30°C for 30 minutes, and extrusion in a Brabender twin screw extruder at a product exit temperature of 115°C.

Extruded xylanase-treated rye meal was prepared by mixing and kneading rye meal (62 %), endoxylanase preparation (about 0.7%) and water (37 %) followed by incubation at 30°C for minutes, and extrusion in a Brabender twin screw extruder at a product exit temperature of 115°C. After extrusion, the products were dried with a fluid bed drier. The dried extruded products were milled to a fine powder in a hammer mill.

Animal trial conditions.

6-week-old male rats (Wistar) were purchased from Elevage Janvier (Le Genest-St-lsle, France) and randomly assigned to 8 groups of 10 rats each. The rats were housed in stainless steel wire-bottom cages (2 rats per cage) in an environmentally controlled room (22°C) with a 14-10 h light-dark cycle. The rats were randomly assigned to one of 8 different treatment groups (10 rats/group), and the groups were each given free access during 21 days to pellets (10 mm) of one of the 8 diets described in Table 6. Rats were given free access to water and to pellets of the appropriate diet.

Animals were weighed and feed intake was measured 3 times per week. After 21 days of treatment, all animals were weighed and euthanized by carbon dioxide asphyxiation.

Thereafter, the animals were dissected to collect the colon content.

Analysis. All analyses were performed as in Example 1.

Results Feeding a rye containing diet supplemented with AXOS results in the production of colonic butyric acid and the stimulation of the presence of bifidogenic bacteria in the gut flora next to a strong reduction of the intestinal protein fermentation. These effects are most clearly in the rats fed the diet comprising the xylanase treated rye and AXOS.

TABLES

Table 1 Composition of the basic diet used in the rat experiments1 Ingredient g/kg diet Corn starch, pregelatinized 204.75 Wheat meal (whole grain) 150.0 Sucrose 130.0 Casein 80.0 Whey powder 50.0 Poultry meal 150.0 Fish meal 10.0 Pork lard 50.0 Beef tallow 60.0 Butterfat 60.0 Cellulose 10.0 Monocalcium phosphate 18.0 Calcium carbonate 10.0 Sodium chloride 3.0 Magnesium oxide 2.0 Choline chloride 2.0 Butylhydroxytoluol 0.25 Vitamin / Trace element premix2 10.0 1 Analytical components of dry matter were: crude proteins, 21.6%; crude fat, 20.3%; crude ash, 6.6%; carbohydrates, 47.6% (energy 18.7 MJ/kg; Snniff).

2 Supplied the following (to provide mg/kg diet, except as noted): all-trans retinol acetate, 4.5; cholecalciferol, 25 ig; all-rac-a-tocopheryl acetate, 100; menadion sodium bisulfite, 5; thiamin hydrochloride, 12; riboflavin, 20; pyridoxine hydrochloride, 15; cyanocobalamin, 100 pg; calcium DL-pantothenate, 30; nicotinic acid, 60; folic acid, 6; D-biotin, 300 pg; myo-inositol, 100; Fe, 100; Cu, 5; Zn, 50; Mn, 30; Co, 2; I, 2; Se, 0.10.

Table 2 Compositional and structural properties of different test preparations and dosages used in the different diets Inulin or (A)XOS (%dm)1 oligofructose2(% avDP avDAS Dosage (%)3 dm) AXOS-61 -0.58 70.5 na.4 61 0.58 5.48 AXOS-12-0.69 89.1 n.a. 12 0.69 4.49 AXOS-15-0.27 72.8 n.a. 15 0.27 5.26 AXOS-5-0.27 74.9 n.a. 5 0.27 5.15 AXOS-3-0.26 81.2 n.a. 3 0.26 4.44 XOS 83.9 n.a. 3 0.09 4.61 FOS 0.0 95.0 4 n.a. 4.21 Inulin 0.0 100.0 25 n.a. 4.21 1 dm, dry matter.

2 As defined by the supplier.

Dosages used are those of the preparations as such and thus yielding 3.6% to 4.0% of pure (A)XOS in the diets.

"n.a., not applicable.

Table 3: Composition and characterisation of the preparations of AXOS, WS-AX and WU-AX. Composition parameters are expressed as % (wiw) on dry weight basis. AIX ratio: arabinose to xylose ratio or the average degree of arabinose substitution of arabinoxylan; avDP: average degree of polymerisation of arabinoxylan.

AXOS WS-AX WU-AX

preparation preparation preparation

ARAB INOXYLAN

-bound xylose 68.2 45.8 28.2 -bound arabinose 14.6 21.5 15.5 -total arabinoxylan 85.2 67.3 43.7 -AIX ratio 0.21 0.47 0.55 -avDP 5 146 >200

OTHER CARBOHYDRATES

-non-cellulosic boundglucose 12.5 16.8 1.2 -bound galactose 0.6 0.9 0.2 -bound mannose 0.2 0.7 1.5

MONOSACCHARI DES

-xylose 1.2 0.2 <0.1 -arabinose 0.2 0.5 <0.1 -glucose 0.2 5.2 <0.1

OTHER COMPONENTS

-protein 0.4 3.5 10.4 -ash 0.5 0.2 4.4 Table 4: Composition of the different rat diets (in g per 100 g). The concentrations of AXOS, WU-AX and WS-AX preparations indicated between brackets were corrected for their purity as calculated by their total AX/AXOS content.

control WU-AX WUAX + WS-AX+ WU AX � Corn starch (pre-gelatinised) 7350 66.30 63.28 6816 65.94 58.74 AXOS preparation --2.22 (1.80) -2.22 (1.80) 2.22 (1.80) WU-AX preparation -9.14 (3.60) 9.14 (3.60) --9.14 (3.60) WS-AX preparation ---5.34 (3.60) 5.34 (3.60) 5.34 (3.60) Soy protein isolate 1080 9.30 9.30 1080 10.80 9.30 Wheat gluten 500 5.00 5.00 500 5.00 5.00 Soybean oil 350 3.20 3.20 350 3.50 3.20 L-Lysine 045 0.50 0.50 045 0.45 0.50 DL-Methionine 015 0.15 0.15 015 0.15 0.15 L-Cystine 008 0.07 007 008 0.08 007 L-Threonine 0.13 0.15 0.15 0.13 0.13 0.15 L-Tryptophan 0.07 0.07 0.07 0.07 0.07 0.07 Vitamin premix 1.00 1.00 1.00 1.00 1.00 1.00 Mineral/trace elem. premix 4.20 4.20 4.20 4.20 4.20 4.20 Calcium carbonate 0.70 0.50 0.50 0.70 0.70 0.50 CrO3 0.20 0.20 0.20 0.20 0.20 0.20 Choline chloride 0.20 0.20 0.20 0.20 0.20 0.20 Butylhydroxytoluol 0.02 0.02 0.02 0.02 0.02 0.02 Table 5: Composition and characterisation of the preparations of AXOS used in the experiment of Example 3.

AXOS

preparation

ARAB INOXYLAN

-bound xylose 67.0 -bound arabinose 13.0 -total arabinoxylan 82.3 -A/X ratio 0.19 -avDP 5

OTHER CARBOHYDRATES

-non-cellulosic bound glucose 12.7 -bound galactose 0.5 -bound mannose 0.2

MONOSACCHARIDES

-xylose 0.4 -arabinose 0.1 -glucose 0.1

OTHER COMPONENTS

-protein 0.6 -ash 1.0 Table 6: Composition of the different rat diets (in g per 100 g).

rye5o%+ rye5o% wheat, wheat, rye 25%, rye 25%, lye 0%. lye 50%, Maltodextrin AXOS maltodextrin AXOS:maltodextnn: xylanase, xylanases.

______________________________________ ___________ __________ __________ ___________ __________ __________ maltodextrin: __________ Maltodextrin 2.00 0,00 2,00 0,00 2.00 0,00 2,00 0,00 AXOS preparation 0.00 2,00 0,00 2,00 0,00 2,00 0,00 2,00 Extruded wheat flour 78.00 78,00 58,50 58,50 39,00 39,00 0,00 0,00 Extruded rye meal 0.00 0,00 19,50 19,50 39,00 39,00 0,00 0,00 Extruded xylanase-treated wheat flour 0.00 0,00 0,00 0,00 0,00 0,00 39,00 39,00 Extruded xylanase-treated rye meal 0,00 0,00 0,00 0,00 0,00 0,00 39,00 39,00 Soy protein isolate 9,46 9,46 9,46 9,46 9,46 9,46 9,46 9.46 Soybean oil 3,50 3,50 3,50 3,50 3,50 3,50 3,50 3,50 L-Lysine 0,45 0,45 0,45 0,45 0,45 0,45 0,45 0,45 DL-Methionine 0,15 0,15 0,15 0.I 0,15 0,15 0,15 0,15 L-Cystine 0,08 0,08 0,08 0,08 0,08 0,08 0,08 0,08 L-Threonine 0,13 0,13 0,13 0,13 0,13 0,13 0,13 0,13 L-Tryptophan 0,07 0,07 0,07 0,07 0,07 0,07 0,07 0,07 Vitamin premix 1,00 1,00 1,00 1,00 1,00 1,00 1,00 1,00 MineraL/trace elem. premix 4,20 4,20 4,20 4,20 4,20 4,20 4,20 4,20 Calcium carbonate 0.70 0.70 0,70 0,70 0.70 0.70 0,70 0,70 CrO3 0,04 0,04 0,04 0,04 0,04 0,04 0,04 0,04 Choline chloride 0.20 0,20 0,20 0,20 0,20 0,20 0,20 0,20 Butylhydroxytoluol 0.02 0,02 0,02 0,02 0,02 0,02 0,02 0,02

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Claims (1)

1. CLAIMS1. A feed or food product enriched with both AXOS and WS-AX characterized in that said food product comprises on a dry weight basis between 0.75 and 15% (wlw) of WS-AX and between 1.0 and 15% AXOS.