A double–blind, placebo-controlled, randomised cross-over study was undertaken to determine the potential benefits of AXOS-enriched bread on the gut. Bacteriology within faeces, faecal volatile fatty acid profiles and concentrations of sIgA in saliva, bowel habit and food intake of healthy volunteers was monitored throughout the feeding study.
During the study, 3 volunteers were withdrawn due to difficulties in eating 180 g bread daily. Based on the volunteer diaries, between 4 to 9 volunteers had difficulty in taking product in each of the periods. However, the breads did not lead to adverse symptoms and compliance was excellent with 99.24% of breads issued to the volunteers consumed. Volunteers were observed to be maintaining their diets throughout the study, with no significant changes in energy, protein, carbohydrate, fibre, non-starch polysaccharide, fat or iron consumption. The observation of increased stool number (stools/day) and an increased energetic feeling following placebo treatment (Table 5) was not observed following AXOS treatment. In the study of Cloetens et al.,  AXOS extracted from wheat bran resulted in increased flatulence, this was not observed in the current study when AXOS was consumed at a lower dose within bread. Hence in the current study the AXOS-bread was well-tolerated.
Following AXOS treatment, a significant increase in faecal butyrate was observed (Table 4). Such an increase is regarded to be of benefit to the host, as butyrate is a major intestinal epithelial cell energy source, associated with potential anticancer activities observed as reducing cellular malignancy through stimulation of apoptosis in malignant cells . Butyrate has also been observed to beneficially effect oxidative stress in the human colonic mucosa  and to induce immune-modulatory effects . Furthermore, a trend for increased combined concentrations of acetate, propionate and butyrate, which are potentially beneficial to the host, was observed following AXOS-bread consumption compared to pre-AXOS treatment.
Protein fermentation is considered to be a non-beneficial process within the colon . Following AXOS-bread consumption, a trend for reduced iso-valerate and reduced combined iso-butyrate, iso-valerate, valerate and caproate concentrations was observed (Table 4), which can therefore be viewed as a potentially positive effect as these organic acids are associated with protein fermentation . This potentially beneficial modulation of fermentation of AXOS observed through VFA changes has been supported by previous research in vitro, within rats  and in humans . It was therefore concluded that the AXOS-breads led to potential benefits following consumption.
Selective stimulation of indigenous beneficial gut genera is characteristic of a prebiotic . Some potentially beneficial micro-organisms including Lactobacillus paracasei, Bifidobacterium adolescentis and Bifidobacterium bifidum[47–49] are able to breakdown AXOS. Previous studies have indicated that oat bran containing high levels of AX play a role in stimulating Lactobacillus and Bifidobacterium. Moreover in research conducted by Cloetens et al. (2010) , AXOS (10 g/d) produced by partial enzymic hydrolysis of wheat AX led to a significant Bifidobacterium increase. Therefore, in the current study increases in numbers of faecal Lactobacillus and Bifidobacterium were expected. However, it was observed that levels of Bifidobacterium increased significantly from baseline following consumption of the control white breads (Table 3). It has previously been noted that white flour can be rich in fructans (up to 2.8%, ), and are largely available for fermentation in the large intestine  therefore it is likely that within the control white bread, with 180 g consumed daily, were sufficient fructans to have an effect on the faecal microbiota. The fructan content of rye flour has been observed to be as high as 4.5% . The lack of observed bifidogenic effect, following consumption of the active breads, could therefore be attributable to the naturally occurring fructans within all bread products, thus masking any AXOS related effects. In a prebiotic dose related study , it was observed that a 10 g dose of galactooligosaccharide did not give rise to an enhanced bifidogenic effect when compared to a 5 g dose, it was therefore considered that above this threshold, the prebiotic dose did not determine the prebiotic effect. In terms of the current study this could explain that, although additionally 2.2 g AXOS were available within the treatment AXOS-bread, the required prebiotic dose for bifidogenicy had already been achieved in the control and placebo breads; thus any added effect was not apparent.
The bacteria numbers in the Lactobacillus – Enterococcus group were observed to significantly increase following the consumption of the placebo and AXOS-breads compared to pre-treatment breads (Table 3). Both of these breads were rye based, therefore it would seem that rye was influencing the microbiota. This has been supported by the use of rye grains commercially with lactobacilli for the production of sourdough [54, 55]. This therefore indicates that rye may be considered as an appropriate substrate for lactobacilli.
Consumption of the placebo breads led to significant increases in numbers of bacteria in the E. rectale group, the Roseburia and the Faecalibacterium prausnitzii cluster compared to the pre-placebo treatment (Table 3). This was expected as AX, which would be found naturally occurring in the rye flour, is known to be non-digestible  and also stimulates the growth of organisms that possess microbial endoxylanase enzymes, such as bacteria within E. rectale
Roseburia groups  and Faecalibacterium prausnitzii. In previous human studies on AXOS, bacteria in the E. rectale group have not been stimulated following 10 g AXOS , thus this lower dose was not expected to stimulate growth of these microorganisms. Futhermore, Roseburia growth was not stimulated in vitro by AXOS , thus the AXOS bread may not be expected to stimulate growth of such microorganisms . More research into this area would be required to determine this.
AXOS-breads were stimulatory to the microbiota, as observed through a significant increase in total bacteria compared to pre-AXOS treatment (Table 3). Previous intervention studies with a high dose (10 g/d) fructooligosaccharides have led to similar results . An increase in the total bacteria numbers indicates that the AXOS breads have an impact on the microbiota. The butyrogenic effects then observed could relate to the direct activities of bacteria that have been stimulated, or from a cross feeding network taking place within the microbiota components  (Belenguer et al., 2006). There was no direct increase of bacteria within the E. rectale
Roseburia and F. prausnitzii groups which contain some known butyrate producers, however there are many other bacteria able to produce butyrate within the microbiota through both cross-feeding networks and also direct stimulation  (Louis and Flint 2007). An increase in butyrate is considered to be a beneficial effect to the host, therefore the AXOS breads lead to this endpoint, whilst the placebo breads did not.
Bacteroides constitutes a large proportion of the healthy adult gastrointestinal tract, that have an ability to adapt well to limited substrate availability . In the current study, a significant increase in numbers of Bacteroides – Prevotella was observed following intervention with AXOS and placebo breads. This is in agreement with previous findings where both AXOS and AX have been observed to increase levels of Bacteroides spp. [19, 59]. Bacteroides can grow on carbohydrates or on proteins; depending on the overall diet, proteolytic bacteroides are considered detrimental , whereas saccharolytic are not, because they produce potentially beneficial VFA . In the current study as there is more carbohydrate likely to persist to the large intestine following the AXOS and placebo treatments, these changes are likely to be positive.
It has recently been observed that probiotics can lead to increased salivary secretory IgA levels . In the current study, no changes in salivary sIgA were observed (Table 6). It is worth noting that sIgA has been seen to be elevated following a stressful work-session , and examination periods . Furthermore, sIgA concentrations have declined in athletes following vigorous exercise . Therefore, there are many other parameters that may be impacting on salivary sIgA results.
In situ enrichment of AXOS in breads provides an enhanced approach to straightforward fortification. This process utilises naturally occurring AX – furthermore xylanases are already used within the baking industry. The production of these breads may be subject to some variations, e.g. as rye and wheat composition may vary amid intrinsic and extrinsic factors. However, providing the level of AX within the grain is in excess of 2%, acceptable levels of AXOS generation would still be possible . Therefore, it is unlikely that changes in grain will pose a problem in production of AXOS enriched breads.
Whilst shifts in microbial groups that are generally considered to be of benefit (bifidobacteria, lactobacilli) to the host were not observed following AXOS consumption, there were changes in the fermentation characteristics observed in terms of increased butyrate production and a trend for less protein fermentation markers. These results are important within a human population as similar shifts have been observed in animal studies and in vitro. Furthermore, the effect of such doses on the microbial community within humans has not been tested in such a population and whilst a (typically) beneficial bacterial shift could not be directly observed a fermentation shift showing positive potential was observed. This was not observed with the placebo products, therefore, although fermentation of the placebo was apparent, additional benefits from consumption of the AXOS breads – in terms of a fermentation shift are evident.