D. JANSSENS
B. GAETHOFS
Nutrex, Lille, Belgium
Earlier research on prebiotic compounds mainly focused on inulin and fructo¬oligosaccharides. Recent investigations involving in vivo studies in monogastric animals and in vitro culture fermentations, as well as the availability of more powerful fractionation techniques, have shown the existence of other potential polysaccharide based sources with prebiotic properties. Their effectiveness seems to be related to their molecular structure and the purity of the preparation. This implies that production techniques will require concentrating on optimal yield and efficient recovery of specific molecular weight fractions. Arabinoxylans are abundantly present in cereals. Both the water-soluble as the water-¬insoluble arabinoxylans exert well-described anti-nutritive effects in monogastrics. Endo¬xylanases with a pronounced capability to hydrolyze the insoluble fraction into soluble oligosaccharides will convert an omnipresent polysaccharide into beneficial substances. The effect of different EC-registered enzyme preparations, based on endo-xylanase activity, on different xylan substrates was investigated.
I. Prebiotics - Plenty of substances
The crucial role of the intestinal microbiota in the general health status of man and animal is becoming more and more acknowledged. In animal production, good health is a prerequisite for high performance. Recent years, following the footsteps of human nutrition, considerable effort has been made to reveal the possible effects and working mechanisms of prebiotics, non-digestible substances that provide a beneficial physiological effect on the host by selectively stimulating the favourable growth or activity of a limited number of indigenous bacteria (Gibson and Roberfroid, 1995).
Prebiotics are fermented by microorganisms in the distal part of the gastro-intestinal tract. Hereby the composition and/or the activity of the microbiota is altered, leading to secondary effects such as: increased gas production (short chain fatty acids - SCFA, lactic acid, hydrogen, carbon dioxide, hydrogen sulphide); a drop in pH; and decreased metabolic activity, (e.g. the activity of 7-α-deshydroxylase, which converts primary bile salts into carcinogenic secondary bile salts, Marteau et al., 2004). This shift in microbiota may be translated into macro-effects like an increased resistance against pathogens or an improved growth performance.
So far, most research has been performed on inulin and fructooligosaccharides. However, emphasis has widened towards a complete range of oligosaccharides including galacto-oligosaccharides, beta-glucans, soybean oligosaccharides and xylo-oligosaccharides. Up to now, the major part of research on these prebiotics has been performed in vitro, despite of the known limitations. Recent molecular techniques indicate that only 20 to 50 % of the bacterial species in the intestines can be cultured (Patterson and Burkholder, 2003). In order to fully assess the potential of a prebiotic substance, trials in the intestinal environment are necessary, where substrate availability, nutrient competition, cross-feeding, and bacterial population levels and niches will influence bacterial metabolism, population dynamics and functional effects in the host (Crittenden et al., 2002).
The structure of the prebiotic substance largely determines its effect. Inulin is known to be fermented faster (resulting mainly in the production of butyric acid) than xylo-¬oligosaccharides, which are slowly fermented, generating acetic, propionic and lactic acids. There is a large diversity in the responsiveness of the different bacteria. Differences have been noticed between bacterial genera, species and strains (Crittenden et al., 2002). Xylo-¬oligosaccharides tend to stimulate bifidobacteria, but effects are influenced by the degree of polymerisation (DP), the degree of substitution, and the character of possible side-chains.
II. The manufacture of xylo-oligosaccharides
Recently considerable effort has been put into the refinement of the manufacturing process of xylo-oligosaccharides. One possibility of obtaining these is to use an enzymatic de novo synthesis process, starting from mono- and oligosaccharides (Rastall et al., 2002). Up until now costs of production are high, resulting in an expensive product that cannot be used at a large scale in human and animal nutrition.
On the other hand, plant cell wall polysaccharides, rich in arabinoxylans, are abundantly present in nature. This makes them an excellent substrate for producing xylo¬-oligosaccharides, in a chemical and/or enzymatic way.
The xylo-oligosaccharides obtained this way consist of a diversity of components, which need refining. Multiple purification steps (solvent extraction, ultra filtration techniques) may be necessary in order to obtain a high-purity end-product. (Moure et al., 2006).
Looking for an alternative, we considered the fact that feeds for poultry and pigs are mostly based on corn, wheat and/or barley. All of them are raw materials rich in arabinoxylans, a known anti-nutritional factor, countered by the standard addition of endo¬xylanase. In the gastro-intestinal tract, the endo-xylanase breaks down the arabinoxylan chains into smaller fragments, with potential prebiotic properties.
III. Characterisation of the hydrolysates formed by endo¬xylanase
a) Materials and methods
Purified xylo-oligosaccharides with an average degree of polymerization of respectively three, four and five, were purchased from MegaZyme, Ireland. Three enzyme preparations containing mainly endo-xylanase activity were compared in the trial: one originating from Bacillus subtilis (xylanase-B), one from Trichoderma Longibrachiatum (xylanase-T) and one from Aspergillus oryzae (xylanase-A). All three preparations are registered for use in the EC.
The synthetic xylo-oligosaccharides with three, four and five xylose units were incubated in the presence of the different endo-xylanase preparations. A negative control group was subjected to the same incubation conditions, without endo-xylanase addition. The average degree of polymerisation (DP) of the hydrolysates was determined.
b) Results and discussion
Figure one illustrates the average DP values of the xylo-oligosaccharides of the 12 treatments: all possible combinations of three different xylo-oligosaccharide substrates (DP of three, four and five) and four enzyme treatments (three commercial endo-xylanase preparations and a negative control group).
In every substrate, the DP of the negative control group showed a slight deviation compared to the theoretical value. Therefore, the DP values of the three groups treated with enzymes are compared to the measured values of the DP of XOS in hydrolysate corresponding negative control. The endo-xylanase of Bacillus subtilis did not affect the DP when the substrate contained four xylose units or less, so the bacterial endo-xylanase requires a minimal chain length of five xylose units in its substrate prior hydrolytic action to carry on.
The fungal xylanases (-T and -A) started hydrolyzing xylo-oligosaccharides of smaller size, theoretical DP values of respectively three and four were needed minimally before hydrolytic action was initiated. One can conclude that after complete hydrolysis of an arabinoxylan substrate, xylanase-B will result in fragments with an average DP of not less than three and without the concomitant generation of xylose monomers that exert a metabolic (osmotic) stress in monogastric animals. These results were in accordance with prior findings that the in vitro degradation of rye water-soluble arabinoxylans by xylanase-B, after 24 hours of incubation, generated hydrolysates of 550 to 1.730 Dalton, corresponding to an average DP of respectively three to 12 (unpublished data, Puratos, Belgium).
Measured average degree of polymerization of the fragments formed after enzymatic breakdown of xylotriose, xylotetraose and xylopentaose
Courtin and Delcour (2001) carried out similar research on xylanase-B and an endo¬xylanase preparation originating from Aspergillus aculeatus. They incubated a standardised water-insoluble arabinoxylan substrate with each of the endo-xylanase preparations and screened the generated solubilised fragments (Table one). Courtin and Delcour determined the amount of reducing sugars, as a measure of the number of fragments solubilised, but did not observe large differences between the two preparations. On the other hand, the total amount of xylose units present in the solubilised fraction was much higher in the xylanase-B hydrolysate (35 times). This can only be explained by the fact that the average size (and thus the DP value) of the xylanase-B hydrolysate was much higher than in the other xylanase preparation. This suggests the following in vivo benefits from the use of xylanase-B: a fast decrease of intestinal viscosity, absence of generated xylose monomers and the conversion of water-insoluble arabinoxylans, naturally present in the feed, into substances with prebiotic potential.
c) Conclusion
Most animal feeds contain relatively high amounts of arabinoxylans, present in the fibre fraction of cereals.
Diets with corn or wheat inclusions of 50% or more contain a water-insoluble arabinoxylan fraction of at least 30 g per kg feed. Through the use of a selective endo-xylanase, the in situ generation of xylo-oligosaccharides with a specific DP range is possible. This hydrolysis and its resulting hydrolysate product(s) will be characterised by a sequence of parameters such as reaction time, pH, availability and structure of the substrate. An in vitro experiment closely matching intestinal conditions could provide more insight into the structure of the hydrolysates and their potential prebiotic effect.
References
Courtin, C.M. and Delcour, J.A. (2001). Journal of Cereal Science, 33:301-312.
Crittenden, R., Karppinen, S., Ojanen, S., Tenkanen, M., Fagerström, R., Mättö, J., Saarela, M., Mattila-Sandholm, T. and Poutanen, K. (2002). Journal of the Science of Food and Agriculture, 82:781-789.
Gibson, G.R. and Roberfroid, M.B. (1995). Journal of Nutrition, 125:1401-1412.
Marteau, P., Seksik, P., Lepage, P. and Doré, J. (2004). Mini-reviews in Medicinal Chemistry, 4:889-896.
Moure, A., Gullón, P., Domínguez, H. and Parajó, J.C. (2006). Process Biochemistry,
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Rastall, R.A. and Maitin, V. (2002). Current Opinion in Biotechnology; 13:490-496.
Patterson, J.A. and Burkholder, K.M. (2003). Poultry Science; 82:627-631.
From Proceedings of the "19th Australian Poultry Science Symposium", New South Wales, Australia.
