Probiotics from corn cobs

INTRODUCTION
In the recent years, fructo-oligosaccharides and inulin have been the main sources of prebiotic ingredients used in the food industry. However, some other classes of functional oligosaccharides, such as xylo-oligosaccharides (XOS), are also emerging since they possess important technological advantages, such as pH and temperature stability. In 2002, the International Scientific Association for Probiotics and Prebiotics (ISAPP) produced a report where it was stated that biomass residues from forestry, agriculture and agro-industries would constitute an economically attractive substrate for the production of novel candidate prebiotics and that the development of a second generation of prebiotics would demand alternative manufacturing technologies for the processing of raw materials and separation of oligosaccharides. At DB – INETI (Department of Biotechnology from the National Institute of Engineering, Technology and Innovation, Portugal) an important effort has been made to set-up a process (autohydrolysis) for the production of a range of complex XOS suitable as food ingredients from lignocellulosic by-products, as a result from a previous joint European project (4th FWP, FAIR CT98-3811). More recently our research efforts were enlarged to focus an integrated approach for XOS, comprising not only the production but also the evaluation of the prebiotic potential of the mixtures obtained. Accordingly, in our paper of LWT - Food Science and Technology, we produced short-chain XOS by autohydrolysis of corn cobs and tested their capability to support the growth of bifidobacteria and lactobacilli strains.

XOS and AUTOHYDROLYSIS
Oligosaccharides are molecules containing a small number of monosaccharide units connected by glycosidic linkages. XOS in particular are made up of a main backbone of xylose units which can be more or less substituted by sugar units, uronic acids and acetyl groups, depending on the feedstock origin and manufacturing process. Commercially, XOS are produced from xylan-rich materials, such as corn cobs and cotton husk bran in a two stage process of alkaline extraction followed by enzymatic hydrolysis of the dissolved xylan. Autohydrolysis can be considered as an alternative environmentally friendly process, aiming XOS production from xylan-rich materials. This process is based on the hydrolytic degradation of lignocellulosic materials at temperatures usually ranging from 150 to 230 ºC. The hydronium ions generated both from water autoionisation and acetic acid released from hemicellulosic acetyl groups act as catalysts in the hydrolytic reaction. The process mainly promotes the depolymerisation of hemicelluloses into oligo- and monosaccharides, leaving both cellulose and lignin essentially as undegraded polymers. The operational conditions can be optimised for maximizing XOS production. These crude XOS hydrolysates can be further separated by gel filtration chromatography (GFC) and pooled according to the desired average molecular weight range. This possibility enables the preparation of “tailor-made” XOS mixtures, for example short-chain XOS similar in degree of polymerisation (DP) to commercial preparations or longer chain XOS with increased molecular weight, theoretically more capable to persist throughout the gastrointestinal tract.

FERMENTATION BY Bifidobacterium and Lactobacillus
For the evaluation of the prebiotic potential of XOS produced by autohydrolysis we conducted in vitro fermentation studies by Bifidobacterium and Lactobacillus strains. Two XOS mixtures with a relative distribution of 66% (w/w) of DP4 to DP6 and 51% (w/w) of DP3 and DP4 were chosen for comparison with a commercial product containing 54% of xylobiose. The strains tested were Bifidobacterium adolescentis, B. longum, Lactobacillus brevis and L. fermentum.

The best strains
By comparison of the maximal optical densities obtained in growth experiments with the selected XOS, it was observed that B. adolescentis and L. brevis exhibited a higher capacity to grow on XOS than B. longum and L. fermentum. Comparing between the first two strains, the specific growth rates of B. adolescentis in XOS-supplemented media were substantially higher than those of L. brevis, indicating a better efficiency on XOS utilisation.

Influence of DP on specific growth rate and XOS consumption
The increase on the average chain length of the XOS mixtures led to a slight decrease on the specific growth rate attained by B. adolescentis. However, the values were still high and comparable to the ones attained on glucose, indicating that B. adolescentis was able to rapidly consume XOS with DP up to 6. The extension to which B. adolescentis consumed one of the XOS mixtures obtained by autohydrolysis (51% DP3+DP4) was similar to commercial XOS, confirming the extreme ability of the strain to use XOS with DP higher than 2. No xylose accumulation was detected in the fermentation media of B. adolescentis as a result of XOS consumption. L. brevis displayed a diauxic growth behaviour on XOS substrates probably resulting from the coexistence of residual monomers and more complex carbohydrate structures in the fermentation media. This strain exhibited a preference for XOS with smaller DP, essentially xylobiose. However, the accumulation of free xylose in the media suggests some difficulties on XOS uptake.

Conclusions
With our study we aimed to evaluate autohydrolysis as an alternative process for the production of prebiotic XOS. We observed that B. adolescentis was capable to grow and utilise XOS mixtures from autohydrolysis mainly constituted by xylotriose and xylotetraose as well as commercial XOS. Conversely, L. brevis consumption pattern was more dependent on XOS average chain length, with preference for the commercial mixture.

THE FUTURE
The use of an environmental-friendly process - autohydrolysis - for the production of unique XOS structures from forestry, agro-industrial or agricultural by-products, associated with the possibility of selecting oligosaccharides with a desirable DP, certainly opens a world of research opportunities. The possibility of implementing a downstream separation step allowing the selection of structurally diverse XOS also brings an important advantage, since the type, degree and pattern of substitution are determinant for XOS functionality. Thereby, the ultimate goal of our research is to explore autohydrolysis as a suitable and scalable technology for the production of XOS with potential prebiotic effect, proven by a solid scientific methodology.

LWT – Food Science and Technology, Vol. 40, Issue 6, p. 963-972, 2007. “In vitro fermentation of xylo-oligosaccharides from corn cobs autohydrolysis by Bifidobacterium and Lactobacillus strains”
Patrícia Moura, Rosário Barata, Florbela Carvalheiro, Francisco Gírio, Maria C. Loureiro-Dias and M. Paula Esteves