Obtención enzimática, caracterización y propiedades prebióticas de oligosacáridos empleados en leches infantiles

Resumen

Prebiotics are selectively fermented ingredients that allow specific changes in composition and/or activity in the gastrointestinal microflora that confer benefits upon host well-being and health. In this field, the attention is mainly focused on fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS) due to their current use in many foods and their importance in human health. Human milk oligosaccharides (HMOs) supply all the nutritional needs to the baby and they are essential to the proper development and growth of the neonate. Their components provide bioactive compounds that confer protection against pathogens and diseases, modulate the immune system, stimulate desired bacteria like Bifidobacterium and Lactobacillus, influence in cognitive development, and establishment of the intestinal microbiota. Due to the complexity of these HMOs, it is a common practice the supplementation of FOS/GOS in infant formulas as alternative sources of prebiotic ingredients to provide similar bifidogenic effects to breast fed babies. In this work several enzymes were studied for their use in prebiotic oligosaccharide production; three ß-galactosidases from Kluyveromyces lactis, Bacillus circulans and Aspergillus oryzae for GOS synthesis and one ß- fructofuranosidase from Aspergillus aculeatus for FOS production. The major products synthesized by B. circulans ß-galactosidase contained only ß(1,4) bonds, whereas the enzyme from K. lactis and A. oryzae displayed a clear tendency to form ß(1,6) linkages. B. circulans and K. lactis ß-galactosidases produce nearly 45-50% (w/w) GOS, whereas the A. oryzae enzyme produced less than 30% (w/w) GOS. Regarding product distribution, B. circulans enzyme synthesized the highest amount of trisaccharides whereas K. lactis and A. oryzae would be a better choice to synthesize a product with content in di- and trisaccharides. The ß-fructofuranosidase from Aspergillus aculeatus formed basically a mixture of 1-kestose, nystose and 1F-fructosylnystose. It is reported that the chemical structure of the obtained oligosaccharides (composition, number of hexose units and types of linkages between them) may affect their fermentation pattern by probiotic bacteria in the gut. To verify this effect, fermentation selectivity of some purified carbohydrates, with different polymerization degree and distribution, was estimated using in vitro fermentation systems inoculated with human fecal slurries. Changes in bacterial populations were measured by fluorescence in situ hybridization (FISH) and HPLC analysis of short-chain fatty acids (SCFA) produced by oligosaccharide fermentation. All of the tested GOS could be considered bifidogenic under the assayed conditions with selectivity index (SI) slightly lower to that determined for a commercial GOS mixture. A challenging objective of this work was to obtain milk with a low content of lactose and a significant amount of GOS. GOS concentration obtained with K. lactis ß-galactosidase using skim milk was close to the HMOs content in breast milk. Pasteurization after controlled enzymatic treatment could result in a product with a low lactose content and with the extra benefit of a significant presence of prebiotic GOS. In order to scale-up the processes to synthesize prebiotic oligosaccharides, immobilization in calcium alginate gel beads was successfully performed for both ß-galactosidase from B. circulans and ß- fructofuranosidase from A. aculeatus. The controlled dehydration of alginate beads resulted in novel biocatalysts (DALGEEs, Dry ALGinate Entrapped Enzymes) with improved properties with respect to the wet beads. DALGEEs displayed higher volumetric activity, reduced volume and better storage. The operational stability of DALGEEs containing ß-fructofuranosidase in a fixed-bed reactor was extraordinary with a nearly constant FOS composition during 700 h.