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24Resistance to gastrointestinal conditions is a requirement for bacteria to be 25 considered probiotics. In this work, we tested the resistance of six different Lactobacillus 26 strains and the effect of carbon source to four different gastrointestinal conditions: presence 27 of α-amylase, pancreatin, bile extract and low pH. Novel galactooligosaccharides 28 synthesized from lactulose (GOS-Lu) as well as commercial galactooligosaccharides 29 synthesized from lactose (GOS-La) and lactulose were used as carbon sources and 30 compared with glucose. In general, all strains grew in all carbon sources, although after 24 31 h of fermentation the population of all Lactobacillus strains was higher for both types of 32 GOS than for glucose and lactulose. No differences were found among GOS-Lu and GOS-33La. α-amylase and pancreatin resistance was retained at all times for all strains. However, a 34 dependence on carbon source and Lactobacillus strain was observed for bile extract and 35 low pH resistance. High hydrophobicity was found for all strains with GOS-Lu when 36 compared with other carbon sources. However, concentrations of lactic and acetic acid 37 were higher in glucose and lactulose than GOS-Lu and GOS-La. These results show that 38 the resistance to gastrointestinal conditions and hydrophobicity is directly related with the 39 carbon source and Lactobacillus strains. In this sense, the use of prebiotics as GOS and 40 lactulose could be an excellent alternative to monosaccharides to support growth of 41 probiotic Lactobacillus strains and improve their survival through the gastrointestinal tract. 42 43

A detailed study was performed to compare the in vivo ileal digestibility and modulatory effects in fecal microbiota of novel galacto-oligosaccharides (GOS) derived from lactulose [GOS-Lu; degree of polymerization (DP) ≥2, 14.0% trisaccharides] and commercial GOS derived from lactose (GOS-La; DP ≥3, 35.1% trisaccharides) in growing rats (5 wk old). Rats were fed either a control diet or diets containing 1% (wt:wt) of GOS-Lu or GOS-La for 14 d. Quantitative analysis of carbohydrates from dietary and ileal samples demonstrated that the trisaccharide fraction of GOS-Lu was significantly more resistant to gut digestion than that from GOS-La, as indicated by their ileal digestibility rates of 12.5 ± 2.6% and 52.9 ± 2.7%, respectively, whereas the disaccharide fraction of GOS-Lu was fully resistant to the extreme environment of the upper digestive tract. The low ileal digestibility of GOS-Lu was due to the great resistance of galactosyl-fructoses to mammalian digestive enzymes, highlighting the key role played by the monomer type and linkage involved in the oligosaccharide chain. The partial digestion of GOS-La trisaccharides showed that glycosidic linkages (1→6) and (1→2) between galactose and glucose monomers were significantly more resistant to in vivo gastrointestinal digestion than the linkage (1→4) between galactose units. The absence of GOS-La and GOS-Lu digestion-resistant oligosaccharides in fecal samples indicated that they were readily fermented within the large intestine, enabling both types of GOS to have a potential prebiotic function. Indeed, compared with controls, the GOS-Lu group had significantly more bifidobacteria in fecal samples after 14 d of treatment. The number of Eubacterium rectale also was greater in the GOS-Lu and GOS-La groups than in controls. These novel data support a direct relationship between patterns of resistance to digestion and prebiotic properties of GOS.

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