As studies on gut microbiota are being accumulated, needs for the materials such as prebiotics, probiotics, and synbiotics to modulate gut microbiota are growing rapidly. Human milk oligosaccharides are mostly fucosylated, the fucosylated oligosaccharides are considered as functional food like prebiotics. Moreover, there is growing interest in industrially useful microorganisms to improve the production efficiency and prebiotic effect of prebiotics.
In this thesis, we reported the purification of bifidobacterial α- and β-galactosidases from recombinant E. coli and investigated the optimal synthesis conditions of novel prebiotics using bifidobacterial α- and β-galactosidases purified from recombinant E. coli. The optimal pHs for the hydrolytic activity of purified α- and β-galactosidases were found to be pH 6.5 and pH 8.5, respectively, when investigated in the pH range of 5.7-7.6 and 7.4-8.9. At the optimal pH, the maximal temperatures for the hydrolytic activity of purified α- and β-galactosidases were found to be 31.5℃ and 40℃, respectively. The maximum production of α- and β-prebiotics was observed at pH 6.5 and pH 8.5. At the optimal pH, the optimal production of stachyobifiose occurred in the temperature range of 22-32°C. On the other hand, the optimal production of β-prebiotics occurred at 45℃.
We synthesized novel α-galactooligosaccharides (α-GOS) using bifidobacterial α-galactosidase purified from recombinant E. coli. The structure of the novel α-GOS has not yet been reported in other articles, and the term stachyobifiose was assigned to it. In addition, we reported the synthesis of β-fucosylated galactose (fucosyl-gal), which is thought to have prebiotic effect and has not yet been reported in other articles, using bifidobacterial β-galactosidase purified from recombinant E. coli. The stachyobifiose was identified as α-D-galactopyranosyl-(1→6)-O-α-D-glucopyranosyl-(1→2)-[α-D-galactopyranosyl-(1→6)-O-β-D-fructofuranoside] by NMR. The fucosyl-gal was identified as β-D-fucopyranosyl-(1→3)-O-D-galactopyranose by NMR.
Moreover, we investigated the prebiotic effects of the fucosyl-gal (in vitro). The 16 strains of non-probiotics except Prevotella intermedia KCTC 5694, Bacteroides fragilis KCTC 5013, Enterococcus faecalis KCTC 3511, and Clostridium perfringens KCTC 3269 grew less on fucosyl-gal than on β-GOS. On the other hand, all of the tested Lactobacillus strains except L. delbrueckii subsp. bulgaricus KCTC 3635 and most of the tested Bifidobacterium strains grew better on fucosyl-gal than on β-GOS. In particular, Bifidobacterium infantis KCTC 3249, which is known to be most abundant in breast-fed infants, grew best on fucosyl-gal. Therefore, in vitro study showed that fucosyl-gal was selectively used by probiotics over non-probiotics, and this prebiotic effect was superior to commercial β-GOS.
Finally, we confirmed whether fucosyl-gal contributes to beneficial changes in gut microbiota when administered orally to mice using the next generation sequencing (NGS) method. The study was conducted in 6 groups as follows: Control group (C group; Complex probiotics gavage group (P group); β-GOS gavage group (G group); fucosyl-gal gavage group (F group); Complex probiotics + β-GOS gavage group (GP group); Complex probiotics + fucosyl-gal gavage group (FP group). The alterations in phylum level of 4 day and final day fecal microbiota appeared in F group and FP group compared to C group, respectively. The alterations in phylum level showed the increase in Firmicutes and Actinobacteria and the proportional decrease in Bacteroidetes. The Firmicutes/Bacteroidetes ratio of 4 day fecal microbiota in F group compared to C group was increased. Moreover, the Firmicutes/Bacteroidetes ratio of final day fecal microbiota in FP group compared to G group was significantly different. In final day fecal microbiota, members of bacterial taxa in the Bacteroidetes were enriched in G group, whereas those in the Firmicutes were enriched in FP group. At the family level, Lactobacillaceae and Streptococcaceae were prevalent in FP group, whereas Porphyromonadaceae and Verrucomicrobiaceae had significantly higher relative abundances in G group and P group, respectively. At the level of genus, Lactobacillus and Lactococcus, which are known as beneficial bacteria, were prevalent in FP group, whereas Parapedobacter and Lachnospira had significantly higher relative abundances in P group. Clostridium, which is known as harmful bacteria, was prevalent in C group.
There was no significant difference, but Bifidobacterium was increased in F group compared to C group in 4 day fecal samples. Bifidobacterium and Lactobacillus were increased in GP and FP group compared to C group in final day fecal samples albeit not significantly. On the other hand, Ruminococcus and Desulfovibrio, which are known to be associated with inflammatory bowel disease, were decreased in F group and FP group compared to C group in 4 day and final day fecal samples, respectively. There was no significant difference, but PI was the highest in F group in 4 day fecal samples and in FP group in final day fecal samples. The total SCFA in F group was significantly higher than the total SCFA in C group. Among total SCFA, the acetate, propionate, isobutyrate and lactate in F group was higher than that in C group.
Relative abundance of Actinobacteria, Bifidobacteriaceae and Bifidobacterium exhibited significant positive correlations with concentrations of cecal acetic acid. Relative abundance of Porphyromonadaceae and Dysgonomonas was positively correlated with concentrations of cecal propionic acid. Moreover, higher concentration of Clostridiaceae and Lachnospira was associated with higher concentrations of cecal butyric acid. Relative abundance of Lactococcus was positively correlated with concentrations of cecal isobutyric acid.
In conclusion, a novel fucosyl-gal contributes to beneficial changes in the gut microbiota, showing primary potential as prebiotics, and further is expected to contribute to the prebiotic effect associated with disease treatment.