This study aimed to investigate the fermentative metabolism of Bifidobacterium adolescentis
MB 239 on glucose, galactose, and lactose and on a mixture of GOS composed of oligosaccharides with DP ranging between 3 and 9 (58.3%), lactose (19.4%), galactose (3.5%), and glucose (18.8%). An unstructured unsegregated mathematical model that accurately described the kinetics for batch cultures was successfully developed. The kinetic parameters were calculated by means of a Matlab algorithm using experimental data from batch cultures as proposed by Boonmee et al. (4
Based on the 95% confidence limits, accurate values were obtained for all the calculated parameters. Carbohydrate preferences were established by comparing maximum specific growth rates, cellular yields, and saturation constants. Galactose led to the highest growth rate and cellular yield, whereas glucose was the poorest carbon source for growth. GOS and lactose were both worse growth substrates than galactose but better than glucose, in agreement with a study demonstrating that B. adolescentis
MB 239 preferred oligosaccharides over glucose (1
Previous papers dealing with fermentation of mono- and oligosaccharides by bifidobacteria report wide differences in sugar preferences among oligosaccharides and their monomeric constituents. In fact, monosaccharides were preferred over oligosaccharides in a few cases (19
), while growth rates and cell yields were higher on disaccharides and oligosaccharides than on their monomeric moieties in many others (1
Even if the half-saturation constant cannot be used as a direct measure of microbial affinity for substrates, KS values confirmed that galactose was preferred over lactose and lactose over glucose. The KS value was significantly higher on GOS, since the mixture components were consumed with different kinetics and carbohydrates with lower affinity, which were used at the end of the growth phase, negatively affected the KS estimation. In fact the mathematical model regarded the GOS mixture as a single chemical species, and total carbohydrate concentration (C-mM) was used by the calculation algorithm.
MB 239 exhibited a stringent selectivity based on the DP since the oligomers with DP 2 (lactose) and 3 were the first to be consumed, and longer oligosaccharides were simultaneously utilized after lactose depletion. The different consumption kinetics of the mixture components are consistent with a decreasing specific growth rate. Polyauxic consumption can be explained by the activity of different enzymes and/or transporters or by the different affinities of enzymes and/or transporters toward the oligomers with different DPs. Unlike the vast majority of documented cases in which monosaccharides are the substrates preferred by microorganisms in a mixed-carbon source environment (6
), B. adolescentis
MB 239 did not show any preference for glucose that was contained in the commercial mixture, in agreement with a previous study concerning its behavior on mixtures of mono- and oligosaccharides (1
). The biochemical effects of lactose on galactose uptake and/or metabolism by bifidobacteria have never been investigated, but a study demonstrated that lactose led to the repression of a glucose-H+
symporter gene, glcP
, in B. longum
NCC2705, thus explaining the lactose-over-glucose preference in that strain (23
) and possibly in B. adolescentis
Throughout the batch fermentations on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that β(1-4) galactosides can be hydrolyzed before they are taken up by B. adolescentis
MB 239 and that the rate of galactose production was higher than the rate of galactose uptake, leading to a net accumulation into medium. Even if galactose supported the fastest growth, it was not used up before more complex sugars were, in agreement with previous chemostat and batch experiments on single and mixed carbohydrates which also demonstrated that similar extracellular hydrolysis by B. adolescentis
MB 239 can occur for raffinose and fructooligosaccharides as well (1
). In order to assess activity and location of β-Gal, intracellular, surface, and extracellular specific activities were assayed during batch cultures on GOS, lactose, galactose, and glucose. On all the carbohydrates, very low β-Gal activity was found in the supernatant. On glucose, intracellular β-Gal was scarce. On galactose, the intracellular β-Gal activity that was found in the early hours of cultivation probably came from the corresponding preculture; then it was not produced during exponential phase, or it was produced at a lower rate than biomass was. At the end of growth, when galactose ran out, β-Gal was restored. As expected, lactose was the best inducer of intracellular β-Gal and high levels were also found during growth on GOS. It is remarkable that β-Gal production was not strongly repressed during growth on GOS even if the commercial mixture contained a high glucose concentration. β-Gal activity was found on the surface of B. adolescentis
MB 239 during growth on galactose, lactose, and GOS, confirming that both intracellular and extracellular hydrolysis are involved in the utilization of β(1-4) galactosides.
In bifidobacteria, oligosaccharides and polysaccharides are depolymerized down to their monomeric constituents, which are incorporated into the fructose-6-phosphate shunt, giving lactic and acetic acids as the major products and ethanol and formic acid as the minor ones (2
). Hexoses are broken down throughout the shunt so that the carbon flux is equally divided into two-carbon and three-carbon molecules. In response to different ATP or NAD+
cellular requirements, pyruvate can be diverted from forming lactate to forming acetylphosphate and formate by the phosphoroclastic reaction, and acetylphosphate can be reduced to ethanol at the expense of acetate production (Fig. ) (2
). Moreover, part of the carbon flux is subtracted to fermentation and is directed to anabolic processes for biomass production. The relative amounts of fermentation products reflect cellular metabolic conditions that are anything but invariable, and it should not be expected that the theoretical lactate/acetate ratio of 1:1.5 is generally obtained. Wide differences in end product formation were actually reported in literature, depending on the strain, the carbon source, the cultural medium, and the growth conditions (22
In the present study, the yields of fermentation products on glucose, galactose, lactose, and GOS are discussed with respect to ATP gains. Two-carbon molecules (acetate plus ethanol) always accounted for about the 55% of the carbon flux toward products on all the carbon sources, whereas lactate accounted for the remaining 45%. No formate production was observed, suggesting that the phosphoroclastic reaction did not occur in B. adolescentis MB 239; hence, the imbalance between two-carbon and three-carbon products could be due to the efflux of metabolic intermediates from the fermentative shunt toward the anabolic pathways. The enzymes involved in the phosphoroclastic reaction in bifidobacteria are still not biochemically characterized. It is likely that they are formate acetyltransferase (EC 220.127.116.11) and phosphate acetyltransferase (EC 18.104.22.168). The putative genes coding for both the enzymes are present in the genomes of B. adolescentis ATCC 15703 and B. adolescentis L2-32. Nevertheless, this does not ensure that they are expressed by B. adolescentis MB 239 in the experimental conditions of the present study. It is remarkable that different ethanol yields were obtained: no ethanol was produced on galactose and low yields were obtained on lactose and GOS, whereas glucose led to the highest ethanol yield. Ethanol production caused a lower amount of acetylphosphate to be converted into acetate; thus, ATP production was lower, too. Based on the shunt stoichiometry and on the end product yields, ATP production was 25, 8, and 5% lower on glucose, GOS, and lactose, respectively, than on galactose. Sugar uptake mechanisms in B. adolescentis are not known; thus, energetic expenses for transport of glucose, galactose, lactose, and β-galactosides could not be included in calculations. Nevertheless, there was a correspondence among ethanol production, low ATP yields, low maximum specific growth rate, and low biomass yield.
This study extends the knowledge of Bifidobacterium
interactions with such important prebiotic carbohydrates as GOS. Moreover it demonstrates that carbohydrate preferences in Bifidobacterium
result not only from the efficiency of substrate transport systems as previously suggested (1
) but also from different distributions of carbon fluxes through the fermentative pathway.