Page 41 - Read Online

Klaassens et al. Microbiome Res Rep 2024;3:38 https://dx.doi.org/10.20517/mrr.2024.13 Page 7 of 19

ammonium), an FDR of 0.1 was used.

Graphs and statistical analyses were generated in Prism version 9.2 (GraphPad, San Diego, CA, US).
Graphical abstract was created with BioRender.com.

RESULTS
Matrix-dependent effects on probiotic growth and activity in vitro
When probiotic strains are combined with IF, the surrounding formula matrix (i.e., the sum of prevailing
prebiotics, as well as carbohydrates, fats, and proteins) might have an effect on probiotic growth and
activity.

The effect of different formula matrices, or the presence of GOS, on the growth rates of Lf and Bb
(combined in a 1:1 co-culture) was tested in vitro. The addition of GOS increased the growth rates of both
strains (Figure 1A; iPF vs. iPF + GOS). The eHF + GOS matrix showed strain-specific effects as indicated by
differing CFU/mL kinetics between Lf and Bb in the respective formula matrices (Figure 1A, iPF + GOS vs.
eHF + GOS). In the formula containing extensively hydrolyzed protein and GOS (eHF + GOS), Lf showed
significantly higher growth rates compared to the iPF and GOS (iPF + GOS; after 12 h: 8.7 vs. 8.0 log10
CFU/mL respectively; P < 0.001). For Bb, no growth-supportive effect of the eHF + GOS, compared to iPF +
GOS, was seen (8.8 vs. 8.8 log10 CFU/mL, respectively; n.s.).

Probiotic activity was visible through the formation of different acids such as acetate, propionate, butyrate,
and lactate along the fermentation process. To evaluate the respective amount of SCFA which is produced
by either Lf, Bb, or the combination of the strains, the probiotics were either grown as single strains or
simultaneously (combined in a 1:1 co-culture). Bacterial growth was comparable between single and co-
culture setups (data not shown). In single culture, Bb produced high acetate levels, especially in a matrix
with intact protein (Figure 1B; open bars). Acetate production was independent of GOS availability, as
shown by the non-significant differences between iPF and iPF + GOS setups. Co-culture of Bb and Lf
showed the lowest acetate levels in all tested formula setups and time points (filled bars).

In contrast, propionate production was significantly higher in the presence of GOS, when Bb and Lf were
added in a 1:1 ratio (filled bars), independent of the formula matrix [Figure 1C]. Butyrate, however, was
highest in formula without prebiotic or eHF, especially for Lf [Figure 1D]. Lactate formation was highest in
the eHF + GOS matrix [Figure 1E].

The production of SCFA and microbial fermentation can be associated with gas formation in vitro, as
shown for Lf in single culture [Figure 1F]. Interestingly, the combined growth with Bb significantly
abrogated the amount of gas detected after 48 h, despite growth and associated gas production of Lf in a
matrix with iPF. This effect was specific for the iPF only, as gas production in the eHF is not diminished,
probably due to a massive decrease in the growth of Bb in eHF + GOS at this time point [Figure 1A].

It could be shown that in vitro, both strains were growing well in single and co-cultured conditions. The
presence of prebiotics and the protein form influenced the growth and metabolic activity of the probiotic
strains in vitro. Culturing the strains in single- and co-cultured conditions indicated the presence of cross-
feeding mechanisms, as seen in different levels of SCFA and gas volume. Therefore, probiotic activity and
abundance of Lf and Bb in the presence of a complex microbiota were assessed in the SHIME model.
®