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Klaassens et al. Microbiome Res Rep 2024;3:38 https://dx.doi.org/10.20517/mrr.2024.13 Page 15 of 19

Deciphering the presence of a probiotic strain in a complex infant gut microbiome at strain-level resolution
[50]
is a challenge. In a previous study on the development of the infant microbiota , analysis of strain-level
resolution was used to demonstrate the transfer of microbes between mother and infant. Strain-level
taxonomic profiling is also important for the medical and pharmaceutical field, since research has shown
that strains belonging to the same species can have very different phenotypes and functional properties [51-53] .
Finally, the food industry has a high interest in analyzing ecosystem data on a strain-level resolution to
demonstrate the possible success of a probiotic product and its temporal persistence in the gut. The use of a
validated infant-specific database for taxonomic profiling of the samples made it possible to identify
members of the microbiota at the strain level. This knowledge is important for the development of health-
promoting probiotics and the evaluation of their performance in different matrices.

Infant formula based on partially or extensively hydrolyzed protein has been used for many years for infants
at risk of allergy development who cannot be exclusively breastfed [12,54-56] . However, there is still uncertainty
[56]
regarding the mechanism behind this assumed protection . Recently, the presence of already pre-
hydrolyzed protein in the IF raised the question of an effect on the overall microbial ecosystem and
[57]
subsequent effects on the immune maturation of the infants . Especially for infants in their first year of life,
the microbiota is still developing and has not yet reached an equilibrium. Therefore, a clear definition of a
healthy microbiota for this age group is difficult. However, bacterial species from the family of
Lactobacillaceae and Bifidobacteriaceae are assumed markers of a healthy microbiota and are often added to
infant formula or food supplements [58-60] . While the strain-specific effects of probiotics are well known,
knowledge about the effect of the surrounding - i.e., co-administered - matrix is scarce. IF with hydrolyzed
protein is tested for its suitability to support infant’s growth, as well as putative allergy preventative effects.
Recently, the allergenic activity of Lf in the respective formulae was tested in vitro in a basophil activation
experiment, revealing attenuated basophil degranulation in combination with eHF. As this attenuation was
absent in the combination with iPF, the protein matrix might have a significant effect on the metabolic
activity and survival of probiotic strains .
[17]

The present study showed that the eHF induced only minor effects on the overall composition of the
bacterial ecosystem in the ex vivo model. One reason might be the presence of prebiotic GOS in both
matrices, which might be overriding any minor effects based on the hydrolyzed protein per se. A recent
study by Heppner et al. also showed the more pronounced effect of GOS supplementation compared to
probiotic supplementation .
[61]
There are several limitations to be considered when using ex vivo models with complex microbial
ecosystems and food matrices to characterize the effects of food composition on the gut ecosystem and host
health. The biggest limitation can be seen in the one-time dosage of the probiotics as well as infant formulae
to the ex vivo model. While gut models surpass the issue of compliance and confounders of human
intervention trials, the long-term effects of repeated and continuous application of pro- and prebiotics are
still a challenge. Therefore, assessing higher numbers of infant microbiota donors and assessing them at
several different time points and after repeated application of the infant formula might have given a more
realistic model and should be tested in the future. The infant gut microbiota is known to be very variable, so
a larger number of donors would be needed, as each infant has an individual response toward the different
probiotics and protein matrices used in this study. Particularly at this early age, the mode of birth can also
have a significant effect on the composition of the microbiome and, therefore, on probiotic colonization and
activity. Therefore, a larger number of infants born either vaginally or by CS would be needed to elucidate
the mode-specific effects in order to draw more reliable conclusions. In addition, a greater range in age of
the infants providing the donor microbiota and more variety in their feeding history (i.e., exclusively