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Page 2 of 19 Klaassens et al. Microbiome Res Rep 2024;3:38 https://dx.doi.org/10.20517/mrr.2024.13

Results: Both protein forms (i.e., intact or extensively hydrolyzed protein) in IF supported infant gut microbial
metabolic activity equally, as evidenced by similar levels of short-chain fatty acids (SCFAs). Interestingly, gut
microbial metabolic activity was found to be differently activated in a strain-dependent manner. Taxonomic
profiling of the microbiome at the strain level enabled monitoring of the prevalence and abundance of both
probiotic strains, even in a complex ecosystem.

Conclusion: Food matrix and host microbiota interactions should be considered when evaluating strain-specific
probiotic effects in the future.

Keywords: Infant microbiome, metagenomics, probiotics, hydrolyzed, Limosilactobacillus, Bifidobacterium

INTRODUCTION
In the first days of life, neonates are rapidly colonized by different microorganisms at different body sites.
The composition of newly formed microbial ecosystems is mostly influenced by the mode of birth,
[1,2]
administration of antibiotics, and nutrition . By disturbing the microbiota in this early time, the initiation
of a physiologic interaction of colonizing microorganisms with the infant’s immune system might be off-
balanced , and consequently, the risk of infections and allergies might increase .
[4]
[3]
Human milk (HM) is the best nutrition for infants as it ensures optimal growth and development, while
also supporting the infant’s microbiota maturation with its probiotic and prebiotic compounds . Breastfed
[5]
infants have a lower risk for infections, metabolic diseases, and other pathologies, presumably also
attributed to an age-tailored microbiota . The composition of HM macronutrients, micronutrients, and
[6]
[7-9]
microorganisms changes over the lactation period . HM is also a source of microorganisms that can
subsequently be found in the infant’s intestinal microbiota. However, the extent to which these HM-derived
strains contribute to the infant’s microbiota over time is still under debate . This might also be due to
[10]
missing tools to follow these microorganisms at the strain level.

When exclusive breastfeeding is not possible, infant formula (IF) is a dietary substitute for infants. The IF
aims to support the nutritional needs, as well as the development of the infant and its developing intestinal
microbial ecosystem. Adding probiotic and prebiotic compounds to IF is a possible way to enhance the
healthy development of the infant gut microbiota. The safety of the probiotic strain has to be proven and the
beneficial effects should be traced back to the specific strain(s) . When exclusive breastfeeding is not
[11]
possible, and the infant is at higher risk for allergy development (e.g., by parental predisposition), some
guidelines recommend feeding an IF with hydrolyzed protein as a possible substitute . Recently,
[12]
compounds in IF other than probiotics and prebiotics, such as hydrolyzed protein, were studied for their
effect on the infant’s microbiota or immune system [13-17] . Additionally, the effect of different IF matrices on
the respective formulas’ allergenic activity was tested in an in vitro basophil activation experiment. This
experiment revealed that probiotic-induced reduction in basophilic degranulation was strongest when the
probiotic strain was combined with extensively hydrolyzed protein IF. Thus, these results demonstrate that
the protein matrix can have a significant effect on the viability and metabolic activity of the probiotic
strains .
[17]
Being aware of the complex dynamics between the gut microbiota, probiotics and macronutrients such as
proteins, we investigated the effects of IF based on extensively hydrolyzed protein and IF with intact (i.e.,
non-hydrolyzed) proteins, as well as their interactions and colonization benefits to the added probiotics
Bifidobacterium breve (B. breve) DSM 32583 (Bb) and Limosilactobacillus fermentum (L. fermentum) CECT
5716 (Lf). The two probiotics isolated from HM were studied in vitro for their growth and metabolic activity