Differences in non-probiotic fed and probiotic fed infants

One of the main goals of this study was to reveal marked differences in the fecal metabolome between the infants, consuming the interventional formula or the placebo formula, which might be indicators for similar effects between breast milk and probiotic formula. Therefore, in this chapter the focus was to evaluate and to find differences of the two formula-fed groups, including the breastfed group. The breastfed group was considered as well to be able to compare the two formula diets to breastfeeding and to find possible similarities between the breastfed and interventional formula-fed group. This may lead to an approximation of the fecal metabolome due to probiotics towards the breastfed infants.

Nevertheless, mass signals similarly impacted through the interventional formula and breast milk or the placebo formula and breast milk were taken into account as well.

Already the previously performed month by month PLS-DA (Chapter 3.4.2.2) revealed some mass signals to be affected by either interventional formula or placebo formula. In order to detect further mass signals to be affected by the two formulas differently, both groups were evaluated by month taking the matrix of each month separately of the PCA plots in Figure 3.4-4 and Figure 3.4-5 respectively. Several mass signals were observed to be altered differently in the placebo or the probiotics group. For identification (+) TOF MS/MS experiments of all F+ and F- discriminating metabolites were performed, whereas only one could be identified. This comprised one mass signal, which was increased in the placebo formula group (F-) and was detected in both ionization modes. Namely the mass signal at retention time 6.1 minutes with m/z 440.2844 in (+) ESI mode and m/z 438.2606 in (-) ESI mode.

Through the annotation and following MS/MS experiments, it could be constrained to a lysophosphatidylethanolamines (LysoPE) 15:0/0:0 (Bazanella et al. 2017), a glycerophospholipid derived from phosphatidyethanolamindes through partial hydrolysis of one fatty acid group (Gregory et al. 2013). The experimental MS/MS spectrum is shown in Figure 3.4-20 and was compared manually against the METLIN database with an error of 0.01 Da. The function of LysoPE (15:0/0:0) in the human gut microbiome of infants and the role in exclusively formula fed without probiotics in comparison to probiotic formula fed infants remains unclear.

However, it was observed that the intensity level in the F+ group was more similar to the breastfed one than the F- group. LysoPE 15:0/0:0 (Bazanella et al. 2017) was only significantly changed in month 1 and in no other months between F+ and F- up to month 12.

Figure 3.4-20: Lysophosphatidylethanolamine LysoPE(15:0) significantly increased in F- infants.

Boxplot of (A) Lysophosphatidylethanolamine LysoPE(15:0) significantly increased (B vs. F-: p-value = 0.00033, F- vs. F+: p-value = 0.09) in infants fed with the placebo formula (red) compared to breast fed (blue) and interventional formula-fed (green) infants, analyzed in (+) ToF-MS. B: The experimental (+) TOF MS/MS spectra of m/z 440.2844, collision energy 20 eV.

Another two unknown metabolites could be observed, whereas the mass signal m/z 644.4008 was significantly increased in month 1 and month 3 in the F- group, as well as m/z 445.3707 in month 1 (Figure 3.4-21 A/B/C). The mass signal with m/z 445.3707 showed an increase over time in both formula-fed groups, but was not significantly different anymore between those two groups after month 1. Again, it was remarkable that the intensity levels of all three mass signals in the probiotics group were similar to the breastfed group, whereas the intensities in the placebo group were rather high.

The mass signal m/z 510.3339 was discriminative for the F+ group (Figure 3.4-21 D, MS/MS spectra in Figure 5.2-10). Also, m/z 537.3863 showed increased intensities in the F+ group, even if the changes are not significant. However, this two mass signals, changed in the F+ group showed similar fragmentation patterns (MS/MS spectra in Figure 5.2-10), assuming they are structure-related compounds.

0 1.0 2.0 3.0

Intensity x 10⁵

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155.0103 216.0629

268.2636 299.2583

379.2240 440.2766

0 20 40 60 80 100 Intens. [%]

100 200 300 400 m/z

422.2658

Figure 3.4-21: Mass signals at early life significantly changed through placebo or interventional formula.

Boxplots of mass signals significantly increased in placebo formula-fed (A, B, C, red) infants or interventional fed ones (D, E, green) compared to breast fed infants (blue), analyzed in UHPLC-(+)-ToF-MS mode. A/B: m/z 644.4008 significantly increased in F- fed infants (# p-value < 0.027) in month 1 and 3. C: m/z 445.3707 significantly increased in F- fed infants (# p-value < 0.05). D: m/z 510.3339 significantly increased in F+ fed infants (* p-value < 0.042) and E: m/z 537.3863 (*p-value < 0.022). Significance was tested through the post hoc Kruskal-Nemenyi test; further details are listed in Table 6.2-18.

Further mass signals differing between the probiotics group and the placebo group were observed in the negative ionization mode. This comprises LysoPE (15:0/0:0), which already was identified in the positive ionization mode and increased in infants fed with the placebo formula. Further unknown metabolites with m/z 516.3156, m/z 573.3827 were significantly changed in the F- group. One mass signal with m/z 541.3335 was increased in the F+ group. All significant metabolites for either the F+ or the F- group were only increased in month 1 – except m/z 644.4008 ((+) ESI). All shown metabolites revealed a clear distinction between the two formula-fed groups, and as time passed, those metabolites decrease in the groups or level with the breastfed and/or probiotics group. Additionally, it could be observed that some of those metabolites showed similar patterns in the breastfed and the interventional formula-fed group. This observation could lead to assume that the probiotic formula had the same impact on specific fecal metabolites as breast milk.

Intensity 10⁴

breastfed F- Placebo formula F+ Interventional formula

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#

*

Month 1 Month 3 Month 1

0

Figure 3.4-22: Mass signals at month 1 significantly changed through placebo or interventional formula.

Boxplots of mass signals significantly increased in placebo formula-fed (A, B, C, red) infants or interventional fed ones (D, green) compared to breast fed infants (blue), analyzed in UHPLC-(-)-ToF-MS mode. A: m/z 438.2606 significantly increased in F- fed infants (# p-value < 0.005) in month 1. B: m/z 516.3154 significantly increased in F- fed infants (# p-value < 0.029). C: m/z 573.3827 significantly increased in F- fed infants (# p-value < 0.024) and D: m/z 541.3335 significantly increased in F+ fed infants (*p-value < 0.061). Significance was tested through the post hoc Kruskal-Nemenyi test; further details are listed in Table 6.2-18.

Unfortunately, it was not possible to identify the other unknown metabolites or to compare the experimental MS/MS spectra with any database. All mass spectra were compared against METLIN, HMDB and in-silico MS/MS platforms (e.g. MetFrag (Ruttkies et al. 2016)), but did not provide sufficient information for classification or either identification. The applicable experimental MS/MS spectra are displayed in the supplement in chapter 5.2.1.4.

Several changes significantly differed in the F+ and the F- groups were also observed later on in life.

This included the mass signals with m/z 378.2958 (month 5) significantly increased in the interventional group (Figure 3.4-23 C). On the contrary, mass signals with m/z 403.2689 (month 9) and m/z 298.1122 (month 12) were significantly increased in the placebo group (Figure 3.4-23 A, B), but their identity

breastfed F- Placebo formula F+ Interventional formula

m/z 516.3154 m/z 573.3827 m/z 541.3335 m/z 438.2606

#

#

#

*

Figure 3.4-23: Mass signals significantly changed through placebo or interventional formula later in life.

Boxplots of mass signals significantly increased in placebo formula-fed (A, B, red) infants or interventional fed ones (C, green) compared to breast fed infants (blue), analyzed in UHPLC-(-)-ToF-MS mode. A: m/z 403.2689 significantly increased in F- fed infants (# p-value < 0.038) in month 1. B: m/z 298.1122 significantly increased in F- fed infants (# p-value < 0.032). C: m/z 378.2958 significantly increased in F+ fed infants (# p-value < 0.033).

Significance was tested through the post hoc Kruskal-Nemenyi test; further details are listed in Table 6.2-18.

To conclude, in the first year of life, differences in the fecal samples of infants either fed with the placebo formula or the interventional formula could be observed, displaying a few mass signals, which were significantly altered through different formula. It was remarkable that the intensity levels of almost all mass signals in the probiotics formula group leads to an approximation towards the breastfed infants.

All those mass signals emerged as very important, wherefore not only the identification of those metabolites has top priority, but also further research and long-time studies are needed to evaluate the possible health promoting effect of probiotics on the development of the infant’s fecal metabolome.

Intensity x 10⁴

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Intensity x 10⁴

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breastfed F- Placebo formula F+ Interventional formula Intensity x 10⁴

3.4.4 Correlation studies between OTUs and fecal metabolites of breast- and formula-fed