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Sasaki et al. Microbiome Res Rep 2023;2:12 https://dx.doi.org/10.20517/mrr.2023.08 Page 11 of 15

Figure 5. In vitro assimilation test of β-L-Arap-(1→3)-L-Ara and larch AGP. Bifidobacterium pseudocatenulatum (B. pse) and B.
kashiwanohense (B. kas) strains were used for the in vitro assimilation test. A: Gene clusters containing AAfase in Bifidobacterium species
used in the growth test (left). Amino acid sequence identity and coverage between B. pseudocatenulatum MCC10289 and other strains
using sequences from B. pseudocatenulatum MCC10289 as a query sequence (right table); B: The growth profile of β-L-Arap-(1→3)-L-
Ara (left) (n = 1) and larch AGP (right) (n = 3). The absorbance of media of β-L-Arap-(1→3)-L-Ara was monitored at 0, 17, 24, 41, and
48 h, and that of larch AGP was monitored at 0, 18, 24, 42, and 48 h. The absorbance of the growth medium was calculated by
subtracting the absorbance of the medium in the absence of bacteria. Error bars indicate standard deviation (n = 3). The black triangle
indicates the sample collection point for residual sugar analysis via HPAEC-PAD or TLC; C: HPAEC-PAD analysis of the culture
supernatant of β-L-Arap-(1→3)-L-Ara after incubation for 41 h. β-L-Arap-(1→3)-L-Ara was used as a standard.

To examine bacterial AAfase activity, the precipitates of bacterial cells and supernatant of the culture
medium containing larch AGP were collected and incubated with larch AGP [Supplementary Figure 6].
TLC analysis showed bacterial AAfase activity in the supernatant of B. pseudocatenulatum MCC10289 but
not in the precipitate, indicating that AAfase is a secreted enzyme. This finding is reasonable based on the
predicted domain structure of the enzyme with the SP sequence but no transmembrane domain
[Figure 1B]. No enzyme activities were detected using other AAfase-carrier strain bacterial cells or
supernatant.