The functional capacity of the microbial community was estimated from the 16S rRNA sequence results with the PICRUSt software (version 1.1.4, https://picrust.github.io/picrust/index.html )(32 (link)) using reference genomes to predict functional pathways for experimental groups that showed significantly altered tissue strength. In our previous study, vitamin K was linked to decreased tissue quality(4 (link)), and therefore PICRUSt results were analyzed using a biased pathway-focused approach focused on vitamin K biosynthesis pathways.
Cecal vitamin K content (phylloquinone and menaquinones 4–13 (MK4-13)) was measured and characterized (n=6/group, the same animals used for fecal microbiota analysis) using high-performance liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS), previously described(33 ). The characterization system consisted of an Agilent 6130 Quadrupole MSD with an APCI source connected to an Agilent series 1260 HPLC instrument (Agilent Technologies, Santa Clara, CA, USA). The limit of detection for phylloquinone and each type of menaquinone were as followed: phylloquinone and MK4, 30 pmol/g; MK6, 10 pmol/g; MK5, MK7-MK9, MK11-13, 5 pmol/g; MK10, 1 pmol/g. For findings below the detection limit a value of one half the detection limit was used when making statistical comparisons.
Cecal vitamin K content (phylloquinone and menaquinones 4–13 (MK4-13)) was measured and characterized (n=6/group, the same animals used for fecal microbiota analysis) using high-performance liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS), previously described(33 ). The characterization system consisted of an Agilent 6130 Quadrupole MSD with an APCI source connected to an Agilent series 1260 HPLC instrument (Agilent Technologies, Santa Clara, CA, USA). The limit of detection for phylloquinone and each type of menaquinone were as followed: phylloquinone and MK4, 30 pmol/g; MK6, 10 pmol/g; MK5, MK7-MK9, MK11-13, 5 pmol/g; MK10, 1 pmol/g. For findings below the detection limit a value of one half the detection limit was used when making statistical comparisons.
