Protocol detail

Analyzing Cecal Microbiome and Metabolites

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Bacterial genomic DNA was extracted from cecal digesta using an Isofecal DNA extraction kit (Nippon Gene, Co., Ltd., Tokyo, Japan) according to the manufacturer's instructions. The cecal microflora was analyzed using a terminal restriction fragment length polymorphism method as described previously (18 (link)). Cecal organic acids were measured as described previously (19 (link)). Fecal acidic sterols were analyzed using an internal standard (nor-deoxycholic acid; Steraloids, Wilton, NY, USA) by gas chromatography as described previously. The total IgA concentration in feces was measured using an ELISA quantitation kit (Bethyl Laboratories Inc., Montgomery, TX, USA). Mucins were extracted according to the method of Bovee-Oudenhoven et al (20 (link)) and quantitated using a fluorometric assay (21 (link)). The activities of harmful fecal enzymes, such as tryptophanase, β-glucuronidase and β-glucosidase, were determined as described previously (22 (link)).

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YANG Y., SITANGGANG N.V., OKAZAKI Y., TOMOTAKE H., ARITA K., ASHIDA T, & KATO N. (2015). Supplemental fermented plant product (‘Manda Koso’) reduces succinate and deoxycholate, as well as elevates IgA and mucin levels, in rats fed a high-fat diet. Biomedical Reports, 3(6), 787-791.

Publication 2015

ELISA quantitation kit

Acidic Assay Bacterial dna Cecal Deoxycholic acid Elisa Enzymes activities Fecal Fluorometric Gas chromatography Gene Genomic Glucuronidase Mucins Restriction fragment length polymorphism Sterols Tryptophanase β glucosidase

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Variable analysis

independent variables

Bacterial genomic DNA extraction method (Isofecal DNA extraction kit)
Terminal restriction fragment length polymorphism (T-RFLP) method for analyzing cecal microflora
Measurement of cecal organic acids
Analysis of fecal acidic sterols using an internal standard (nor-deoxycholic acid) and gas chromatography
Measurement of total IgA concentration in feces using an ELISA quantitation kit
Extraction and quantitation of mucins using a fluorometric assay
Determination of activities of harmful fecal enzymes (tryptophanase, β-glucuronidase, and β-glucosidase)

dependent variables

Cecal microflora composition
Cecal organic acid levels
Fecal acidic sterol levels
Total IgA concentration in feces
Mucin levels
Activities of harmful fecal enzymes (tryptophanase, β-glucuronidase, and β-glucosidase)

control variables

Not explicitly mentioned

controls

No positive or negative controls were explicitly mentioned in the provided information.

Annotations

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