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The purpose of this study was to examine which component in the microbial protease-resistant fraction of Katsuobushi (KBR), smoke-dried bonito, is hypocholesterolemic in ovariectomized rats (OVX-rats). KBR contains two major components: oil and protease-resistant protein. Oil extracted from KBR (EX) was rich in palmitic, oleic and docosahexaenoic acids. OVX-rats were fed one of the following diets for 28 d: diets containing casein as the protein source (C or C+EX diet), a diet containing KBR as the protein source (KBR diet) or diets containing degreased KBR as the protein source (DF/KBR or DF/KBR+EX diet). The C and DF/KBR diets contained soybean oil as the oil source. In the C+EX, KBR and DF/KBR+EX diets, soybean oil was replaced by oil extracted from KBR (EX). Plasma total- and low density lipoprotein-cholesterol concentrations in the C+EX, KBR and DF/KBR+EX groups, but not in the DF/KBR group, were significantly lower than that in the C group. Fecal bile acid excretion was significantly greater in the C+EX, KBR, DF/KBR and DF/KBR+EX groups in comparison to the C group, whereas excretion in the KBR and DF/KBR+EX groups was significantly greater than in the C+EX and DF/KBR groups. Cholesterol 7alpha-hydroxylase activity was higher in the C+EX, KBR, DF/KBR and DF/KBR+EX groups than the C group. In OVX-rats fed C, C+EX or KBR for 28 d, bile acid flux into the small intestine increased in KBR and C+EX groups in comparison to the C group. The hypocholesterolemic effect of KBR in OVX-rats reflected in increased fecal bile acid excretion may be mediated by increased bile acid flux caused by EX and the binding of bile acids by protease-resistant proteins.