Osteoporosis is a major skeletal disease associated with estrogen deficiency in postmenopausal women. Kefir-fermented peptides (KPs) are bioactive peptides with health-promoting benefits that are produced from the degradation of dairy milk proteins by the probiotic microflora in kefir grains. This study aimed to evaluate the effects of KPs on osteoporosis prevention and the modulation of the composition of the gut microbiota in ovariectomized (OVX) mice. OVX mice receiving an 8-week oral gavage of 100 mg of KPs and 100 mg of KPs + 10 mg Ca exhibited lower trabecular separation (Tb. Sp), and higher bone mineral density (BMD), trabecular number (Tb. N) and bone volume (BV/TV), than OVX groups receiving Ca alone and untreated mice, and these effects were also reflected in bones with better mechanical properties of strength and fracture toughness. The gut microbiota of the cecal contents was examined by 16S rDNA amplicon sequencing. α-Diversity analysis indicated that the gut microbiota of OVX mice was enriched more than that of sham mice, but the diversity was not changed significantly. Treatment with KPs caused increased microbiota richness and diversity in OVX mice compared with those in sham mice. The microbiota composition changed markedly in OVX mice compared with that in sham mice. Following the oral administration of KPs for 8 weeks, the abundances of Alloprevotella, Anaerostipes, Parasutterella, Romboutsia, Ruminococcus_1 and Streptococcus genera were restored to levels close to those in the sham group. However, the correlation of these bacterial populations with bone metabolism needs further investigation. Taken together, KPs prevent menopausal osteoporosis and mildly modulate the structure of the gut microbiota in OVX mice.
Introduction
Kefir is an acidic and alcoholic fermented milk product with multiple health‐promoting benefits. A previous study demonstrated that kefir enhanced calcium absorption in intestinal Caco‐2 cells. In this study, kefir‐fermented peptide‐1 (KFP‐1) is isolated from the kefir peptide fraction, and its function as a calcium‐binding peptide is characterized.
Methods and Results
KFP‐1 was identified as a 17‐residue peptide with a sequence identical to that of κ‐casein (residues 138–154) in milk protein. KFP‐1 is demonstrated to promote calcium influx in Caco‐2 and IEC‐6 small intestinal cells in a concentration‐dependent manner. TRPV6, but not L‐type voltage‐gated calcium channels, is associated with the calcium influx induced by KFP‐1. An in vitro calcium binding assay indicates that the full‐length KFP‐1 peptide has a higher calcium‐binding capacity than the two truncated KFP‐1 peptides, KFP‐1∆C5 and KFP‐1C5. Alexa Fluor 594 labeling shows that KFP‐1 is taken up by Caco‐2 cells and interacts with calcium ions and TRPV6 protein. Moreover, KFP‐1 is found moderately resistant to pepsin and pancreatin digestions and enhanced calcium uptake by intestinal enterocytes in vivo.
Conclusion
These data suggest that KFP‐1, a novel calcium‐binding peptide, binds extracellular calcium ions and enters Caco‐2 and IEC‐6 cells, and promotes calcium uptake through TRPV6 calcium channels. The present study is of great importance for developing kefir‐derived metal ion‐binding peptides as functional nutraceutical additives.
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