Bone tissue maintains a dynamic balance between bone resorption by osteoclasts and bone formation by osteoblasts. Disruption of this balance leads to osteoporosis, a condition characterized by decreased bone mass and deterioration of bone microarchitecture, significantly increasing fracture risk, particularly in postmenopausal women. As global populations age, osteoporosis is becoming an increasingly significant public health challenge. Dietary factors play an important role in bone health, and fermented foods are gaining attention due to their rich content of bioactive compounds. This study systematically investigated the effects of two traditional Japanese fermented food products—sake lees and rice koji extracts—on bone metabolism using both in vitro cell models and a postmenopausal osteoporosis mouse model. The results indicate that these extracts promote osteoblast differentiation, enhance alkaline phosphatase activity and collagen accumulation, and suppress osteoclast differentiation. Furthermore, both interventions alleviated bone loss in ovariectomized mice. These findings suggest that components present in sake lees and rice koji may offer potential as nutritional strategies for preventing or managing osteoporosis, although further research is required to clarify their active compounds and optimal application.
Background and Experimental Design
Bone tissue is a dynamic equilibrium system in which osteoclasts are responsible for bone resorption, while osteoblasts promote bone formation. Osteoporosis occurs when this balance is disrupted, leading to reduced bone mass and deterioration of bone microstructure, thereby increasing the risk of fractures, especially among postmenopausal women.
With population aging, the number of osteoporosis patients is increasing each year, making it a major public health challenge. Diet is an important factor influencing bone health, and fermented foods have attracted growing attention because they contain abundant bioactive substances. In the traditional Japanese diet, sake lees and rice koji, as fermentation products, are not only rich in proteins, vitamins, and minerals, but also contain key metabolites such as folate and S-adenosylmethionine (SAM), which may exert bone-protective effects by regulating bone cell function.
This study systematically explored for the first time the effects of extracts from two traditional Japanese fermented foods—sake lees and rice koji—on bone metabolism. Using in vitro cell models and a postmenopausal osteoporosis mouse model, the study found that both extracts promoted osteoblast differentiation, increased alkaline phosphatase (ALP) activity and collagen accumulation, and simultaneously inhibited osteoclast differentiation. These effects effectively slowed bone loss in mice, suggesting that they may serve as novel nutritional interventions for the prevention and treatment of osteoporosis.
The research team obtained sake lees and rice koji from the Hakusan Sake Brewery in Japan and prepared extracts for both cellular and animal experiments.
In the in vitro experiments, mouse osteoblast precursor cells MC3T3-E1 were used to measure ALP activity, collagen accumulation, and mineralization levels, while Western blot analysis was performed to examine the expression of osteogenesis-related proteins Hsp47 and Sec23IP.
For the osteoclast model, RAW264.7 cells were used to evaluate the effects of the extracts on osteoclast differentiation and cell viability. In the in vivo experiments, 12-week-old female C3H/HeJ mice underwent ovariectomy (OVX) to simulate postmenopausal osteoporosis. The animals were then given diets containing freeze-dried sake lees or orally administered rice koji extract. After four weeks, micro-computed tomography (micro-CT) was used to analyze bone microstructural parameters of the distal femur.
Effects on Osteoblast and Osteoclast Activity
The results showed that extracts of sake lees and rice koji significantly increased ALP activity, collagen accumulation, and mineralization in MC3T3-E1 cells. The protein levels of Hsp47 and Sec23IP were also increased, indicating enhanced collagen maturation and secretion. Both extracts significantly inhibited the differentiation of RAW264.7 cells into osteoclasts, while having limited effects on cell viability.
Effects of sake lees extract or rice koji extract on osteoblast differentiation
In the OVX mouse model, diets containing 20% and 40% freeze-dried sake lees significantly suppressed the decline in bone volume fraction (BV/TV) and trabecular volume (Tb.V), while the 40% group also slowed the loss of trabecular thickness (Tb.Th). Oral administration of rice koji extract similarly suppressed decreases in BV/TV, Tb.V, and trabecular number (Tb.N). In cellular experiments, folate and SAM promoted collagen secretion from osteoblasts, suggesting that they may represent active components responsible for the effects of sake lees.
Effects of sake lees extract or rice koji extract on osteoclast viability and differentiation
Overall, the study demonstrates that extracts of sake lees and rice koji not only enhance osteoblast function but also inhibit osteoclast formation, thereby helping restore the balance of bone remodeling and slowing the progression of osteoporosis. The upregulation of Hsp47 and Sec23IP indicates that the extracts promote the correct folding and secretion pathway of collagen from the endoplasmic reticulum to the Golgi apparatus, thereby improving the efficiency of bone matrix formation. In animal experiments, freeze-dried sake lees and rice koji improved trabecular bone microstructure, demonstrating their bone-protective potential, particularly in the early stages of osteoporosis.
Folate and SAM, as central components of one-carbon metabolism, participate in DNA methylation and protein modification processes and regulate osteoblast differentiation, suggesting that they are key active substances. Although the observed effects were slightly weaker than those of traditional isoflavones, the composition of sake lees and rice koji extracts is complex and may involve synergistic actions of multiple components. Future studies are needed to further identify active compounds and optimize dosage and administration methods.
This study also has several limitations, including a limited sample size, incomplete standardization of extract composition, and the absence of dynamic bone histomorphometry analysis. In addition, the bones of 12-week-old mice are not yet fully mature, which may influence experimental outcomes. Future research is recommended to employ mature animal models, extend observation periods, and incorporate measurements of bone formation and bone resorption rates to comprehensively evaluate their effects on bone metabolism.