Phosvitin Extraction from Egg Yolk and Its Potential as a Functional Food Ingredient for Improving Bone Health

Abstract: Osteoporosis is a common disease afflicting a quarter of the population of 50 years old and above worldwide. The osteoporotic fracture brings great pain/inconvenience to the patients, and sometimes causes immobility or even mortality. Drugs are available for treating osteoporosis but are associated with serious side-effects. There is an interest in searching food components for improving bone health. Egg yolk phosvitin is one of the most phosphorylated proteins in nature with potential for bone health. The overall objectives of this thesis were to develop a scalable method of phosvitin extraction and to study the effects of phosvitin and phosvitin hydrolysate on bone cells as well as osteoporosis rat model. Since inflammation stimulates bone cells and causes bone loss, the effects of phosvitin and phosvitin hydrolysate on bone cells under inflammatory stimulation were investigated. The first experiment studied the effects of pH, low centrifugal force, use of industrial separators and different water dilution factors on the extraction. The results suggested that decreasing pH from neutral to acidic range and increasing water dilution ratio from 1/1 to 1/4 (yolk/water, weight/weight) increased phosvitin purity and recovery. Low centrifugal force resulted in low phosvitin recovery, although the purity remained at high level. None of the industrial separators (decanter and disc stack separators) were sufficient to separate pellet from egg yolk and thus resulted in significantly lower yield (~20-30%), while the purity was comparable (~70%). Phosvitin and its hydrolysate promoted osteoblast differentiation by upregulating expression of runt-related transcription factor 2 (RUNX2), alkaline phosphatase, osteocalcin production and collagen synthesis. Tumor necrosis factor alpha (TNF-ɑ) induced production of inflammatory proteins including regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1) and cyclooxygenase-2 (COX-2). Phosvitin and phosvitin hydrolysate inhibited the production of these proteins in osteoblast cells. In another experiment, both phosvitin and phosvitin hydrolysate inhibited receptor activator of nuclear factor kappa-Β ligand (RANKL) induced osteoclastogenesis in RAW264.7 cells. Inflammatory biomarkers, such as TNF-ɑ, MCP-1, RANTES, and inducible nitric oxide synthases (iNOS), were also significantly suppressed by phosvitin and phosvitin hydrolysate. In the animal study with ovariectomized (OVX) rats, 2% phosvitin hydrolysate in the diet improved bone micro structure and prevent bone loss measured by Micro computed tomography. Higher concentrations of serum osteocalcin (OCN) and N-terminal propeptide of type 1 collagen (P1NP) and lower serum Tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) were also detected in the same animal group, indicating the activities of phosvitin hydrolysate might be a combination of both anabolic and anti-resorptive effects. The findings of this thesis suggest that phosvitin and phosvitin hydrolysate have good potential to be incorporated into functional foods to reduce bone loss and/or promote bone growth.