Effect of high-hydrostatic pressure on the digestibility of egg yolk and granule.

The impact of high hydrostatic pressure (HHP) on protein digestibility of egg yolk and egg yolk granule was evaluated by static in vitro digestion using the standardized INFOGEST 2.0 method. The degree of hydrolysis (DH) and the phospholipid content were determined during digestion, and the protein and peptide profiles were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse phase-high pressure liquid chromatography (RP-HPLC). The results showed that HHP induced protein aggregation in egg yolk and granule, mainly by disulfide bridges, which were not disrupted in the oral phase. Proteolysis during the gastric phase improved egg yolk and granule protein solubility, regardless of whether HHP was applied. However, the extent of the samples' digestibility was not affected, with DH values ranging from 15% to 20%. During the intestinal phase, the DH of egg yolk protein (∼40%) was higher than that of the granule (∼25%), probably due to the denser structure of the granule reducing the accessibility of intestinal enzymes. The DH, peptide, and protein profiles of control and HHP-treated egg yolk showed similar protein digestion behaviors for both gastric and intestinal phases. Among the different proteins, only the digestibility of ß-phosvitin in HHP-treated granule was enhanced. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin with the potential to generate bioactive phosvitin-derived phosphopeptides. PRACTICAL APPLICATION: High hydrostatic pressure, mainly used as a preservation process, did not impair the nutritional quality of the egg yolk and granule proteins but improved the susceptibility of phosvitin (protein contained in egg yolk) proteolysis to produce bioactive phosphopeptides. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin.

Promoting effect of phosvitin in the mineralization of eggshell inner membrane with the application in osteogenic induction scaffold.

Exploring affordable and easily prepared inorganic-organic hybrid membrane materials has attracted a great interest in the bone repair field. This study is based on biomimetic mineralization technique to study the role of phosvitin (PV) in the mineralized process of eggshell inner membrane. Results showed that PV promoted the formation of hydroxyapatite on the eggshell inner membrane surface, and the phosvitin content in the simulated body fluid was decreased during the mineralization process. Besides, in vitro preosteoblast experiments indicated that mineralized membrane with PV exhibited more conducive to cell proliferation and differentiation than that mineralized membrane without PV. Interestingly, with the increase of mineralization time, the stimulating ability of mineralized membranes with PV on adhesion, proliferation, alkaline phosphatase activity and collagen type I content gradually improved. In summary, the eggshell inner membrane composites mineralized with PV obtained by biomimetic mineralization might be potential scaffold materials for bone repair.

Phosvitin-based hydrogels prepared in AmimCl under magnetic field treatment: Structural characteristics, biological functions, and application in skin wound healing.

Due to the serious bacterial infection of skin and the waste of petroleum-based materials, there is an urgent need to develop natural biodegradable wound dressings with high antibacterial activity. Phosvitin (PSV) has shown its natural antioxidant and antibacterial properties, making it an excellent material for preparing wound healing dressings. In this study, we investigated the effect of magnetic field on the preparation of PSV-Microcrystalline Cellulose (MCC) composite hydrogels in 1-Allyl-3-methylimidazolium chloride (AmimCl) system. The results showed that the prepared hydrogels exhibited homogeneous surface structure, suitable swelling capacity and elasticity modulus, and sufficient thermal stability. The excellent antibacterial and antioxidant activities of hydrogels were mainly resulting from AmimCl and PSV, respectively, and the properties were enhanced after magnetic field treatment. The proteomics analysis indicated that AmimCl can readily penetrate the biological membranes of Staphylococcus aureus (S. aureus), upsetting the metabolism and reducing the virulence. The hydrogels showed great blood compatibility. Compared with the commercial materials, the 5 mT-treated hydrogels presented a comparable wound healing rate in the full-thickness skin injury model. On day 7, the wound healing rate of the 5 mT group reached approximately 84.40 %, which was significantly higher than that of the control group, 72.88 % (P < 0.05). In conclusion, our work provides experience for the development of biodegradable materials combined in ionic liquids and magnetic field, and explores their applications in wound healing dressings.

Preparation, characterization, and bioavailability evaluation of antioxidant phosvitin peptide-ferrous complex.

BACKGROUND: Iron deficiency anemia (IDA) is one of the commonest global nutritional deficiency diseases, and the low bioavailability of iron is a key contributing factor. The peptide-iron complex could be used as a novel iron supplement to improve iron bioavailability. RESULTS: In this study, antioxidant low molecular weight (<3 kDa) phosvitin peptide (named PP-4) was separated to prepare a phosvitin peptide-ferrous complex (named PP-4-Fe); then the structural conformation of PP-4-Fe was characterized and its bioavailability by in vitro digestion was evaluated. The results showed that PP-4 had good ferrous-binding activity with 96.14 ± 2.86 µg Fe2+ mg-1 , and had a strong antioxidant effect with 995.61 ± 79.75 µmol TE mg-1 in 2,2'-azinobis'3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 62.3 ± 3.95 µmol FeSO4 mg-1 in ferric ion reducing antioxidant power (FRAP). After ferrous binding, the FRAP activity of PP-4-Fe, enhanced by 1.8 times, formed a more ordered structure with an increase in α-helix and decrease in γ-random coil. The ferrous binding sites of PP-4 involved were the amino, carboxyl, imidazole, and phosphate groups. The PP-4-Fe complex displayed excellent gastrointestinal stability and antioxidant effects during digestion. The iron dialysis percentage of PP-4-Fe was 74.59% ± 0.68%, and increased to 81.10% ± 0.89% with the addition of 0.25 times vitamin C (VC). This indicated that PP-4-Fe displayed excellent bioavailability and VC in sufficient quantities had a synergistic effect on improving bioavailability. CONCLUSIONS: This study demonstrated that antioxidant phosvitin peptide was an efficient delivery system to protect ferrous ions and suggested that the phosvitin peptide-ferrous complex has strong potential as a ferrous supplement. © 2023 Society of Chemical Industry.

The elastase and melanogenesis inhibitory and anti-inflammatory activities of phosvitin phosphopeptides produced using high-temperature and mild-pressure (HTMP) pretreatment and enzyme hydrolysis combinations.

This study aimed to determine the skin protective effect of egg yolk phosvitin phosphopeptides (PPPs). Phosvitin was separated from the egg yolk, and PPPs were produced using high-temperature and mild-pressure (HTMP) pretreatment and enzyme-sterilization hydrolysis combinations. The elastase and melanogenesis inhibitory activities and anti-inflammatory effects of egg yolk PPPs were determined. All PPPs significantly inhibited elastase activity, but the PPPs prepared with HTMP pretreatment and trypsin-sterilization (HTMP-T-S) combination suppressed the tyrosinase activity the most. PPPs (3 mg/mL) inhibited the α-melanocyte-stimulating hormone-induced melanin production in B16F10 melanoma cells by 31.18 to 38.58%. In addition, PPPs effectively inhibited nitric oxide (NO) production in the LPS (lipopolysaccharide)-stimulated RAW 264.7 macrophages, and the PPPs from HTMP-T-S exhibited the highest inhibitory activity. The protein expressions of pro-inflammatory enzymes, inducible nitric oxide synthase, and cyclooxygenase-2 were down-regulated by the PPPs from the HTMP-T-S. Therefore, PPPs could be used as an anti-melanogenic, anti-elastase, and anti-inflammatory agent for humans and skin care products.

Vitellogenin Ab structure of the amazonian Arapaima gigas.

The vitellogenin is composed by polypeptides that are precursors of egg yolk proteins that provides embryo and larvae nutrition. The mRNA encoding for vitellogenin Ab (Vtg-Ab; 4,536 bp long and 1,512 amino acids) were obtained by RNA-Seq library sequencing of pirarucu gonads. The Vtg-Ab sequences had high homology with Vtgs of other three teleosts species of the order Osteoglossiformes. The transcript of ovarian Vtg was identified based on structural criteria, and so we classify the Vtg of pirarucu as Vtg-Ab due to the truncated or shortened phosvitin (N-terminal end) and phosvitinless domain (C-terminal end). The Vtg-Ab of pirarucu present two major deletions with 133 amino acids in the Lipovitellin I domain and 89 amino acids in the truncated or shortened Phosvitin domain, both located in the N-terminal end region. The three-dimensional (3-D) structure Vtg-Ab protein shows the presence of a typical 4α-helices bundle protein that runs in anti-parallel. In general, the characterization of Vtg-Ab may be the useful elucidation of the hormonal regulation of vitellogenesis and improve the production of pirarucu for broodstock management in aquaculture and preparation of Vtg antibody production (species-specific) for sex identification.

Binding mechanism and bioavailability of a novel phosvitin phosphopeptide (Glu-Asp-Asp-pSer-pSer) calcium complex.

Phosvitin has excellent calcium binding capacity, related to its phosphopeptides. The phosphopeptides may be used as functional ingredients for improving calcium bioavailability, but the calcium-binding mechanism is unclear. In this study, a novel phosvitin phosphorylated pentapeptide (Glu-Asp-Asp-pSer-pSer, EDDpSpS) was selected to prepare an EDDpSpS calcium complex (EDDpSpS-Ca), and the calcium-binding mechanism and bioavailability investigated. The calcium-binding capacity of EDDpSpS was up to 468 ± 152.80 mg/g. Calcium ions prompted the folding of the EDDpSpS structure to form spherical nanoparticles. The calcium binding sites of EDDpSpS involved peptide bonds, carboxyl, amino, and phosphate groups. Molecular forces involved in these interactions were electrostatic in nature. Moreover, EDDpSpS-Ca had excellent bioavailability when compared to CaCO3, calcium lactate, and d-calcium gluconate. This study revealed the calcium-binding mechanism of phosvitin phosphopeptide, and suggested that EDDpSpS-Ca has the potential to be a novel, efficient, and promising calcium supplement.

Covalent and Noncovalent Complexation of Phosvitin and Gallic Acid: Effects on Protein Functionality and In Vitro Digestion Properties.

To investigate the effects of different binding modes on the structure, function, and digestive properties of the phosvitin (Pv) and gallic acid (GA) complex, Pv was covalently and noncovalently combined with different concentrations of GA (0.5, 1.5, and 2.5 mM). The structural characterization of the two Pv-GA complexes was performed by Fourier transform infrared, circular dichroism, and LC-MS/MS to investigate the covalent and noncovalent binding of Pv and GA. In addition, the microstructure of the two Pv-GA complexes was investigated by super-resolution microscopy and transmission electron microscopy. The particle size and zeta potential results showed that the addition of GA increased the particle size and the absolute potential of Pv. The determination of protein digestibility, polyphenol content, SH and S-S group levels, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and antioxidant capacity of the digests indicated that noncovalent complexes had greater antioxidant and protective effects on polyphenols. Molecular docking revealed that GA was conjugated with Pv through hydrogen bond interactions.

Interaction research of resveratrol and phosvitin based on fluorescence spectroscopy and molecular docking analysis.

Phosvitin (PV) is the main phosphoprotein in egg yolk, with the highest degree of phosphorylation known in nature. The PV and resveratrol (Res) can form a complex, thus effectively improve the solubility of Res. In this work, the interaction between Res and PV was investigated by the fluorescence spectroscopy and molecular docking. The fluorescence emission intensity of PV became weak along with a red shift when it interacted with Res and the antioxidant activity was enhanced. The quenching constants of the interaction systems were 1.12×104  M-1 and 9.40×103  M-1 at 25°C and 35°C, respectively, which indicated the presence of static quenching phenomena between them. The binding constant was 1.80×104  M-1 , and the number of corresponding binding sites was approximately equal to one. The thermodynamic results revealed the combination was spontaneous, and the change of enthalpy and entropy was ∆H = 53.50 kJ/mol, ∆S = 261.00 J/mol·K, respectively. It indicated that the interaction forces between Res and PV were mainly hydrophobic interaction and hydrogen bonding. Molecular docking showed the binding mode, which was consistent with the experiment results. The research on the interaction between Res and PV provided theoretical guidance for the application of Res in food. PRACTICAL APPLICATION: PV is the most highly phosphorylated protein in nature and has pro-calcium absorption effects. Res is a polyphenol with strong antioxidant and anti-inflammatory activity, but its poor solubility limits its application. In this study, the solubility of Res was considerably enhanced by compounding Res and PV, and the antioxidant activity of Res was well retained. It increases the value of Res in food and other applications and opens up new possibilities for processing and utilization of PV.

Reversible, ß-sheet-dependent self-assembly of the phosphoprotein phosvitin is controlled by the concentration and valency of cations.

The hyperphosphorylated protein phosvitin (PV) undergoes a pH-dependent transition between PII- and ß-sheet secondary structures, a process deemed crucial for its role in the promotion of biogenic apatite formation. The transition occurs surprisingly slowly (minutes to hours). This is consistent with a slow aggregation process involving ionic interactions of charged groups on the protein surface. Herein, we determined the associated transition pK values and time constants through matrix least-squares (MLS) global fitting of a series of pH- and time-dependent circular dichroism (CD) spectra recorded in the presence of different mono-, bi- and trivalent cations. Supporting our results with dynamic light scattering data, we clearly identified a close correlation of ß-sheet transition and the formation of small aggregates at low pH. This process is inhibited in the presence of all tested cations with the strongest effects for trivalent cations (Fe3+ and Al3+). In the presence of Ca2+ and Mg2+, larger higher-order particles are formed from PV in the ß-sheet conformation, as identified from the interpretation of differential scattering observed in the CD spectra. Our observations are consistent with the existence of a multi-step equilibrium between aggregated and non-aggregated species of PV. The equilibrium is highly sensitive to the environment pH and salt concentration with exceptional behavior in the presence of divalent cations such as Ca2+ and Mg2+.

In vitro and in vivo wound healing-promoting activities of phosvitin-derived peptide Pt5-1c.

The closure of skin wounds is indispensable for resistance against pathogens, and fibroblast plays a critical role in skin wound healing. Our previous study demonstrates that the phosvitin-derived small peptide Pt5-1c not only possesses broad-spectrum antimicrobial activity but also exhibits synergistic effect and antibiofilm activity with traditional antibiotics against bacteria, including multi-drug resistant (MDR) strains. Here we provided the first evidence that Pt5-1c promoted the wound closure of surrogate scratch "wounds" of fibroblasts in vitro, and speeded up the healing and re-epithelialization of murine dermal wounds in vivo. We also showed that Pt5-1c activated migration of fibroblasts via a combined action of inducing migratory phenotype and trans-activating epidermal growth factor receptor (EGFR). Moreover, Pt5-1c accelerated attachment and proliferation of fibroblasts in vitro. Interestingly, Pt5-1c was able to promote collagen contraction through activation/differentiation of fibroblasts into myofibroblasts. These data together suggest that Pt5-1c is a promising candidate with therapeutic potential to promote wound healing.

Janus effects of NaCl on structure of egg yolk granules.

Egg yolk granules are supramolecular assembly of high-density lipoproteins and phosvitin driven by calcium bridges. However, applications of granules are severely restricted by the large particle size and poor water dispersibility. This study revealed the Janus effects of NaCl on structure of granules at varied pH values. Addition of 0.3-0.5 M NaCl led to the dissociation of at pH 5.0-7.0. At pH 5.0-10.0, dissociated granules demonstrated good colloidal stability with NaCl because of the adsorption of highly hydrated Na+ and Ca2+, which provided strong hydration repulsion when electrostatic repulsion was screened. In contrast, at pH 2.0 and 3.0, dissociated granules were positively charged with adsorption of poorly hydrated Cl- as counterions. Cl- failed to give sufficient hydration repulsion, leading to the phase separation with 0.3-0.5 M NaCl. Similar effects have been also found in LiCl, KCl, and CsCl, but Li+ might be less effective to disrupt calcium bridges.

Isolation of a novel calcium-binding peptide from phosvitin hydrolysates and the study of its calcium chelation mechanism.

A new peptide with strong calcium binding capacity was isolated from phosvitin hydrolysates. Taking calcium chelating rate as an indicator, phosvitin hydrolysates were separated gradually by anion-exchange chromatography, gel filtration chromatography and reversed-phase high performance liquid chromatography. A peptide with a molecular weight of 1106.44402 Da was identified by liquid chromatography-electrospray/mass spectrometry (LC-ESI/MS), and its amino acid sequence was DEEENDQVK, the calcium binding capacity reached 151.10 ± 3.57 mg/g. Its chelating mechanism was investigated. Results showed that, the ß-sheet structure of peptide increased after adding calcium ion, and the main binding sites were carboxyl oxygen atom and amino nitrogen atom. In vitro simulated digestion experiments showed that, the solubility and dialysis rate of calcium in peptide-calcium chelate were higher than those in CaCO3 and D-calcium gluconate. This finding would promote the development of calcium supplements from food resources.

Phosvitin phosphopeptides produced by pressurized hea-trypsin hydrolysis promote the differentiation and mineralization of MC3T3-E1 cells via the OPG/RANKL signaling pathways.

Phosvitin (PV) from egg yolk is an excellent substrate for the production of phosphopeptides, which have a strong calcium chelating capacity and promoting calcium absorption and bone mineralization. This study investigated the effect of PV hydrolysates produced using a effective preparation method (high temperature (121°C) and mild pressure (0.1 MPa), HTMP) or HTMP pretreatment and trypsin hydrolysis combination (HTMP-PV18) on the physiology of an osteoblast MC3T3-E1 cells line. The proliferation, apoptosis, and differentiation of MC3T3-E1 cells were analyzed using the CCK-8, flow cytometry, and RT-PCR reactions, respectively. Both the HTMP-PV and HTMP-PV18 increased the proliferation, and inhibited the apoptosis of MC3T3-E1 cells significantly. The HTMP-PV increased the proliferation of MC3T3-E1 cells by 147.12 ± 2.11% and the HTMP-PV18 by 136.43 ± 4.51%. In addition, the HTMP-PV and HTMP-PV18 effectively promoted the expression of genes related to the OPG/RANKL signaling channel during cell differentiation. This indicated that both the HTMP-PV and HTMP-PV18 have the potential to promote bone mineralization by improving the proliferation and differentiation of osteoblastic cells.

Phosvitin-wheat gluten complex catalyzed by transglutaminase in the presence of Na2SO3: Formation, cross-link behavior and emulsifying properties.

Phosvitin (PSV) is considered as a good emulsifier, although it has a low proportion of hydrophobic regions and steric hindrance. Wheat gluten (WG) possesses excellent hydrophobicity and macromolecular network structure. In this work, WG was subjected to a series of Na2SO3 solution, followed by cross-linking with PSV under transglutaminase (TGase) catalyzation. The results showed that Na2SO3 could break disulfide bonds of WG and increase its solubility from 7.33% to 42.82% with 1200 mg/L of Na2SO3. Correspondingly, the cross-linking degree was significantly enhanced. Compared to PSV, the cross-linked PSV-WG exhibited a higher surface hydrophobicity and thermal stability, with a lower zeta potential and apparent viscosity. The emulsifying activity of PSV-WG reached 17.42, 20.63 and 20.28 m2/g with Na2SO3 concentration of 300, 600 and 900 mg/L, which were all higher than that of PSV (15.19 m2/g). This work provided a novel strategy to elevate emulsifying properties of PSV by cross-link reaction.

Phosvitin Derived Phospho-Peptides Show Better Osteogenic Potential than Intact Phosvitin in MC3T3-E1 Osteoblastic Cells.

Phosphorylated proteins from food sources have been investigated as regulators of bone formation with potential benefits in treating osteoporosis. Egg, a cheap and nutritious food, is also the source of various proteins and bioactive peptides with applications in human health. Egg yolk is rich in phosvitin, the most phosphorylated protein in nature. Phosvitin has been shown to improve bone health in experimental animals, although the molecular mechanisms and its specific effects on bone-forming osteoblastic cells are incompletely understood. Previous work in our group has identified pancreatin-generated phosvitin phospho-peptides (PPP) as a potential source for bioactive peptides. Given this background, we examined the roles of both phosvitin and PPP in the function of osteoblastic cells. Our results demonstrated their potential to improve bone health by promoting osteoblast differentiation and proliferation, suppressing osteoclast recruitment and the deposition of extracellular matrix, although PPP appeared to demonstrate superior osteogenic functions compared to phosvitin alone.

Bioactivities of hen's egg yolk phosvitin and its functional phosphopeptides in food industry and health.

Phosvitin, one of the most noteworthy bioactive components of hen egg yolk, is an amphiphilic protein that stands out with its unique composition and functionality in the food industry and health. Phosvitin consists of 4% of egg yolk dry matter and 11% of egg yolk proteins. It is considered as the most phosphorylated protein with 10% phosphorus. Besides, some potential novel phosphopeptides containing clusters of phosphoserines can be derived from hen's egg yolk phosvitin. Phosvitin, which has many functional features thanks to its unique structure, is known primarily for its metal bonds binding (iron, calcium, etc.) feature. On the other hand, its phosphopeptides may increase the bioavailability of metals compared to phosvitin. Although this feature of phosvitin may partially decrease the bioavailability of especially iron in the egg, it allows the phosvitin to have many bioactivities in the food industry and health. Lipid oxidation, which is a serious problem in the food industry, can be inhibited by adding phosvitin and its derived phosphopeptides to the food production chain via inhibiting bivalent iron. Because phosvitin is an amphiphilic protein capable of chelating, it also shows potential antibacterial effects against the Gram-negative bacteria. Moreover, the literature has recently been attempting to define the promising relationship between phosvitin and its phosphopeptides and plenty of health-promoting activities such as immune-enhancing, melanogenesis inhibitor, anti-ageing, and anticancer. In this review, current information on the hen's egg yolk phosvitin and its phosphopeptides and their bioactivities in the food industry and health are discussed and some future directions are given.

Fabrication and Characterization of a Microemulsion Stabilized by Integrated Phosvitin and Gallic Acid.

The purpose of this work was to conjugate phosvitin (Pv) with gallic acid (GA) to explore a new emulsifier that had both good emulsifying properties and antioxidant activity. The Pv-GA complex was prepared at a GA concentration of 1.5 mg/mL with pH 9.0. The Pv-GA complex obtained was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and characterized with infrared, ultraviolet, and fluorescence spectra. The emulsifying activity and stability of the Pv-GA complex were slightly improved, and antioxidant activities was significantly enhanced. Furthermore, the Pv-GA complex was used to load conjugated linoleic acid (CLA) for microemulsion preparation. Results showed that the Pv-GA complex could increase the viscosity and lipid antioxidant capacity of Pv-GA/CLA microemulsion. The Pv-GA/CLA microemulsion had remarkable emulsifying activity, emulsifying stability, pH, and thermal stability and poor salt stability.

High hydrostatic pressure induced extraction and selective transfer of ß-phosvitin from the egg yolk granule to plasma fractions.

Egg yolk phosvitin is of particular interest due to its functional and biological properties. Recently, it was demonstrated that high hydrostatic pressure (HHP) (400 MPa for 5 min) induced the transfer of folic acid and phosvitin from the egg yolk granule to the plasma fraction. A granule fraction (Gin) produced by egg yolk centrifugation was pressure-treated at 400 and 600 MPa for 5 and 10 min, and centrifuged to generate granule fractions (GP1 to GP4) and plasmas (PP1 to PP4). Iron and phosphorus contents were also increased in PP1 to PP4 fractions, confirming the transfer of phosvitins from pressure-treated granule to plasma. Pressurization drastically improved phosvitin recovery in PP fractions, specifically at 600 MPa for 10 min, which had the highest value of phosvitin/100 mg of dry plasma at 33.3 ± 4.39 mg. Consequently, HHP represents an alternative approach for phosvitin transfer and recovery in the egg yolk soluble fraction.

Anti-elastase and anti-hyaluronidase activity of phosvitin isolated from hen egg yolk.

1. Phosvitin, a major phosphoprotein found in egg yolk, has strong antioxidant activity. Activation of elastase, collagenase, and hyaluronidase by reactive oxygen species are related to the degradation of ECM and skin aging. The objective of this study was to determine the anti-elastase and anti-hyaluronidase activity of phosvitin.2. Elastase from porcine pancreas and hyaluronidase from bovine testes were used to study the inhibitory activity of phosvitin. To elucidate the mechanism of enzyme inhibition, a Lineweaver-Burk plot was constructed.3. Phosvitin inhibited elastase and hyaluronidase activity in a dose-dependent manner. The IC50 value of phosvitin was 31.6 µg/ml and 1,270 µg/ml against elastase and hyaluronidase, respectively. The analysis of elastase and hyaluronidase kinetics indicated that the apparent Michaelis constant (appKm) was increased by phosvitin but the Vmax value was not affected.4. In conclusion, phosvitin exhibited competitive inhibitory activity against elastase and hyaluronidase. Thus, phosvitin could be used as a natural anti-aging agent in the cosmetics industry.