Novel anti‐cariogenic phosvitin‐phosphopeptides produced by hydrostatic pressure combined with enzymatic hydrolysis
Authors: Yoo Heejoo 
Published: November 2023

Purpose

Phosvitin (PV) is known as a metal binding protein in egg yolk with unique amino acid composition (>55% serine) associated with a large proportion of phosphates. Many attempts to use PV as a potential source of anti‐microbial agent were made but it turns out be no feasible due to its insolubility and poor stability in aqueous media. Our preliminary data show the enzyme‐treated PV phosphopeptides (PV‐P) could be dissolved. On the other hand, the yield of PV‐P was extremely low due to high serine‐phosphate resistant to enzymatic hydrolysis. This study was to increase the yield of PV‐P by using an innovative processing platform technology of high hydrostatic pressure combined with enzymatic hydrolysis (HHP‐EH). Further study was to evaluate the iron‐chelating capacity of PV‐P. The main objective of this research is to produce PV‐P with iron binding capacity using HHP‐EH technology and study the possible correlation between iron binding capacity and antimicrobial activity of PV‐P against bacteria causing dental caries.

Methods

PV was isolated from egg yolk residues after IgY antibody extraction. PV‐P fractions (pH6.0) were produced by HHP‐EH with various proteases (Alcalase, Trypsin, Bromelain, Papain, Thermolysin, Elastase, Flavourzyme, Visozyme, and Savinase), in single, double or triple combinations, at E:S ratio of 1:50, under 100 MPa, at 37–50ºC for 12–24 h. The optimization of HHP‐EH was evaluated by TNBS method for degree of hydrolysis (DH) of PV. Mw distribution of PV‐P was monitored by SDS‐PAGE, MALDI‐TOF and HPLC techniques. Iron‐chelating capacity of PV‐P fractions (> or <3 kDa) vs PV was measured by spectrophotometric detection of ferrous ions. Selected PV hydrolysates with highest iron chelating capacity were used in antimicrobial assays. Streptococcus sobrinus was grown in appropriate media at 37 ºC. PV, PV‐P and fractions were added to the cell suspension at different concentrations. A 100 μL portion was spread over Mitis‐Salivarius agar (Difco) plates. The number of colonies formed after incubation at 35 ºC for 24 h was measured to calculate the survival ratio. Samples were analyzed in triplicate and results were plotted in student t‐test.

Results

Triple combination of Alclase, Elastase and Flavourzyme showed the highest DH (89%) at E:S ratio of 1:50 in each enzyme, under 100 MPa, at 37ºC for 24 h. The results of SDS‐PAGE and MALDI‐TOF, HPLC showed that PV Mw bands (48 and 37 kDa) were hydrolyzed into PV‐P at 30, 23, 17 kDa and <3 kDa. The highest iron‐chelating capacity was observed in PV‐P fraction (<3 kDa, 30%), compared to PV‐P fraction (>3 kDa, 13.5%), regardless of enzymatic and pressure treatment, indicating an efficient iron‐chelating capacity of <3 kDa PV‐P fraction. The antibacterial effect of PV‐P (<3 kDa fraction) on S. sobrinus strain showed a growth inhibition effect.

Conclusion

We optimized parameters of HHP‐EH processing to increase the yield of PV‐P. These short phosphopeptides (< 3 kDa) show high iron‐chelating capacity used in anti‐microbial agent.

Support or Funding Information

MITACS Graduate Internship Program and Canada Food Innovators Grant