Progress 04/01/19 to 03/31/22
Outputs
Target Audience:Egg processing industry, egg producers, a wide range of food, nutraceutical, cosmeceutical, and pharmaceutical industries, national and international researchers in egg and food science areas, governmental agencies related to egg processing and bioactive ingredients, students in food and poultry sciences, and consumers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has created training opportunities for graduate students to understand how to approach the production of highly bioactive peptides from structurally solid, compact proteins such as phosvitin and acquire the analytical capacities to evaluate the structural and functional characteristics and biological activities such as the anti-cancer activity of bioactive phosphopeptides. Understanding and research experience obtained from the project will be a great asset for the students to perform their future biopeptide research that can improve human health. How have the results been disseminated to communities of interest?The results of the project have been shared with related industries such as egg processing, food, nutraceutical industries, governmental agencies, and scientific communities mainly via publications and presentations in international meetings. We have published 2 articles in peer-reviewed journals (1 published and 1 under review) and 2 presentations at international conferences. In addition, we developed a research proposal on the development of the calcium delivery system using the phosvitin hydrolysate based on the results of this Seed Grant project and submitted it to the USDA/NIFA AFRI Competitive Grants Program in 2021. It was not funded. We are improving the proposal and have a plan to re-submit it in 2022. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Phosphopeptides are one of the most promising bioactive compounds because of their ability to enhance human nutrition as essential mineral delivery systems to address their deficiencies and prevent various chronic diseases such as cancer. Casein, the major milk protein, has been used for the commercial production of phosphopeptides. Phosvitin, the major egg yolk protein, is a highly phosphorylated protein more than casein and therefore has potential as the source of bioactive phosphopeptides. However, because of its compact structure, phosvitin is not easily hydrolyzed by enzymes and therefore has not been used for phosphopeptide preparation. The overall goals of the project were to demonstrate the effectiveness of the proposed, novel combination technology of high-temperature, mild-pressure (HTMP) pretreatment and two-enzyme hydrolysis to produce small sizes of bioactive phosphopeptides (<3 kDa in molecular sizes) and to evaluate the structural and functional properties of the phosvitin hydrolysates. Objective I of the project was to determine the optimum conditions of the HTMP pretreatment (pressure and heating duration) and identify proteinases for the two-enzyme hydrolysis to produce the phosvitin hydrolysate with the maximum amounts of the small phosphopeptides (<3 kDa). The phosvitin solution (50 mg/mL deionized, distilled water (DDW)) was treated with the HTMP pretreatment at 1.2 or 1.5 atm at 121 °C for 30 or 60 min. Subsequently, food-grade proteinases were added to the solution alone or in combination to hydrolyze phosvitin for 6 hours (phosvitin to enzyme ratio - 50:1): trypsin (pH 8.0, 40 °C), Protex-6L (alkaline serine endopeptidase, pH 7.0, 60 °C), Multifect-14L (mainly thermolysin, pH 8.0, 70 °C), trypsin+Protex-6L, and trypsin+Multifect-14L. For two-enzyme hydrolysis, trypsin was first added for the hydrolysis, followed by the hydrolysis by Protex-6L or Multifect-14L. The hydrolysates were centrifuged, supernatants were freeze-dried, and lyophilized hydrolysates were stored at - 20 °C until analysis. SDS-PAGE and electrophoretic pattern image analysis were used to determine the degree of hydrolysis (DH) of phosvitin to evaluate the effects of the HTMP pretreatment and two-enzyme hydrolysis on the production of small peptides (< 3 kDa). The results showed that The HTMP pretreatment caused the degradation of phosvitin into smaller peptides and facilitated subsequent enzyme hydrolysis to produce small peptides (<3 kDa). The HTMP pretreatment at 121 °C at 1.2 atm for 30 min significantly degraded the phosvitin molecules into peptides, but the intact phosvitin was still visible in the electrophoretic image. Increases in the pressure and heating duration of the HTMP pretreatment to 1.5 atm and 60 min, respectively, accelerated the degradation of phosvitin with the majority of the peptides lower than 10 kDa in molecular size and facilitated subsequent enzyme hydrolysis, resulting in the production of peptides more than 85% of which were smaller than 3 kDa. These results indicated that the HTMP pretreatment can cause the disintegration of the phosvitin structure and its breakdown into peptides, resulting in increases in enzyme accessibility and hence the facilitation of subsequent enzyme hydrolysis. Among the proteinases evaluated in this project, trypsin showed the highest DH (23.24%), followed by Protex-6L (17.43%) and Multifect-14L (8.43%). In the two-enzyme hydrolysis system, trypsin+Multifect-14L (26.01%) showed a significantly higher DHvalue than trypsin+ Protex-6L (19.04%), indicating a significant synergistic effect between trypsin and Multifect-14L on phosvitin hydrolysis. This is probably because the hydrolysis by trypsin may expose the hidden cleavage sites for Multifect-14L, facilitating the phosvitin hydrolysis. Therefore, the results suggested that the HTMP pretreatment is the essential step for phosvitin hydrolysis and the use of two enzymes could further enhance phosvitin hydrolysis. The HTMP pretreatment at 1.5 atm and 121 °C for 60 min in combination with the two-enzyme (trypsin+Multifect-14L) hydrolysis was suggested as the optimal condition for phosvitin hydrolysis to produce the small phosphopeptides (< 3 kDa). Objective II was to evaluate the structural and functional characteristics of the phosvitin phosphopeptides in the hydrolysates. The lyophilized phosvitin hydrolysate prepared in Objective I was analyzed to determine the structural characteristics (peptide profiles and amino acid sequences) of the peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Their functional properties were also determined: metal ion (calcium, iron, and copper) binding capacity, antioxidant capacity, angiotensin-I-converting enzyme (ACE)-inhibitory activity, and cytotoxic effects on various cancer cell lines (HeLa (cervix), MCF-7 (breast), AGS (stomach), HT-29 (colon), and LoVo (colon) cell lines). The molecular size of the phosphopeptides identified by the LC-MS/MS analysis ranged between 0.5 and 3.5 kDa. The results showed that the HTMP pretreatment produced 154 peptides varying in molecular size and amino acid sequences. Subsequent enzyme hydrolysis produced more diverse peptide profiles. The hydrolysis by trypsin, Protex-6L, and Multifect-14L in combination with HTMP pretreatment generated 252, 280, and 164 peptides, respectively, while two-enzyme hydrolysis (trypsin+Protex-6L and trypsin+Multifect-14L) produced 158 and 154 peptides, respectively. This was probably because the sizes of many peptides generated by the two-enzyme systems were below the detection limit (<0.5 kDa). The results in Objective I showed that the majority of small peptides produced by the HTMP pretreatment in combination with two-enzyme hydrolysis were concentrated at the bottom of the electrophoretic gel image significantly below the 2 kDa marker. These results suggested that the HTMP pretreatment can hydrolyze native phosvitin at random sites, contributing to the production of diverse peptides. Hydrolysates produced by the HTMP pretreatment alone (HTMP hydrolysate) or in combination with enzyme hydrolysis (Enzyme hydrolysate) showed different functional properties from native phosvitin. Metal ion (calcium, iron, and copper) binding capacities of native phosvitin were significantly higher than those of HTMP and Enzyme hydrolysates probably because the HTMP pretreatment and enzyme hydrolysis can hydrolyze phosvitin and therefore expose its non-phosphorylated core part, resulting in decreases in phosphorous group-concentrated areas on the surface of peptides. Although the metal iron binding capacities of the hydrolysates were lower than that of native phosvitin, they still showed the moderate-to-high binding capacity of metal ions for transportation and release. HTMP and Enzyme hydrolysates more effectively inhibited lipid oxidation compared to native phosvitin and the Two-Enzyme hydrolysates were the most effective. Enzyme hydrolysates showed significantly higher ACE-inhibitory activities than native phosvitin and HTMP hydrolysates probably due to their higher amounts of small peptides. In addition, Multifect-14L and Two-Enzyme hydrolysates showed cytotoxic effects on all cancer cell lines examined. Trypsin+Multifect-14L hydrolysates exhibited highly potent cytotoxic effects on all the cancer cell lines, except for AGC (stomach) cell lines. In conclusion, the project demonstrated that the proposed HTMP pretreatment (at 1.5 atm at 121 °C for 60 min) in combination with two-enzyme (trypsin+Multifect-14L) hydrolysis can produce hydrolysate, the majority of which were small phosphopeptides (<3 kDa), showing the excellent functional properties. The project suggested that the phosphopeptide-rich phosvitin hydrolysate produced by the proposed novel technology has high potential as a bioactive ingredient for food and neutraceutical products to enhance human nutrition and health.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Noh, E., Moon, S. H., Ahn, D. U., and Min, B. R. 2022. Functional characteristics of phosvitin hydrolysates prepared by novel combination technology of high-temperature mild-pressure pretreatment and two-enzyme system. 2022 IFT Annual Meeting. July 10 ~ 13, 2022. Chicago, IL.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
Lee, J. E., Lee, J. H., Min, B., Kim, K. T., Ahn, D. U., and Paik, H. D. 2022. Immunostimulatory effect of egg yolk phosvitin phosphopeptides produced by high-temperature and mild-pressure pretreatment and enzyme combinations in RAW 264.7 cells via TLR2/MAPK signaling pathway. Journal of Functional Foods. (under review)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Noh, E., Moon, S. H., Ahn, D. U., and Min, B. R. 2022. Functional properties of enzyme hydrolysis of phosvitin pre-treated with high temperature and mild pressure. US Korea Conference 2022. August 17-20. Arlington, VA.
|
Progress 04/01/20 to 03/31/21
Outputs
Target Audience:Egg processing industry, egg producers, a wide range of food, nutraceutical, cosmeceutical, and pharmaceutical industries, national and international researchers in egg and food science areas, governmental agencies related to egg processing and bioactive ingredients, students in food and poultry sciences, and consumers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided opportunities to develop protocols to analyze functional properties of enzyme hydrolysates and train graduate students to conduct those analyses. Understanding and experience of these analyses will enable our research team and students to conduct future research on the functional properties of bioactive peptides from various protein sources. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?The cytotoxic effects of the phosphopeptides prepared in Objective I on various cancer cell lines will be determined to achieve Objective II. The findings of the project will be presented at national/international conferences and published in a peer-reviewed journal. The final report for the project will be prepared.
Impacts
What was accomplished under these goals?
Egg consumption in the U.S. has continuously increased over more than two decades primarily because of the increase in the consumption of processed egg white products due to consumer's demands on high protein/low lipid products. Therefore, novel strategies to increase the use of undervalued, underutilized egg yolk should be developed to improve the sustainability and profitability of the U.S. egg industry and provide economic and health benefits for consumers. In recent years, health-beneficial, bioactive ingredients have been exponentially introduced into food products for their health benefits. Among them, phosphopeptides are one of the most promising bioactive ingredients because of their ability to deliver essential metal ions such as calcium and iron and their preventive effects on various chronic diseases. Currently, phosphopeptides made from casein, one of the major milk proteins, are commercially available as a mineral absorption facilitator in Japan and several European countries. Phosvitin, one of the main egg yolk proteins, is a highly phosphorylated protein, more than casein. It has attracted much attention as a high potential source for bioactive phosphopeptide production. However, phosvitin has not been utilized to produce phosphopeptides because the hydrolyzation of phosvitin is extremely challenging due to its structural complexity. The overall goal of this project is to demonstrate the potential of the proposed, novel combination technology of the pressurized heat-pretreatment (PHPT) and two-enzyme system to produce small The objective achieved in this reporting period was to evaluate the functional characteristics of the phosvitin phosphopeptides (Objective II). Lyophilized phosvitin dissolved in deionized, distilled water (DDW, 10 mg/mL) was treated with the pressurized heat-pretreatment (PHPT; at 1.5 atm and 121 °C for 30 min) and subsequently hydrolyzed by food-grade proteinases singly or in combination at their optimal temperature and pH for 6 hours (substrates to enzyme ratio = 50:1): trypsin (type I from bovine pancreas, pH 8.0, 40 °C), Protex 6L (alkaline serine endopeptidase, subtilisin or alcalase, pH 7.0, 60 °C), Multifect 14L (mainly thermolysin, pH 8.0, 70 °C), trypsin + Protex 6L, and trypsin + Multifect 14L. The hydrolyzed solution was centrifuged and the supernatant was lyophilized. The dried hydrolysate was collected and stored in a -20 °C freezer until analysis. The lyophilized hydrolysates were reconstituted into DDW or appropriate buffer to evaluate their functional properties: metal ion (calcium, iron, and copper) binding capacity, antioxidant capacity using an oil emulsion system containing free ionic irons as catalysts for lipid oxidation, angiotensin-I-converting enzyme (ACE)-inhibitory activity, and cytotoxic effects on various cancer cell lines using the MTT assay. Phosvitin and PHPT-treated phosvitin were used as negative controls. Calcium-binding capacity was determined by measuring amounts of calcium ion solubilized by phosvitin hydrolysates in a buffer system. The solubility of calcium chloride (CaCl2) was 1.94 mg Ca/100 mL. Native phosvitin, PHPT-treated phosvitin, and phosvitin hydrolysates considerably increased the amount of solubilized calcium by over 20 times (38.75 - 41.14 mg Ca/100 mL) because of their calcium-binding capacity. The calcium-binding capacity of native phosvitin (41.14 mg Ca/100 mL) was slightly but significantly higher than those of PHPT-treated phosvitin and its hydrolysates (P < 0.05) probably because PHPT and proteinases may break down the phosvitin into phosphopeptides and therefore cause the exposure of the non-phosphorylated core part of phosvitin, resulting in the reduction in phosphorous group-concentrated areas in the surface of peptides. The one-enzyme system did not cause a further decrease in the calcium-binding capacity of the hydrolysates compared to PHPT-treated phosvitin, but the two-enzyme systems significantly decreased the calcium-binding capacities of the hydrolysates (P < 0.05) probably due to higher amounts of small phosphopeptides (< 0.5 kDa). On the other hand, iron and copper-binding capacities of the PHPT-treated phosvitin and its hydrolysates were significantly higher than that of native phosvitin (P < 0.05). While the copper-binding capacities were not different between the hydrolysates produced by one-enzyme and two-enzyme systems, the iron-binding capacities of the two-enzyme system-treated hydrolysates were significantly higher than those of the one-enzyme system (P < 0.05) regardless of the enzymes used. In addition, the two-enzyme system-treated hydrolysates inhibited Fe-catalyzed lipid oxidation in the oil emulsion system more effectively compared to the one-enzyme system (P <0.05). These results indicated that the smaller phosvitin phosphopeptides produced by the two-enzyme system may have a better binding capacity to iron compared to calcium and copper. The enzyme hydrolysates showed significantly higher ACE-inhibitory activities (18.42 - 30.80 µM hippuric acid produced) compared to native phosvitin, and PHPT-treated phosvitin (133.91 and 148.53 µM) (P <0.05). The two-enzyme system-treated hydrolysates showed significantly higher ACE-inhibitory activities than did the one-enzyme system treated (P <0.05) regardless of the enzyme used. In summary, the findings in this reporting period indicated that the novel combination technology of PHPT and two enzyme system may be able to produce the phosvitin hydrolysate (containing high amounts of small phosphopeptides) that has excellent metal ion-binding capacities and ACE inhibitory activities. Their cytotoxic effects on various cancer cell lines using the MTT assay are still analyzed.
Publications
|
Progress 04/01/19 to 03/31/20
Outputs
Target Audience:Egg processing industry, egg producers, a wide range of food, nutraceutical, cosmeceutical, and pharmaceutical industries, national and international researchers in egg and food science areas, governmental agencies related to egg processing and bioactive ingredients, students in food and poultry sciences, and consumers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided opportunities to understand the potentials of the novel combination system with the pressured heat pre-treatment and two-enzyme system to produce small, diverse phosphopeptides from structurally stable proteins such as phosvitin for the production of bioactive peptides. Understanding and experience acquired from the project will be of a great asset for future research for the development and industrialization of bioactive peptides from various protein sources. How have the results been disseminated to communities of interest?The findings of this project were published in the Journal of Agricultural and Food Chemistry in this reporting period. What do you plan to do during the next reporting period to accomplish the goals?The functional characteristics of the phosphopeptides prepared in Objective I, including metal ions-binding capacity and antioxidant capacity, and angiotensin-I-converting enzyme (ACE)-inhibitory capacity, will be determined to achieve Objective II. The findings of the project will be presented in the national/international conferences and published in a peer-reviewed journal.
Impacts
What was accomplished under these goals?
The consumption of processed egg white products has been remarkably increased over the past decades because of their high nutritional quality. However, egg yolk products have been underutilized because of consumers' health concerns on their cholesterols and lipids. Consequently, it is essential to develop novel ways to increase the utilization of egg yolk for balancing the use of egg white and yolk, which can improve the sustainability and profitability of the egg industry and economic advantages for consumers. Phosvitin, one of the major egg yolk proteins, is highly phosphorylated and, therefore, has excellent potential for the source of bioactive phosphopeptides, one of the most promising bioactive ingredients that can improve human health. Phosphopeptides in small sizes (<3 kDa in molecular size) have shown diverse bioactivities such as antimicrobial, antioxidant, anti-inflammatory, and anti-cancer activities. Because of their metal ion binding ability, phosphopeptides can be utilized as an efficient delivery system of nutritionally essential metal ions such as calcium and iron for human health. Currently, milk casein is utilized to produce commercial phosphopeptides that are sold as a nutraceutical or mineral absorption facilitator worldwide. Because of its higher phosphorylation rate, phosvitin can be a better source than milk casein for phosphopeptide production. However, enzymatic hydrolysis of phosvitin to produce small phosphopeptides is extremely difficult because of its structural compactness and stability. The objectives of the project achieved in this reporting period were to identify the optimal condition of PHPT (level of pressure and duration) and types of proteinases for phosvitin hydrolysis for the maximum production of the small phosphopeptides (Objective I) and to evaluate structural characteristics (peptide profiles and amino acid sequence) of the phosvitin phosphopeptides (Objective II). Phosvitin was separated from hen's egg yolk using our economical, solvent-free method and then lyophilized. The purity of phosvitin was over 95%. Lyophilized phosvitin dissolved in deionized, distilled water (DDW, 50 mg/mL) was treated with different PHPT conditions (at 1.2 or 1.5 atm and 121 °C for 30 or 60 min). The PHPT-treated phosvitin was subsequently hydrolyzed using food-grade proteinases singly or in combination at their optimal temperature and pH for 6 hours (substrates to enzyme ratio = 50:1). The proteinases used in this project were trypsin (pH 8.0, 40 °C), Protex 6L (alkaline serine endopeptidase, subtilisin or alcalase, pH 7.0, 60 °C), Multifect 14L (mainly thermolysin, pH 8.0, 70 °C), trypsin + Protex 6L, and trypsin + Multifect 14L. Heat-pretreated phosvitin (at 100 °C and atmospheric pressure for 60 min) was prepared as a control. The hydrolyzed solution was centrifuged and the supernatant was lyophilized. The dried hydrolysate was collected and stored in a -20 °C freezer until analysis. Degree of hydrolysis (DH) of phosvitin was determined using SDS-PAGE and electrophoretic pattern image analysis to determine the optimal condition for PHPT and the best proteinase for enzymatic hydrolysis. The peptide profile and amino acid sequence of the phosphopeptides were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). PHPT significantly improved the enzyme hydrolysis of phosvitin into small phosphopeptides (<3 kDa in molecular size). The heat-pretreated phosvitin without higher pressure (Control) produced the majority of peptides larger than 10 kDa in molecular size after enzyme hydrolysis. PHPT at 1.5 atm for 30 min showed better phosvitin hydrolysis into the small phosphopeptides, compared to that at 1.2 atm. Moreover, an increase in PHPT duration from 30 min to 60 min at 1.5 atm further enhanced the phosvitin hydrolysis. PHPT at 1.5 atm and 121 °C for 60 min was able to partially disintegrate phosvitin structure and break down phosvitin into peptides in a wide range of molecular sizes, resulting in the increase in enzyme accessibility to the protein and consequently the enhancement of phosvitin hydrolysis. The results showed that the phosphopeptides smaller than 3 kDa in molecular size accounted for over 85% of peptides produced by PHPT in combination with enzyme hydrolysis, regardless of enzyme types and application methods. Therefore, the results suggested that PHPT be the essential step for phosvitin hydrolysis and the condition of PHPT at 1.5 atm and 121 °C for 60 min is the optimal condition for phosvitin hydrolysis. The subsequent proteinase treatments further hydrolyzed larger polypeptides generated by PHPT into small peptides (<3 kDa). When singly applied, trypsin performed the best (DH 23.24%), followed by Protex 6L (17.43%) and Multifect 14L (8.43%). Multifect 14L showed the poorer phosvitin hydrolysis compared to trypsin and Protex 6L probably because of its substrate specificity. When Protex 6L or Multifect 14L were applied after trypsin hydrolysis, the DHvalues improved significantly (19.04 and 26.01%, respectively). Unexpectedly, the combined use of trypsinMultifect 14L showed higherDHvaluesthantrypsin+Protex6L despite the poorer performance of Multifect 14L in its single application. This phenomenon was difficult to explain and needed further investigation. The results suggested the application of trypsin + Multifect 14L in combination with PHPT for phosvitin hydrolysis to produce small, functional phosphopeptides. The number and size of phosphopeptides produced from phosvitin varied considerably, depending on enzyme type and application method. The molecular size of the phosphopeptides identified ranged between 0.5 kDa and 3.5 kDa. PHPT alone produced 154 peptides identified by the LC-MS/MS system. The results showed that PHPT was able to break down peptides bonds of phosvitin at random sites to generate diverse phosphopeptides with different sizes and amino acid sequences and disintegrate the phosvitin structure to facilitate the subsequent enzyme hydrolysis. Enzyme hydrolysis of the PHPT-treated phosvitin using different proteinases produced more diverse phosvitin peptides with varying sizes and amino acid sequences, compared to those produced by PHPT alone. The numbers of identified peptides generated from PHPT-treated phosvitin by trypsin and Protex 6L were 252 and 280, respectively, which were significantly higher than that by PHPT alone. However, those by Multifect 14L and two-enzyme system (trypsin+Protex 6L and trypsin+Multifect 14L) were 164, 158, and 154, respectively, that were similar to that generated by PHPT alone. This could be because the sizes of many peptides generated by the two-enzyme systems were below the detection limit (<0.5 kDa). It should be noted that a considerable number of small peptides generated from the PHPT phosvitin by enzyme hydrolysis could be left undetected, considering that most of the peptides were concentrated at the bottom part (MW <1 kDa) of the electrophoretic gel. In summary, the findings in the project indicated that PHPT at 1.5 atm and 121 °C for 60 min is essential to facilitate subsequent enzyme hydrolysis for the production of diverse phosphopeptides in small sizes. Trypsin + Multifect 14L as the two-enzyme system was recommended for the hydrolysis of PHPT-treated phosvitin to produce small, diverse phosphopeptides (<3 kDa) with various functionalities.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Huang, X., Moon, S. H., Lee, J., Paik, H., Lee, E. J., Min, B., and Ahn, D. U. 2019. Effective preparation method of phosphopeptides from phosvitin and the analysis of peptide profiles Using Tandem Mass Spectrometry. Journal of Agricultural and Food Chemistry. 67: 14086-14101
|