BIOCONJUGATION OF PHENOLIC COMPOUNDS TO FOOD BIOPOLYMERS FOR THE ENHANCED ANTIOXIDATIVE ACTIVITY IN FOOD SYSTEMS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1018502
• Grant No.: 2019-67018-29186
• Cumulative Award Amt.: $199,040.00
• Proposal No.: 2018-06615
• Project Start Date: Feb 15, 2019
• Project End Date: Feb 14, 2022
• Grant Year: 2019
• Program Code: [A1361]- Improving Food Quality

Recipient Organization
NORTH DAKOTA STATE UNIV
1310 BOLLEY DR
FARGO,ND 58105-5750

Performing Department
Plant Sciences

Non Technical Summary
The FDA's new legislation of banning partially hydrogenated oils in human foods and other health organization's recommendations of increasing consumption of polyunsaturated fatty acids (PUFA) and decreasing consumption of saturated fats have shifted American diet toward unsaturated fat enriched food. However, the incorporation of PUFA into foods and beverages is often challenging, as they are more susceptible to oxidation than the oils having little to no double bonds. The oxidation of PUFA can not only result in the loss of their potential benefits, but also cause the development of rancidity and the formation of potentially toxic compounds. Food lipid oxidation has long been a major reason for global food loss and food waste due, in large measure, to the sensory deviations of oxidized suboptimal foods. Thus, preventing the oxidation of chemical labile PUFA is the foremost goal to maintain their benefits and minimize the loss of food they incorporated. Although some strategies, such as the application of antioxidants and microencapsulation of PUFA in a matrix, have been commercially used to control lipid oxidation, it is still difficult to completely prevent the formation of unpleasant rancid odors as most of the aldehydes formed in PUFA oxidation can be detected by human at a very low concentration. In other words, current available strategies cannot completely prevent lipid oxidation and retard rancidity in reformulated food with an increased amount of PUFA. In response to this need, the project will biosynthesize dual effect antioxidants that bear both free radical scavenging and metal chelating properties to effectively prevent lipid oxidation and extend the shelf-life of PUFA enriched food.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
502	5010	2000	100%

Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
5010 - Food;

Field Of Science
2000 - Chemistry;

Keywords

lipid oxidation

phenolic compounds

bioconjugation

beta-lactoglobulin

pectin

laccase

Goals / Objectives
The overall objective of this project is to evaluate the concept of dual effect antioxidants design through laccase cross-linked phenolic compound/food biopolymer bioconjugates, whereby a hetero-cross-linked antioxidant with enhanced activity is achieved to inhibit lipid oxidation and extend the shelf-life of high PUFA foods. There are two specific aims of this project: (1) biosynthesize and characterize novel dual effect antioxidant bioconjugates; (2) evaluate antioxidative activity of bioconjugates to inhibit oxidation in fish oil-in-water emulsions.

Project Methods
In this research, we will use 2,5-dihydroxybenzoic acid (DHBA) as representatives of phenolic compounds. Beta-lactoglobulin (b-Lg) and amidated low methoxyl (ALM) pectin will be selected as the representatives of functional food biopolymers with proven metal chelating properties. Fish oil-in-water emulsions will be prepared as model heterogeneous food systems. These compounds will be purchased from Sigma Aldrich and commercial food ingredients suppliers. For all the results, statistical analysis will be conducted using statistical software, SAS version 9.4 (SAS institute Inc. Cary, NC). One-way analysis of variance (ANOVA) followed by Tukey's test will be conducted with significant difference being defined at p?

Progress 02/15/19 to 02/14/22

Outputs
Target Audience:The target audiences include food and cereal researchers, graduate students, food products and ingredients industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this project, one postdoctoral researcher with inorganic chemistry backgroup worked on this project and received advanced training on food protein chemistry, lipid oxidation, and biopolymer conjugation. How have the results been disseminated to communities of interest?The results were disseminated through publications of multiple papers and presentations to national scientific meetings and other research meetings such as ACS, IFT, and AOCS. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In order to effectively tackle food loss and waste, two indices, i.e. Food Waste Index (FWI) and Food Loss Index (FLI), have recently been proposed by UN Environment and FAO. Food loss and waste to be measured explicitly by the two separate indicators will unequivocally specify the future goal for the food industry on reducing both food loss and waste occurred during the food supply chain. Whereas a number of factors can cause food loss and waste, both microbiological and chemical food spoilage (e.g., lipid oxidation) remain to be the major challenges in the food industry's efforts to confront food waste. Such food spoilages render discoloration, off-flavor, and texture change of food products, not only impacting human health but also inducing huge economic loss. Thus, intervening both microbiological and lipid oxidation has been an essential focus of modern food science/engineering, suppressing both of which has been considered effective in food preservation. In this project, we have developed an efficient strategy for massive and controllable production of dual-functional bioactive food biopolymers through the conjugation of functional proteins (beta-lactoglobulin and lysozyme) with phenolic compounds (gentisic acid and rosmarinic acid). The first step of this project was to design, synthesize and characterize the conjugates. We then assessed the antioxidant activity and antimicrobial properties of the conjugates. Furthermore, the antioxidant activity of BLG-phenolics on hemp oil-in-water emulsion was systematically evaluated. The findings indicated that the conjugated BLG may lower the emulsification properties as compared to the native BLG albeit the oxidative stability of the emulsions stabilized by the conjugates was greatly improved. The antimicrobial activity of lysozyme-phenolics conjugated was largely determined by the feeding ratio. Such surprising findings prompted us to unravel the structural fundamentals of lysozyme upon conjugating with various sizes of phenolics using site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy. The major contribution of the research is the understanding of the protein dynamics at the residue level upon conjugation and the final functionalities relationship, which provides the guidance to control the protein functionalities modification through conjugated compounds selection.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Hui Li, Yanxiong Pan, Zhongyu Yang, Jiajia Rao, Bingcan Chen. Modification of ?-lactoglobulin by phenolic conjugations: Protein structural changes and physicochemical stabilities of stripped hemp oil-in-water emulsions stabilized by the conjugates.Food Hydrocolloids, 2022,128, 107578.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Hui Li, Yanxiong Pan, Chun Li, Zhongyu Yang, Jiajia Rao, Bingcan Chen. Design, synthesis and characterization of lysozymegentisic acid dual-functional conjugates with antibacterial/antioxidant activities. Food Chemistry, 2022, 370, 131032.
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Hui Li, Yanxiong Pan, Yang Lan, Zhongyu Yang, Jiajia Rao, Bingcan Chen. Molecular interaction mechanism and structureactivity relationships of proteinpolyphenol noncovalent complexes revealed by side-directed spin labeling EPR spectroscopy.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Hui Li, Yanxiong Pan, Zhongyu Yang,Jiajia Rao,Bingcan Chen. Improving Antioxidant Activity of Lactoglobulin by Nature-Inspired Conjugation with Gentisic Acid.Journal of Agricultural and Food Chemistry. 2019, 67, 42, 11741-11751
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Bingcan Chen. Nature-inspired protein-phenolic compounds conjugates for enhancing antioxidant capability. World Congress on Oils & Fats, Feb 9-12, 2020, Sydney, Australia
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Hui Li, Yanxiong Pan, Zhongyu Yang, Jiajia Rao, Bingcan Chen. Emerging applications of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) to study food protein structure, dynamics, and interaction. Trends in Food Science & Technology, 2021, 109:37-50
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Hui Li, Yanxiong Pan, Chun Li, Zhongyu Yang, Jiajia Rao, Bingcan Chen. Lysozyme?dietary phenolic antioxidants bioconjugates as antioxidant and antibacterial bifunctional polymers: Structural Basis Underlying the Dual-function.

Progress 02/15/20 to 02/14/21

Outputs
Target Audience:The target audience during the reporting period including food ingredient suppliers, food and dairy researchers, product developer, and graduate students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One post-doctoral research associate has been trained. How have the results been disseminated to communities of interest?An oral presentation has been given at the 2020Institute of Food Technologist virtual annual meeting. What do you plan to do during the next reporting period to accomplish the goals?We plan to test the oxidative stability of o/w emulsions stabilized by the B-LG bioconjugates with different phenolics.

Impacts
What was accomplished under these goals? Due to the back order of B-LG from the vendor, antimicrobial lysozyme (Lys) was selected as a model protein. In order to further elucidate the mode of action for the dual effect conjugates, we seleced two phenolic compounds, including gentisic acid (GA) and rosmarinic acid (RA). Then, via controllable chemical conjugation, we established from the previous year, the compound avoids the destabilization often seen in physical mixing of Lys and two polyphenols with different size, number of phenolic hydroxyl groups, and hydrophilicity. We endorse Lys antioxidant activities in the conjugates. Meanwhile, conjugates with GA well-retained the antibacterial ability of Lys; while the antibacterial capability was suppressed slightly in conjugates with RA. The antibacterial activity discrepancy of two conjugates was attributed to the number and position of phenolic compound grafted on Lys, as revealed by a series of molecular level studies. Our work proves the principle of precisely conjugating different natural products to create bioactive preservatives with antibacterial/antioxidant dual functionalities. The structural features observed may represent a general mechanism for the fabrication of bioactive protein-phenolic conjugates to mitigate the lipid oxidadtion and growth of microorganisms. The revealed mechanisms are meaningful for guiding the design of future functional food ingredients based on functional proteins and natural antioxidants.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Hui Li, Yanxiong Pan, Zhongyu Yang, Jiajia Rao, Bingcan Chen. Emerging applications of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) to study food protein structure, dynamics, and interaction. Trends in Food Science & Technology, 2021, 109:37-50

Progress 02/15/19 to 02/14/20

Outputs
Target Audience:The target audience during the reporting period including food and dairy researchers, product developer, graduate students, and food products and ingredients industry. Information was delivered through laboratory research, classroom instruction, short course, conference, and publications. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One post-doctoral research associate was trained. How have the results been disseminated to communities of interest?Research results have been published in the prestigious journal and presented at international conferences via presentation. What do you plan to do during the next reporting period to accomplish the goals?We are looking at how the dual effect bioconjugates could impact the physical and oxidative stability of oil-in-water emulsions. We will analyze the dynamic changes of gentisic acid during the storage of emulsions. We will also investigate if modification of β-lactoglobulin by gentisic acid would impact the physical stability of the emulsion and how antioxidants combat with radicals generated during the emulsion storage.

Impacts
What was accomplished under these goals? We have synthesized a dual effect antioxidant bioconjugates using a mild, protein-friendly reaction based on EDC/sNHS catalysis. This reaction has allowed us to conjugate a natural antioxidant, gentisic acid (GA), onto an antioxidant protein, β-lactoglobulin (βLG). The goal is to enhance the antioxidant capability of βLG as a functional food ingredients. Upon optimizing the reaction conditions, such as the catalysts and GA contents, we prepared a series of βLG-GA conjugates with different initial GA/βLG ratios. A series of biophysical tools including FT-IR, UV-VIS, and UPLC-ESI-QTOF-MS were employed to confirm the covalent bonding of GA to βLG protein. Then, the UV absorption, surface charge, and secondary structure (CD) of the formed conjugates were characterized to obtain an understanding of how GA impacts the structure and property of βLG. We found that GA conjugation changes the surface charge of βLG to more negative values, which can explain the secondary structural changes quantified by CD spectroscopy. We then quantified the antioxidant capability of the conjugates using EPR spectroscopy. We found that GA can enhance the antioxidant capability of βLG, as expected. Furthermore, an optimal modification condition, initial GA/βLG ratio of 150, was found, which provides the best antioxidant function. Lastly, quantitative data analysis was carried out to calculate the time constants of the DPPH radical signal decay, so that we can reveal deeper insights on the antioxidant mechanism of the conjugates. We found that GA on the protein surface increases the contribution of the first process (first time constant); meanwhile, GA conjugation causes protein conformational changes, which expose more antioxidant amino acids to the protein surface and reduced the second time constant. Overall, our work opens a new route to design functional materials with enhanced functionality and biocompatibility, which find wide applications in the food industry.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Hui Li, Yanxiong Pan, Zhongyu Yang,Jiajia Rao,Bingcan Chen. Improving Antioxidant Activity of Lactoglobulin by Nature-Inspired Conjugation with Gentisic Acid.Journal of Agricultural and Food Chemistry. 2019, 67, 42, 11741-11751
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Bingcan Chen. Nature-inspired protein-phenolic compounds conjugates for enhancing antioxidant capability. World Congress on Oils & Fats, Feb 9-12, 2020, Sydney, Australia

Progress 02/15/19 to 02/14/22

Outputs
Target Audience:The target audiences include food and cereal researchers, graduate students, food products and ingredients industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this project, one postdoctoral researcher with inorganic chemistry backgroup worked on this project and received advanced training on food protein chemistry, lipid oxidation, and biopolymer conjugation. How have the results been disseminated to communities of interest?The results were disseminated through publications of multiple papers and presentations to national scientific meetings and other research meetings such as ACS, IFT, and AOCS. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In order to effectively tackle food loss and waste, two indices, i.e. Food Waste Index (FWI) and Food Loss Index (FLI), have recently been proposed by UN Environment and FAO. Food loss and waste to be measured explicitly by the two separate indicators will unequivocally specify the future goal for the food industry on reducing both food loss and waste occurred during the food supply chain. Whereas a number of factors can cause food loss and waste, both microbiological and chemical food spoilage (e.g., lipid oxidation) remain to be the major challenges in the food industry's efforts to confront food waste. Such food spoilages render discoloration, off-flavor, and texture change of food products, not only impacting human health but also inducing huge economic loss. Thus, intervening both microbiological and lipid oxidation has been an essential focus of modern food science/engineering, suppressing both of which has been considered effective in food preservation. In this project, we have developed an efficient strategy for massive and controllable production of dual-functional bioactive food biopolymers through the conjugation of functional proteins (beta-lactoglobulin and lysozyme) with phenolic compounds (gentisic acid and rosmarinic acid). The first step of this project was to design, synthesize and characterize the conjugates. We then assessed the antioxidant activity and antimicrobial properties of the conjugates. Furthermore, the antioxidant activity of BLG-phenolics on hemp oil-in-water emulsion was systematically evaluated. The findings indicated that the conjugated BLG may lower the emulsification properties as compared to the native BLG albeit the oxidative stability of the emulsions stabilized by the conjugates was greatly improved. The antimicrobial activity of lysozyme-phenolics conjugated was largely determined by the feeding ratio. Such surprising findings prompted us to unravel the structural fundamentals of lysozyme upon conjugating with various sizes of phenolics using site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy. The major contribution of the research is the understanding of the protein dynamics at the residue level upon conjugation and the final functionalities relationship, which provides the guidance to control the protein functionalities modification through conjugated compounds selection.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Hui Li, Yanxiong Pan, Zhongyu Yang, Jiajia Rao, Bingcan Chen. Modification of ?-lactoglobulin by phenolic conjugations: Protein structural changes and physicochemical stabilities of stripped hemp oil-in-water emulsions stabilized by the conjugates.Food Hydrocolloids, 2022,128, 107578.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Hui Li, Yanxiong Pan, Chun Li, Zhongyu Yang, Jiajia Rao, Bingcan Chen. Design, synthesis and characterization of lysozymegentisic acid dual-functional conjugates with antibacterial/antioxidant activities. Food Chemistry, 2022, 370, 131032.
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Hui Li, Yanxiong Pan, Yang Lan, Zhongyu Yang, Jiajia Rao, Bingcan Chen. Molecular interaction mechanism and structureactivity relationships of proteinpolyphenol noncovalent complexes revealed by side-directed spin labeling EPR spectroscopy.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Hui Li, Yanxiong Pan, Zhongyu Yang,Jiajia Rao,Bingcan Chen. Improving Antioxidant Activity of Lactoglobulin by Nature-Inspired Conjugation with Gentisic Acid.Journal of Agricultural and Food Chemistry. 2019, 67, 42, 11741-11751
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Bingcan Chen. Nature-inspired protein-phenolic compounds conjugates for enhancing antioxidant capability. World Congress on Oils & Fats, Feb 9-12, 2020, Sydney, Australia
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Hui Li, Yanxiong Pan, Zhongyu Yang, Jiajia Rao, Bingcan Chen. Emerging applications of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) to study food protein structure, dynamics, and interaction. Trends in Food Science & Technology, 2021, 109:37-50
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Hui Li, Yanxiong Pan, Chun Li, Zhongyu Yang, Jiajia Rao, Bingcan Chen. Lysozyme?dietary phenolic antioxidants bioconjugates as antioxidant and antibacterial bifunctional polymers: Structural Basis Underlying the Dual-function.

Progress 02/15/20 to 02/14/21

Outputs
Target Audience:The target audience during the reporting period including food ingredient suppliers, food and dairy researchers, product developer, and graduate students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One post-doctoral research associate has been trained. How have the results been disseminated to communities of interest?An oral presentation has been given at the 2020Institute of Food Technologist virtual annual meeting. What do you plan to do during the next reporting period to accomplish the goals?We plan to test the oxidative stability of o/w emulsions stabilized by the B-LG bioconjugates with different phenolics.

Impacts
What was accomplished under these goals? Due to the back order of B-LG from the vendor, antimicrobial lysozyme (Lys) was selected as a model protein. In order to further elucidate the mode of action for the dual effect conjugates, we seleced two phenolic compounds, including gentisic acid (GA) and rosmarinic acid (RA). Then, via controllable chemical conjugation, we established from the previous year, the compound avoids the destabilization often seen in physical mixing of Lys and two polyphenols with different size, number of phenolic hydroxyl groups, and hydrophilicity. We endorse Lys antioxidant activities in the conjugates. Meanwhile, conjugates with GA well-retained the antibacterial ability of Lys; while the antibacterial capability was suppressed slightly in conjugates with RA. The antibacterial activity discrepancy of two conjugates was attributed to the number and position of phenolic compound grafted on Lys, as revealed by a series of molecular level studies. Our work proves the principle of precisely conjugating different natural products to create bioactive preservatives with antibacterial/antioxidant dual functionalities. The structural features observed may represent a general mechanism for the fabrication of bioactive protein-phenolic conjugates to mitigate the lipid oxidadtion and growth of microorganisms. The revealed mechanisms are meaningful for guiding the design of future functional food ingredients based on functional proteins and natural antioxidants.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Hui Li, Yanxiong Pan, Zhongyu Yang, Jiajia Rao, Bingcan Chen. Emerging applications of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) to study food protein structure, dynamics, and interaction. Trends in Food Science & Technology, 2021, 109:37-50

Progress 02/15/19 to 02/14/20

Outputs
Target Audience:The target audience during the reporting period including food and dairy researchers, product developer, graduate students, and food products and ingredients industry. Information was delivered through laboratory research, classroom instruction, short course, conference, and publications. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One post-doctoral research associate was trained. How have the results been disseminated to communities of interest?Research results have been published in the prestigious journal and presented at international conferences via presentation. What do you plan to do during the next reporting period to accomplish the goals?We are looking at how the dual effect bioconjugates could impact the physical and oxidative stability of oil-in-water emulsions. We will analyze the dynamic changes of gentisic acid during the storage of emulsions. We will also investigate if modification of β-lactoglobulin by gentisic acid would impact the physical stability of the emulsion and how antioxidants combat with radicals generated during the emulsion storage.

Impacts
What was accomplished under these goals? We have synthesized a dual effect antioxidant bioconjugates using a mild, protein-friendly reaction based on EDC/sNHS catalysis. This reaction has allowed us to conjugate a natural antioxidant, gentisic acid (GA), onto an antioxidant protein, β-lactoglobulin (βLG). The goal is to enhance the antioxidant capability of βLG as a functional food ingredients. Upon optimizing the reaction conditions, such as the catalysts and GA contents, we prepared a series of βLG-GA conjugates with different initial GA/βLG ratios. A series of biophysical tools including FT-IR, UV-VIS, and UPLC-ESI-QTOF-MS were employed to confirm the covalent bonding of GA to βLG protein. Then, the UV absorption, surface charge, and secondary structure (CD) of the formed conjugates were characterized to obtain an understanding of how GA impacts the structure and property of βLG. We found that GA conjugation changes the surface charge of βLG to more negative values, which can explain the secondary structural changes quantified by CD spectroscopy. We then quantified the antioxidant capability of the conjugates using EPR spectroscopy. We found that GA can enhance the antioxidant capability of βLG, as expected. Furthermore, an optimal modification condition, initial GA/βLG ratio of 150, was found, which provides the best antioxidant function. Lastly, quantitative data analysis was carried out to calculate the time constants of the DPPH radical signal decay, so that we can reveal deeper insights on the antioxidant mechanism of the conjugates. We found that GA on the protein surface increases the contribution of the first process (first time constant); meanwhile, GA conjugation causes protein conformational changes, which expose more antioxidant amino acids to the protein surface and reduced the second time constant. Overall, our work opens a new route to design functional materials with enhanced functionality and biocompatibility, which find wide applications in the food industry.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Hui Li, Yanxiong Pan, Zhongyu Yang,Jiajia Rao,Bingcan Chen. Improving Antioxidant Activity of Lactoglobulin by Nature-Inspired Conjugation with Gentisic Acid.Journal of Agricultural and Food Chemistry. 2019, 67, 42, 11741-11751
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Bingcan Chen. Nature-inspired protein-phenolic compounds conjugates for enhancing antioxidant capability. World Congress on Oils & Fats, Feb 9-12, 2020, Sydney, Australia