INVESTIGATING THE EFFECT OF HIGH PRESSURE PROCESSING (HPP) ON MOLECULAR STRUCTURE AND STABILIZATION OF NATURAL HYDROPHILIC COLORANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1022138
• Grant No.: 2020-67017-31274
• Cumulative Award Amt.: $468,000.00
• Proposal No.: 2019-06723
• Project Start Date: Jun 1, 2020
• Project End Date: May 31, 2023
• Grant Year: 2020
• Program Code: [A1364]- Novel Foods and Innovative Manufacturing Technologies

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
Food Science

Non Technical Summary
With the growing demand for clean label and natural ingredients by food industries, there is a critical need to use natural colorants and develop methodologies to stabilize them and investigate their impact on the quality of food and beverages. Color is one of the most critical components of a food product that directly affects the consumer's perception of the quality, freshness, and safety of the food. Over the past century, the food industry has relied on synthetic color additives to provide better color stability and more excellent product attractiveness. However, the growing health awareness of modern consumers has driven the food industry to utilize naturally derived colorants for product coloration. For instance, the concern over the safety of many synthetic colorants, such as Brilliant Blue, Red No. 40, and Yellow No. 5, has led the food industry to increasingly replace them with natural colorants, such as anthocyanins, betalains, phycocyanins, chlorophyllins, and carotenoids, which are extracted from fruit and vegetable sources, such as beetroot, saffron, berries, and annatto. However, the low stability of such natural colorants has limited their application in foods as they tend to degrade when exposed to elevated temperature, pH changes, and light leading to hue changes or complete color loss and the formation of degradation products.To broadly use natural colorants and broaden their application in the food industry, a robust and effective stabilization technique should be in place to not only enhance the stability, but also can be easy employed in large scale as well as being compatible with the food sensory attributes (e.g., taste, smell, and texture). To that end, in this project, we propose to employ high-pressure processing (HPP) as an effective treatment technique to enhance natural colorant stability. First, we will complex these colorants with biopolymers (e.g., polysaccharides and proteins) and biomolecules and ions (e.g., sucrose and copper ions). Then, we will treat the complexed colorants using HPP at pressures between 400-600 MPa (Mega Pascal). Finally, we will investigate the change of the inter/intra-molecular structure of the resulting complexed colorants to comprehensively understand the combinatory effects of HPP and complexation on the stabilization of natural hydrophilic colorants. The stabilized natural colorants qualify as "clean label" ingredients, and we expect they can be used in foods and beverages where highly acidic conditions, low sugar/high water environments, high-temperature thermal processing, and long shelf life are required. This work is designed only to use natural ingredients and employ HPP to strengthen the complexation and enhance the stability of the natural colorants. Moreover, the products will be thoroughly characterized for both molecular level alteration and physiochemical properties associated with sensory appeal and bioavailability of nutrients in various foods.Thus, the outcome of the present work can be considered to promote the clean label through a value-added treatment to natural colorants as well as the inactivation of microorganisms by the HPP treatment. Moreover, the outcome will delineate the role of the HPP process on natural colorants' stability. As a result, the proposed research is of great interest to the community of food science as well as the food industry and yield some insights to promote healthier and higher quality products for the customers.

Animal Health Component

35%

Research Effort Categories

Basic

65%

Applied

35%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	2499	2000	10%
502	2499	3090	60%
502	2499	2020	30%

Knowledge Area
206 - Basic Plant Biology; 502 - New and Improved Food Products;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
2020 - Engineering; 3090 - Sensory science (human senses); 2000 - Chemistry;

Keywords

natural colorants

clean label

complex formation

high-pressure processing

stabilization

Goals / Objectives
The goal of the proposed study is to investigate the effect of the high pressures process (HPP) on natural colorants' stability. The HPP treatment could restructure the weak associative networks (e.g., hydrogen bonding and hydrophobic associations) between natural colorants and complex-forming polysaccharides/proteins. The HPP treatment is allowing them to be preserved under conditions that typically promote rapid degradation, such as thermal treatment at low pH. Thus, the overall goal of the proposed study is the development and characterization of novel systems for colorant stabilization using HPP in order to broaden the use of natural water-soluble colorants, such as chlorophyllin, betalain, and phycocyanin, within the food industry. Additionally, this research could improve the shelf life extension and clean label promotion in the food industry. To achieve this goal, we have categorized our objectives as follows:Objective 1. Complex the hydrophilic colorants (chlorophyllins, phycocyanins, betalains) with biopolymers, small biomolecules, and metal ions, and reinforce the complexation using HPP.Objective 2. Investigation of the HPP effect on the inter/intra-molecular structure of the complexed colorants using circular dichroism (CD) and quartz crystal microbalance with dissipation (QCM-D).Objective 3. Investigation of the HPP effect on the physicochemical properties of the complexed colorants using UV-Vis spectroscopy, fluorimetry, colorimetry, surface/interfacial tension, particle size and surface charge, and Fourier transform infrared (FTIR) spectroscopy to understand the mechanism of the interactions in the complexes with HPP and without HPP treatments.Objective 4. Investigation of the colorant stability against harsh conditions (e.g., thermal, pH, and light) and the incorporation of stabilized colorants into a model beverage system and evaluation of the physical/organoleptic properties.

Project Methods
The proposed study is focused on the development, production, and characterization of novel colorant stabilization systems that can broaden the use of chlorophyllins, betalains, and phycocyanins within the food industry and establish a more significant understanding of how these technologies affect food quality and healthfulness. In order to achieve this goal, we plan to acquire our own natural pigment extracts, develop novel colorant systems according to our strategies, and physically and functionally characterize these colorant systems on their own and in the context of select model food systems, as outlined below.Preparation & Acquisition of Pigment Extracts: All pigments utilized in the studies herein will be natural extracts from common sources of betalains (beetroot), chlorophyllins (alfalfa and mulberry leaf extract), and phycocyanins (spirulina).Preparation of Complexed Colorants with Proteins, Polysaccharides, Sugars, and Copper Ions: The colorants will be individually mixed with aqueous solutions of whey protein, casein, pea protein, sugars, polysaccharides, and copper ions and then homogenized. Homogenized complexed colorants will then be split into two aliquots, one processed using HPP, and another one will not be processed with HPP. The free colorants will be considered as a control.HPP of Complexed Colorants: High-pressure processing will be performed on a Hiperbaric-55 system at the Cornell University HPP Validation Center. Samples will then be treated at 4? for 3 min under 600 MPa of pressure and stored at 4? until further testing.Studying the Real-time Interactions Between the Natural Colorants and Proteins, Polysaccharides, Sugars, and Ions Processed under HPP via Quartz Crystal Microbalance with Dissipation (QCM-D): The effect of HPP on complexation of the colorants with proteins, polysaccharides, sugar, and copper ions will be studied via QCM-D on a Q-Sense Explorer single-module system from Biolin Scientific in our lab.Studying the HPP-Processed Complexed Colorants via Circular Dichroism (CD): To delineate the structural changes in molecules, the CD experiments will be performed. For CD experiments, the HPP-processed complexed colorants will be compared with the unprocessed complexed colorants and free colorants (control).Characterization of HPP-Processed Colorants via Colorimetry and UV-Vis Spectroscopy: All HPP-processed colorants will be investigated using a colorimeter to determine color values (L*, a* and b*). UV-Vis spectroscopy will also be used to obtain the spectra of the colorants before and after complexation and HPP. Minor shifts of the UV-Vis peaks may provide information on the interaction of the polysaccharides/colorants and the effects of HPP on the structural changes, which colorimetry cannot offer.Particle Size and Electrostatic Charge: Particle size distributions of the colorant systems developed within this study will be assessed by dynamic light scattering (DLS), size exclusion (SEC) HPLC, and/or analysis of images acquired by either inverted light microscopy or scanning electron microscopy (SEM).Rheological Properties: Since HPP can modify the rheological properties of polysaccharide and protein solutions, we will evaluate the effect of HPP on the rheology of the complexed colorants.Assessment of the Color Stability in Buffer and Model Beverage Solutions: Thorough colorant stability testing will include dilution of the colorant systems in buffer solutions ranging in pH from 2.0 to 8.0, followed by thermal, light, and storage stability testing in 24- or 96-well microplate formats. Heat treatments of colorant solutions will include standard 1 h heating at 85oC but will also include time monitored testing and lower and higher temperatures ranging from 50-120oC. Sensory Analysis: The colored model beverage will also be subjected to sensory evaluations within the Food Science Department at Cornell and compared with the model beverages colored with FD&C synthetic dyes for visual, texture, and mouthfeel evaluations. A sensory panel with at least 12 panelists will be trained to evaluate the mouthfeel attributes of the beverage, including heaviness, creaminess, sweetness, and aftertaste, as well as the appearance. A consumer acceptance study with at least 60 people will also be conducted at the Sensory Evaluation Center at our department to compare the consumer acceptance of the beverages colored with our naturally stabilized colorants and FD&C synthetic colorants.Statistical Analysis: All the experiments will be carried out in triplicate. Univariate and bivariate descriptive statistics will be calculated, and the mean and standard deviation of the experimental data will be presented. In addition, we will collaborate with the Cornell Statistical Consulting Unit on the statistical analysis for our project, budgeting for 20 hours of their time.

Progress 06/01/20 to 05/31/23

Outputs
Target Audience:In this project,we have developed a portfolio of solutions to overcome the main obstacle ofnatural hydrophilic colorants, especially phycocyanin (cyan to blue), beetroot extract (red), and anthocyanin(pink to red) to commercialization: photo-thermal instability in acidic to neutral media. Based on this, our target audience can be classified into the following categories: Audience in academia: With our efforts in the past four years, we have published nearly 20 papers on natural colorants. Many new findings and theories have been presented, as a result of the use of cutting-edge experimental and analytical methods, such as high-pressure processing (HPP),quartz crystal microbalance with dissipation (QCM-D), and small-angle X-ray scattering (SAXS). Our studies are increasingly gaining worldwide attention, which is evident from the interest in plant-based water-soluble colorants among food academia and our growing citations. Audience in industries: During the period of our study, we have also established beneficial contacts with companies, including PepsiCo, Sensient Technologies, Nanoscience Instruments, etc.Replacing artificial colors with natural ones is an unquestionable trend for the future; knowing this, we shared our research through national meetings and conferences, as well as seminars through the Cornell Institute for Food Systems Industry Partnership Program (CIFS-IPP). We believe that our research will provide solutions for marketable applications and troubleshooting of natural pigments. Audience in international organizations: Throughout our study, we also noted that there are still large energy gaps and food security issues in underdeveloped countries that need to be urgently addressed. With this in mind, we highlighted an algae-extracted blue protein - phycocyanin - that is enriched in algae, sometimes accounting for up to 20% of the dry matter. We would like to promote it to international organizations, such as the Food and Agriculture Organization (FAO) and the World Food Program (WFP), to further facilitate the role of algae as protein supplements. Consumer: We deeply understand the need and dependence of consumers on the visual senses of food; therefore, our research on natural pigments is naturally attractive to consumers. We also expect consumers outside of the food academia to notice and review our research, to enhance their understanding of coloring and brighten their lives. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training activities: With the support of the USDA-NIFA grant, sub-project leaders got a lot of training opportunities and their research proficiency was improved in the long run. For example, Dr. Zhong Zhang and Dr. Ying Li got trained and made proficient in operating thehigh-pressure processing (HPP) system; Dr. Younas Dadmohammadi and Shuang Zhang got the opportunity to contact an external company (Nanoscience Instruments) and learned to operate thequartz crystal microbalance with dissipation (QCM-D) system; Dr. Ying Li and Qike Li reached out to the Cornell High Energy Synchrotron Source (CHESS), and got abundant training on the small-angle X-ray scattering (SAXS) instrument and the corresponding protein modeling software; Qike Li also got a full-course training on the Scanning Electron Microscope (TEM) system, and received a long-term usage permit. In addition, undergraduates were trained in all aspects of research in this project. Professional development: With the support of the USDA-NIFA grant, Qike Li got the chance to attend the Project Director'sconference at UC Davis, where he presented our latest research through a poster, about the reversible disassembly-reassembly process of phycocyanin in pressurization-depressurization cycles, and the possibility of tapping phycocyanin's potential as both a colorant and an emulsifier. How have the results been disseminated to communities of interest? The newest publications are always updated on Dr.AlirezaAbbaspourrad's Google Scholar profile (https://scholar.google.com/citations?hl=en&user=wJg1hOkAAAAJ), and thepublication section on the official website ofAbbaspourrad Lab (https://abbaspourradlab.com/journal-articles/), on which the current projects and recent fundings are briefly introduced (https://abbaspourradlab.com/research/). The paper copies are pinned to a display wall at the door of the lab once published. Posters based on published papers are also presented on the wall outside the lab. The online link to thenewestpublished paper is usually shared by the first author on social media,including LinkedIn and Twitter. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Throughout this project, we have been committed to enhancing the feasibility of replacing synthetic colorants with natural ones, including anthocyanins (red), betalains (red), phycocyanins (blue), and chlorophyllins (green), which are extracted from fruit and vegetable sources, such as berries, beetroot, and alga. Compared with synthetic colorants, natural colorants generally have the advantages of anti-oxidant, anti-inflammatory, and anti-bacterialproperties; however, they are not highly solublein water and/or easily fade under food processing conditions and then lose their original fresh hue. In consideration of this, our team employed approaches such as complexation, encapsulation, and modification, coupled withhigh-pressure processing (HPP), to overcomenatural colorants' main obstacle to commercialization: photo-thermal instability in acidic to neutral media. Representative accomplishments are as follows: Whey protein isolate (WPI), the by-product of cheese production, was employedand upcycledto protect C-phycocyanin from color degradation at pH 3.0 over 5-day storage in light. (https://doi.org/10.1016/j.foodchem.2020.128642) Six representative polysaccharides (κ-/ι-/λ-carrageenans, xanthan gum, high-methoxyl pectin, and guar gum) werechosen to complex and protectC-phycocyaninat pH 3.0 before and after heating (60/80°C, 30 min). The ι-carrageenan-phycocyanin complex exhibits the best colloidal and color stabilities, which is promising for blue beverage processing. (https://doi.org/10.1016/j.foodhyd.2021.106852) Anin-depth study to investigate the mechanism of C-phycocyanin'sphoto-fading under different pH conditions,in which protein structures were modeled with high precision. Intermolecular photo-crosslinking was found at pH 5.0 via di-tyrosine species, which is critical for the light stabilityof C-phycocyanin, and makes it appealing for tumor-targeted photodynamic therapy. (https://doi.org/10.1021/acs.biomac.1c01095) The isoelectric point (pI) and functional properties ofC-phycocyaninwas modified by succinylation and protein-glutaminase (PG) deamidation to avoid its susceptibility to aggregation at and around its isoelectric point (pH 4.0 just as that of acidic food products). As a result, the improved solubility, heat stability, and functional properties between pH 4.0 and 5.0 will allow the modifiedC-phycocyaninto survive in acidic environments and heat processing conditions for many common foods. (https://doi.org/10.1016/j.foodhyd.2022.107994) By conjugating methoxy PEG (mPEG) chains on C-phycocyanin, conjugates exhibited high blue color intensity and improved thermodynamic stability.Techno-functional properties of C-phycocyanin were improved, and its susceptibility to aggregation and color changes upon heating and pH fluctuations was relieved.After PEGylation, the improved functional properties, bioactivity, and color stability against heat and pH fluctuations will facilitate the food and pharmaceutical applications of PC. (https://doi.org/10.1016/j.ijbiomac.2022.09.261) A water-in-oil-in-water (W1/O/W2) double emulsion system was developed to mitigate the discoloration and precipitation issues of C-phycocyanin at pH 3.0 and under light exposure. Through the high-pressure homogenizer (HPH) system, the optimized PC-rich double emulsion exhibited high C-phycocyanin loading capacity and long-term physical stability without leakage and phase separation and retained the vivid blue color with only slight turbidity. During the stress test, the C-phycocyanin-rich double emulsion system demonstrated good thermodynamical, mechanical, and photo-oxidative stabilities against the acidic and light storage conditions that are commonly used during food processing, making C-phycocyanin a widely applicable natural colorant and protein source in foods. (https://doi.org/10.1016/j.colsurfb.2022.112930) A modeling study of the mechanistic aspects of C-phycocyanin. Hydrostatic pressure can reversibly modulate protein-protein and protein-chromophore interactions of C-phycocyanin, under whichC-phycocyanin experience reversible disassembly-reassembly in pressurization-depressurization cycles.Pressure up to 350 MPa was enough to fully disassemble C-PC from trimers to monomers at pH 7.0, or from monomers to detached subunits at pH 9.0. And this finding ispromising to make C-phycocyanin a manipulable biomolecule for further usage. We believe with our effort in this project, plant-based water-soluble colorants, represented by phycocyanin (cyan to blue) and anthocyanin(pink to red), can be of increasing interest to academia, industry, and consumers and are a very important part of the "clean label" initiative. We are now pleased to see that more and more research teams around the world are following our research and that more and more companies are launching products with only natural colorants.With the confidence of knowing that natural pigments will go mainstream, we also noticed the sources of them - plants (especially algae), which are also increasingly receiving global attention, especially from international organizations, to close energy gaps and solve food security issues. We are confident that our findings will contribute towards a food revolution in the near future.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: " Shuang Zhang, Zhong Zhang, Younas Dadmohammadi, Ying Li, Archana Jaiswal, Alireza Abbaspourrad. Whey protein improves the stability of C-phycocyanin in acidified conditions during light storage. Food Chemistry 2020, 344, 128642. https://doi.org/10.1016/j.foodchem.2020.128642
• Type: Journal Articles Status: Published Year Published: 2021 Citation: " Ying Li, Zhong Zhang, Alireza Abbaspourrad. Improved thermal stability of phycocyanin under acidic conditions by forming soluble complexes with polysaccharides. Food Hydrocolloids 2021, 119, 106852. https://doi.org/10.1016/j.foodhyd.2021.106852
• Type: Journal Articles Status: Published Year Published: 2021 Citation: " Ying Li, Richard Gillilan, Alireza Abbaspourrad. Tuning C-phycocyanin photoactivity via pH-mediated assembly-disassembly. Biomacromolecules 2021, 22(12), 5128-5138. https://doi.org/10.1021/acs.biomac.1c01095
• Type: Journal Articles Status: Published Year Published: 2022 Citation: " Ying Li, Zhong Zhang, Alireza Abbaspourrad. Improving solubility and functional properties of phycocyanin under acidic conditions by glutaminase deamidation and succinylation. Food Hydrocolloids 2022, 133, 107994. https://doi.org/10.1016/j.foodhyd.2022.107994
• Type: Journal Articles Status: Published Year Published: 2022 Citation: " Ying Li, Zhong Zhang, Alireza Abbaspourrad. Improved pH stability, heat stability, and functionality of phycocyanin after PEGylation. International Journal of Biological Macromolecules 2022, 222(Part B), 1758-1767. https://doi.org/10.1016/j.ijbiomac.2022.09.261
• Type: Journal Articles Status: Published Year Published: 2022 Citation: " Ying Li, Alireza Abbaspourrad. Phycocyanin-rich water-in-oil-in-water (W1/O/W2) double emulsion with nanosized particles: Improved color stability against light exposure. Colloids and Surfaces B: Biointerfaces 2022, 220, 112930, https://doi.org/10.1016/j.colsurfb.2022.112930
• Type: Journal Articles Status: Submitted Year Published: 2023 Citation: " Ying Li, Qike Li, Richard Gillilan, Alireza Abbaspourrad. Reversible disassembly-reassembly of C-phycocyanin in pressurization-depressurization cycles of high hydrostatic pressure. Biomacromolecules (submitted)

Progress 06/01/21 to 05/31/22

Outputs
Target Audience: Food processing companies interested in adopting new sustainable technologies like HPP Food ingredientcompanies interested in clean-label formulation using natural colorants Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Encouraged by our previously demonstrated stabilization effects of high pressure on food colorants, our group now took a strong and active interest in gaining further insights into the structural and spectroscopic mechanisms of these stabilization effects. Fundamentally, we took advantage of our access to the high-pressure SAXS and spectrophotometers at the Cornell High Energy Synchrotron Source (CHESS), which offered in-situ, visualizable, and quantitative analyses on the high-pressure effects. The project engages students in fundamental research that intend to offer insights into the mechanisms behind the high-pressure effects. Students are trained to do protein structural analysis using instruments such as circular dichroism, fluorimeter, SAXS, etc., and to analyze the data acquired comprehensively in order to offer valid explanations and gain fundamental understandings of the experimental phenomenon they observe. How have the results been disseminated to communities of interest?The project facilitates the future application ofHPPin the food industry and other related fields. From the application point of view, the high-pressure structures and assembly-disassembly pathways revealed by SAXS can be used to predict the stability and aggregation propensity of C-phycocyanin under different product development and processing conditions. Also, irreversible denaturation of C-phycocyanin that was detected at pressures above 400 MPa dictates that the commonly used high-pressure pasteurization techniques, which kill spoilage microorganisms in foods by pressurization at 400-600 MPa, shall not be directly applied to C-PC-containing products without any additional stabilizers. In addition, the demonstrated ability of hydrostatic pressure to control the assembly and folding of C-phycocyanin may be harnessed to design new optical probes and delivery vehicles, and thus propel the application of food colorant in exciting new directions in biomedical and material sciences. What do you plan to do during the next reporting period to accomplish the goals?According to our previous results, HP effects can significantly be affected by solution conditions (e.g., pH and temperatures).Currentresearchin our lab seeks to explore the pressure responses of C-phycocyanin in a more physiologically relevant pH range, as well as to explore the combined effect of pressure and other physical or chemical stimuli, such as temperature, salts, protein denaturants. We expect to further expand theoutcome ofthis workand achieve moreversatile and application-centered turnability from C-phycocyanin. In addition, we plan to apply the HP SAXS and spectroscopic techniques to other natural colorants to explore their dynamic structural changes, reversibility, and interactions with other food ingredients.

Impacts
What was accomplished under these goals? We characterized the dynamic changes in the structure and photochemical properties of C-phycocyanin using high-pressure small-angle X-ray scattering (HP SAXS) and high-pressure spectroscopies (UV-vis and fluorescence). The findings are categorized as follows. First, we revealed that pressure up to 350 MPa was enough to fully disassemble C-phycocyanin from trimers to monomers at pH 7.0, or from monomers to single subunits at pH 9.0, with concomitant unfolding that renders high-pressure structures more extended. Second, pressurized C-phycocyanin showed decayed Q-band absorption and growths in the Soret band, evidencing the folding of tetrapyrroles from linear conformations, in ambient structures; to semicyclic conformations, in unfolded, disassembled high-pressure structures. Blue-shifted Q-bands at pH 9.0 evidence fully decoupled chromophores across the pressure-dissociated subunits. Third, the structural characterizations using circular dichroism, and protein intrinsic fluorescence confirmed the onset of irreversible denaturation of C-phycocyanin at pressures above 400 MPa, particularly at 500 MPa and 600 MPa. Whereas both pH 7.0 and pH 9.0 samples showed the tendency of unfolding at the higher pressures, the unfolded chains at pH 9.0 further reaggregated under the nonspecific binding of the exposed hydrophobic patches. Fourth, the 400-500 MPa pressurized C-phycocyanin showed improved emulsifying and foaming properties to different degrees, demonstrating the effectiveness of high hydrostatic pressure on structural modification of C-phycocyanin.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Tuning C-Phycocyanin Photoactivity via pH-Mediated AssemblyDisassembly
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Improved thermal stability of phycocyanin under acidic conditions by forming soluble complexes with polysaccharides

Progress 06/01/20 to 05/31/21

Outputs
Target Audience:Food industries that use synthetic colorants. Changes/Problems:SAXS and HP SAXS are involved to further characterize the high-pressure effects on phycocyanin structures. The reason for this change is summarized as follows. The proposed conventional protein characterization techniques do not allow adequate quantitative characterization of protein conformational flexibilities (e.g., shapes, folding and unfolding states) that govern the solution behaviors and interfacial activities of proteins; however, this structural information could be crucial in this work to understand how HHP treatments affect the stability and functionalities (e.g., the emulsifying and foaming properties) of phycocyanin. Besides stabilization, we propose to further use HPP for the functionalization of phycocyanin. The food applications of phycocyanin are currently limited to be used as color additives in products like chewing gums, candies, and beverages. Few efforts have been made to further broaden the use of phycocyanin as food emulsifiers, foaming agents, or texture ingredients. Besides, how structural modification and food processing would affect the functionalities of phycocyanin are scarcely understood, highlighting the need for more research in this area. What opportunities for training and professional development has the project provided?The project opens important frontiers in the food and pharmaceutical industries for the processing and stabilization of food colorants. The project indirectly supports several undergraduate and graduate students and post-doctoral scholars, emphasizing cross-training in conceiving experiments, data acquisition, and data analysis. The project engages students in interdisciplinary research. It inspires students to think of more ways to design experiments and understand the mechanisms behind the high-pressure effects on the natural colorants. Students are encouraged to apply their biology, chemistry, and physics knowledge and interdisciplinary skills to promote societal benefit. They are trained to do electrophoresis and use instruments such as colorimeter, CD, FTIR, fluorimeter, and SAXS, etc. How have the results been disseminated to communities of interest?Our research group has demonstrated the remarkable effectiveness of HPP on the extraction, color stabilization, and functionalization of phycocyanin. Particularly, the HP SAXS characterizations provide fundamental insights into the high pressure induced proteins' structural changes; the results help dissect the structural basis behinds the color changes of phycocyanin under HPP. These results have been discussed with our food industry partners and presented in a couple of webinars at Cornell. What do you plan to do during the next reporting period to accomplish the goals?Encouraged by the above achievements, our group is now taking a strong and active interest in gaining further structural insights into the HP effects on phycocyanin in order to comprehensively understand the mechanisms behind the stability and functional changes. Firstly, our previous results given by SDS-PAGE, CD, and intrinsic fluorescence reveal that HHP-induced structural changes on phycocyanin vary according to not only the pressures applied but also the solution conditions (e.g., pH and temperatures). Therefore, we propose to further explore the influence of pH and temperature on the high-pressure effects using HP-SAXS. Secondly, functionalities of proteins (e.g., solubility, emulsifying ability, and foaming ability, etc.) are determined by their assembly and folding-unfolding states. We plan to further investigate the changes in phycocyanin's functionality as a result of the high-pressure treatments.

Impacts
What was accomplished under these goals? Firstly, we have investigated the effect of high-pressure processing (HPP) on the structure and color stability of phycocyanin, phycocyanin-whey protein, and phycocyanin-carrageenan mixtures. Samples at pH 3.0 exhibited minimal color loss and minor structural changes during HPP treatment, whereas the said characteristics at pH 5.0 and 7.0 are not stable under the same HPP treatment. Secondly, we also studied the stability of these HPP-treated samples under natural light exposure. It was found that the light stability of the phycocyanin-whey protein mixture at pH 5.0 was significantly improved after HPP treatment at both 450 MPa and 600 MPa. Thirdly, using high-pressure small-angle X-ray scattering, we have characterized the structural change of phycocyanin during pressurization. It was found that hydrostatic pressures of 300 MPa were sufficient to induce reversible dissociation or unfolding of C-phycocyanin (C-PC).

Publications