UPCYCLING AGRO INDUSTRIAL BYPRODUCTS TO DEVELOP THE NEXT GENERATION OF FOOD COLORANTS DERIVED FROM NATURE.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1030183
• Grant No.: 2023-67017-39862
• Cumulative Award Amt.: $600,000.00
• Proposal No.: 2022-09221
• Project Start Date: May 1, 2023
• Project End Date: Apr 30, 2027
• Grant Year: 2023
• Program Code: [A1364]- Novel Foods and Innovative Manufacturing Technologies

Project Director
Giusti, M. M.

Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691

Performing Department
(N/A)

Non Technical Summary
This proposal is designed to find easy, effective and economic ways to produce better natural or nature derived colorants that the food industry can use as alternatives to the use of synthetic colorants. The food industry needs efficient alternatives to synthetic food colorants to address consumer demand for safer, naturally sourced ingredients and changing regulatory constraints. Yet, most colorants from nature have limited stability, vibrancy, and compatibility with food applications. Our overall objective is to develop a novel class of food colorants derived from nature by upcycling waste materials from agricultural and food processes. In the 3.68-billion-dollar natural colors market, anthocyanins (ACNs)--a class of colored plant antioxidants abundant in nature --emerge as potential replacements with vivid orange, red, purple and blue colors and with health-enhancing properties. However, anthocyanins have limited stability when used to color foods, restricting their applications. Pyranoanthocyanins (PACNs) are ACN-derived pigments with vibrant colors, improved stability to heat, pH, and storage, and higher versatility making them better candidates for food coloring. PACNs are commonly formed during wine processing and aging, and therefore they have been consumed for centuries, and help make the color of wines very stable through time. PACN have also been found in some plant materials, but they are present in very low concentrations. PACN show great potential as food colorants, but their scares availability make them hard to commercialize. Therefore, our first objective is to produce PACN from the reaction of the abundant anthocyanins with other commonly found phenolic compounds. We want to produce PACN efficiently, identifying the more favorable compounds to use for their production (ACN and cofactors) and providing the right environment for their formation. Second, we will characterize color and stability of different PACNs and their interaction with food macromolecules (lipids, proteins, and carbohydrates). Our third objective will develop a method to scale-up PACN-based colorant production from agro-industrial byproducts. We will use different strategies, such as alkaline hydrolysis and lactic acid bacteria, to release ACNs and cofactors from ACN-rich waste streams and favor their interaction.Our proposal will help improve our understanding of the chemical properties of novel food ingredients. By producing food colorants with improved stability, we will be improving the quality, shelf-life, and sensory attributes of novel food ingredients. Improving PACN production will also add value to agro-industrial byproducts, and therefore, develop innovative manufacturing technologies to increase productivity and food quality and minimize waste.Overall, we provide novel, stable, and naturally derived PACN-based food colorants, improving the environmental sustainability, accessibility, and applications of food colorants.

Animal Health Component

40%

Research Effort Categories

Basic

40%

Applied

40%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
501	5010	2000	100%

Knowledge Area
501 - New and Improved Food Processing Technologies;

Subject Of Investigation
5010 - Food;

Field Of Science
2000 - Chemistry;

Keywords

anthocyanins

color

pigment

pyroanthocyanins

waste

Goals / Objectives
The long-term goal of the proposed research is to develop a novel class of food colorants with increased versatility by transforming waste streams into value added ingredients. In doing so, we hope to contribute to improved environmental sustainability, accessibility, and application options for naturally derived food colorants. These colorants will be produced by transforming the natural plant pigments, anthocyanins, using a reaction that naturally occurs in wine, a condensation reaction, to form the more stable pyranoanthocyanins (PACN).The supporting objectives and the hypotheses of the proposed projects are:1. Optimize PACN formation processes by identifying the best reactants and environmental conditions. We hypothesize that certain combinations of ACNs and cofactors will result in faster PACN production and higher yields, especially those that facilitate interaction between reactants (particularly elevated temperatures and agitation). The solution pH will increase abundance of the more reactive ACN and cofactor forms, although the optimal pH will differ depending on starting reactants. Based on our preliminary data, we also hypothesize that decarboxylated cinnamic acids will be more efficient cofactors than their precursor acids.2. Characterize the color, stability, and solubility of PACNs by focusing on the influence of PACN chemical structure and its impact on behavior in food matrices. We hypothesize that each PACN will produce a unique set of colors across pH values, and their unique structures will give different levels of solubility and compatibility in food applications. We believe that PACNs with less sugars and more aromatic rings will have lower solubility in water and may interact better with matrices containing lipids or proteins while precipitating in water. On the contrary, PACNs with more sugars and hydroxyl groups will remain more soluble in water, making them more suitable for water-based matrices. We also predict that PACNs will be able to provide color for a much wider range of pH values (1-9) than their precursor ACNs, and therefore, be better options for coloring a wider range of food applications.3. Develop a novel production process for PACN-based food colorant production by upcycling agro-industrial byproducts using lactic acid bacteria and biotechnology. We hypothesize that insoluble plant residues obtained from agro-industrial processes will contain phenolics trapped in macromolecules that could be released through hydrolysis and fermentation to become the cofactors for PACN formation. Waste streams from colorant production will contain both residual ACNs and the cofactors to use for PACN formation. We hypothesize that a bioreactor can be used to favor the transformations and interactions to produce PACNs from waste streams.

Project Methods
Pyranoanthocyanins (PACNs) are ACN-derived pigments with vibrant colors, improved stability to heat, pH, and storage, and higher versatility making them better candidates for food coloring.Our first task will be to optimize PACN formation efficiency, identifying favorable reactants (ACN and cofactors) and environments. We will choose different anthocyanin sources, and different cofactors, and will incubate them using a bioreactor. We will determine the ideal structures for the reaction as well as the best proportions of anthocyanin:cofactor to increase the production of PACN, the formation rate while minimizing the total pigment loss. We will use spectrophotometric techniques to monitor the changes in color and color loss. We will also use liquid chromatographic techniques (HPLC) coupled to photodiode array and mass detectors to monitor the changes in chemical composition.Second, we will characterize color and stability of different PACNs and their interaction with food macromolecules (lipids, proteins, and carbohydrates). Our hypothesis is that PACN will work well as food colorants, and foods are complex matrices. We need to explore and understand how PACN interact with different macromolecules typically found in foods. PACN extracts will be mixed with different proteins, carbohydrates and lipids and their interactions will be monitored. Major endpoint that will be evaluated include solubility, color expression, color loss and pigment stability.Our third objective will develop a method to scale-up PACN-based colorant production from agro-industrial byproducts. Alkaline hydrolysis and lactic acid bacteria will be used to release ACNs and cofactors from ACN-rich waste streams and favor their interaction. In this objective we will be working with a bioreactor that allows for bacterial growth under controlled temperature and agitation conditions, while monitoring optical density. We will attempt to release anthocyanins and the necessary cofactors for PACN production from agro-industrial by products. We have key collaborators from the food industry that will be providing us with the byproducts so that our results are most relevant to the industry. We will be measuring as endpoints the production of the PACN precursors from the controlled fermentation using lactic acid bacteria as well as the potential production of PACN either from the precursors generated, or from the byproducts themselves directly.

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:Our target audience includes different groups, described below: The Food Colorant Industry is a key target audience as they can directly benefitby our work. We have requested samples from different companies to make sure that the research is relevant to the food industry.We maintained contact with food colorant manufacturer's and several have expressed interest in the technology we are developing, and the potential products we may produce. The food industry in general is an important audience, as they would be the ones to use the colorants we want to produce. They have expressed interest in our work, particularly for certain applications where heat treatments are prolonged, or where ascorbic acid can accelerate color degradation. We have reached the scientific communityas we gather informaiton and prepare to present it at different prefesional conferences. For now, we have presented our workat local and national meetings, but we are getting ready to present at some international venues. Changes/Problems:At this state we have found several challenges that we are working to overcome, but nothing that we think wll force a chance in direction for the project. The process of securing byproducts to evaluate has been slower than anticipated, with some companies being reluctant to share materials with us. Also, we have demonstrated that we can produce PACN from waste materials with the aid of lactic acid bacteria, but the yields are very low. So we will continue exploring different strategies to increase the efficiency of our methods. What opportunities for training and professional development has the project provided?We currently have 4 graduate students working in this project. The post doctoral researcher that helped design the project was recruited to work for the food industry, in part because of his knowledge of this technologies. However, we have been able to maintain a chain of knowledge transfer, with an advanced level PhD candidate leading the bulk of the laboratory work with the aid of the other more junior 3 students, who are now better prepared to continue the work. In addition, we have received 2 undergraduate studetents in the lab, and they are also training in this technology. The knowledge generated in this project is being incorporated into my graduate level class about food colors and pigments, and has already been presented at local conferences with more conference presentations to come. How have the results been disseminated to communities of interest?The knowledge generated in this project is being incorporated into my graduate level class about food colors and pigments, and has already been presented at local conferences. We have submitted abstracts to present additional results at the Annual meetin of the Institute of Food Technologists (IFT), and at the BioColours2024 conference in Finland. We expect to continue to disseminate this knowledge at other scientific conferences, and of course with publication in the near future. What do you plan to do during the next reporting period to accomplish the goals?We will continue to optimize the production process as part of goals 1 (optimization under controlled conditions and reagents) and 3 (optimization using by products from the agro-industry with the aid of lactic acid bacteria). We have contacted different companies to request samples of their waste streams to explore what starting materials might be most effective for our purposes. We are generating a database of the color and stability characteristics of the different pyranoanthocyanins that we are generating. In addition, we are plannign to explore the biological activity of select pyranoanthocyanis. We hypothesize that they may have some health promoting properties similar to their anthocyanin precursors, or even more potent.

Impacts
What was accomplished under these goals? Goal 1: We have strong evidence that hydroxycinnamic acids are very reactive cofactors for pyranoanthocyanin (PACN) formation, much more than other compounds such as pyruvic acid. However, decarboxylation of those hydroxycinnamic acids to form 4-vinyl phenols results in the production of even more reactive cofactors. We have successfully formed PACN from different 4-vinylphenols, and we can detect their presence in just a few hours when the pH and the temperature are favorable (preferably pH close to 3.5 and 45 degrees celcius). In addition we have proven that some anthocyanidins are more prone to form pyranoanthocyanins than others, with malvidin being the most reactive followed by cyanidin. We also evaluated the role of anthocyanin glycosylation, and it seems to be minor as compared to the other factors, Goal 2: Great progress has been made in this area. Different PACN have been produced from the reaction of different anthocyanins, with different glycosylation patterns with different hydroxycinnamic acids. We have demonstrated that most PACN have spectral characteristics that show a hypsochromic shift as compared to their precursor anthocyanin, meaning they look more yellow-orange while the anthocyanins show colors closer to red. In addition, anthocyanin fade in color as pH increases from 1-4, but this fading is not observed with PACN, that keep their color vibrancy while pH increases, finally turning towards a more purple violet color at neutral to alkaline pH. Our data also supports our hypothesis regarding PACN solubility: PACN with di- or tri-glycosylation show more solubility in water than monoglycosylated ones. We think those PACN with less water solubility may be more suitable for lipid based applications, and some preliminary data suggests this may be correct. Goal 3: We have started exploring PACN production from some waste materials. We used grape pomace and blueberry pomace to release hydroxycinnamic acidsfrom complex macromolecules, as well as some residual anthocyanins. We have used alkaline hydrolyses to facilitate this process. We have then used select species of lactic acid bacteria to facilitate the decarboxylation of those hydroxycinnamic acids. Lactic acid bacteria grows best at pH closer to 6 and temperature of around 32 Celcius, not the ideal conditions for PACN production. We are testing the bacteria for growth and decarboxylation ability at lower pH levels. In addition, their growth will further contribute to lower the pH further to levels more favorable for PACN formation. Our preliminary tests suggest we might be able to use complex plant residues containing anthocyanis, and add lactic acid bacteria to favor the release of hydroxycinnamic acids, induce their decarboxylation and then increase the temperature to facilitate PACN formation.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Voss, DM., Giusti, MM. (April, 2024). Glycosylation impacts color and solubility of wine-inspired pyranoanthocyanin colorants. Presented at The Ohio State University College of Food, Agriculture, and?Environmental?Sciences Annual Research Conference. Columbus, OH.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Voss, DM., Giusti, MM. (March, 2024). Evaluating pyranoanthocyanins as innovative, naturally derived colorants for foods. Presented at 38th Edward F. Hayes Advanced Research Forum. Columbus, OH. Placed 3rd.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Porras-Guardado, C., Fan, X., Giusti, MM. (April, 2024). Decarboxylation of Hydroxycinnamic Acids by Lactic Acid Bacteria Strains under Acidic Conditions. Poster presented at The Ohio State University College of Food, Agriculture, and Environmental Sciences Annual Research Conference. Columbus, OH.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: " Giusti, MM (2023). Production of Anthocyanin-Derived Colorants from Byproducts of the AgroIndustry. Virtual presentation at 15 SLACAN, Simp�sio Latino Americano de Ci�ncia de Alimentos e Nutri��o (International Symposium of Food Science and Nutrition). Campinas, Brazil. November 14, 2023. Online.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: " Giusti, MM (2023). La Extraordinaria Estabilidad de los Pigmentos del Vino y su Potencial Comercial (The extraordinary stability of wine pigments, and their commercial potential). Diplomado Internacional de Marketing y Comercializaci�n de Destilados. Universidad Privada San Juan Bautista, Ica, June 23, 2023.