Source: PERSEA NATURALS LLC submitted to NRP
THE GREEN PROCESSING OF A NOVEL FDA EXEMPT ("NATURAL") FOOD COLOR ADDITIVE.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1020202
Grant No.
2019-33610-30183
Cumulative Award Amt.
$650,000.00
Proposal No.
2019-02301
Multistate No.
(N/A)
Project Start Date
Sep 1, 2019
Project End Date
Aug 31, 2021
Grant Year
2019
Program Code
[8.5]- Food Science & Nutrition
Recipient Organization
PERSEA NATURALS LLC
102 OAK POINTE CIR
STATE COLLEGE,PA 16801
Performing Department
(N/A)
Non Technical Summary
Color plays a key role in consumer perceptions of food safety and quality. Synthetic color additives are easy to produce, stable, less expensive and have better coloring properties than natural color additives. Color additives exempt from certification include those derived from plant, animal or mineral sources and synthetic "nature-identical" compounds. Currently, 26 natural color additives are permitted for use as exempt color additives in the United States. Technical advances in natural colors have allowed for better shades, stability and cost. Despite increased interest in natural color additives, there remain unmet needs for certain applications, e.g. the number of red, green, and blue pigments is limited.There isgrowing consumer demand for "clean-label" foodingredients including color additives derived from natural sources. Colorant-related issues represent3 of the top 10 consumer concernsabout food ingredients. Fruit/vegetable-derived anthocyanins, carotenoids, and betalains have been employed as replacements for FD&C yellows and reds in food products. However, there are challenges to the use of these alternatives including poor temperature, light, and oxygen stability; unfavorable solubility (i.e. hydrophobic carotenoids and curcuminoids for hydrophilic applications); and cost of raw materials. Due to the susceptibility of natural plant-based pigments to heat, light, extreme pH and high water activity, convenient solutions for color applications are often highly intricate requiring substantial efforts of research and development.The source material for some natural color additives is expensive. For example, saffron, which contains the pigment crocin, is the hand-harvested stamina of the saffron crocus and its wholesale cost ranges from $500-$5000/lb (ca. $225-$2250/kg) (https://www.thedailybeast.com/in-search-of-the-dollar10000-spice). Some natural colors are derived from animal sources (e.g. carminic acid) and are unacceptable to certain segments of the consumer market such as vegetarians and have a potential risk of allergic reaction. Although artificial color additives have a long history of safe use, consumers increasingly consider them undesirable and major food ingredient companies have made significant investments in the development of alternatives. This change in consumer desire is reflected in the global food colors market, as natural food colors have begun to dominate, increasing from 54.9% market share in 2014 to a predicted 70% by 2020, with particular interest in compounds responsible for yellow, orange, red, and pink colors (Future Market Insights, Inc. 2015, MarketsandMarkets.com 2015).AvoColor® is a red-orange semi-purified avocado (Persea americana, Lauraceae) seed extract with potential application as a natural food color additive.The preliminary safety of AvoColor® has been examined with a 15-d subacute study in male and female CD-1 mice using the dietary route of administration. AvoColor® was well-tolerated in the diet at levels of up to 5% w/w. AvoColor® has been shown to have excellent color value in a wide variety of food products including confections, beverages, and baked goods, and is stable to light, oxygen, and temperature.AvoColor® has the potential to meet consumer demands for "clean-label" food ingredients with appealing aesthetic properties (i.e. vibrant colors) and food manufacturers' requirements for food color additives with excellent stability characteristics. In addition, AvoColor® is produced from avocado seeds, a raw material that has no alternate use or value and is currently being landfilled by avocado processors.Analytical data necessary for the preparation of a color additive petition to FDA will be obtained through validated (HPLC, MS, NMR) and standard methods (e.g. A.O.A.C.). Interpretations of those data and the production/commercialization process improvement will be shared with the target audience through conferences (IACM Global color conference, etc...), video/publication materials in PerseaNaturals website, Scientific publications in peer-reviewed journals, Seminars, interviews, Press releases.
Animal Health Component
30%
Research Effort Categories
Basic
10%
Applied
30%
Developmental
60%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5021099115030%
5021099200030%
5011099202020%
6031099101020%
Knowledge Area
603 - Market Economics; 501 - New and Improved Food Processing Technologies; 502 - New and Improved Food Products;

Subject Of Investigation
1099 - Tropical/subtropical fruit, general/other;

Field Of Science
2020 - Engineering; 1010 - Nutrition and metabolism; 2000 - Chemistry; 1150 - Toxicology;
Goals / Objectives
The overall goal of the proposed studies is to commercialize a "green" process for the safe and efficient preparation of a food color additive derived from avocado (Persea americana) seeds and bring AvoColor® to market in Phase III.The proposed studies are responsive to the SBIR Program Priority topic area 8.5 Food Science and Nutrition and align with research priorities in agriculturally-relatedmanufacturing technology. The proposal supports the objectives of the SBIR program to increase private sector commercialization of new products derived from USDA-supported research and development efforts that enhance the value of foods, and the development of technologies and services that protect or enhance the environment while promoting economic development through sustainable practices and waste reduction.
Project Methods
1. ToxicologystudyThe ongoing 90-dayssub-chronic toxicology study in Sprague-Dawley rats conducted by a GLP-compliant organization (Integrated Laboratory Services Inc.) will benecessary for preparing a Color Additive Petition for submission to the FDA Center for Food Safety and Applied Nutrition.2.Impurity analysisAs with our initial identification of perseorangin (Hatzakis et al. 2019), a combination of NMR and MS methods combined with chromatographic separations will be used to identify the contaminants in AvoColor®. The global biochemical profile of AvoColor® will be elucidated using an integrated LC-MS/NMR approach. We already have a library of masses attributed to the non-purified colored extract (Hatzakis et al. 2019) and some of these masses are expected to appear in the final product. High-resolution MS analysis will provide a list of potential molecules appearing in the product mixture. 1D NMR with an internal standard will be used for quantification and 2D NMR will be applied for structural analysis using concentrated samples. For impurities that appear in very small amounts in the mixture, LC-MS/MS will be applied. If extra structural analysis with NMR is required, the minor impurities will be isolated using large amounts of sample to deal with sensitivity challenges. Several recent publications (Ramos-Jerz 2007; Figueroa et al. 2018A & B; Lopez-Cobo et al. 2016) provide a comprehensive characterization of 100s of compounds present in the seeds of avocado with particular emphasis on phenolics including mass spectra fragmentation patterns and, in some cases, NMR spectra. We will use this extensive compositional data to guide our analyses. To determine if the compound observed in mouse feces at a retention time = 41.5 min was the aglycone, the aglycone of perseorangin will be prepared using b-glucosidase from almonds (Sigma-Aldrich, Cat# G0395). In brief, samples of AvoColor®will be incubated with b-glucosidase at pH 6 - 7 and 37 °C (Freischmidt et al., 2015; Ahn-Jarvis et al., 2017). The conversion will be monitored using our HPLC-UV/Vis method. The identity of the aglycone will be confirmed by LC-MS analysis. LC-MS and NMR data collection will be outsourced but experiment design and data interpretation will be done in-house.3.Techno-economic analysisWe will conduct a techno-economic analysis using SuperPro Designer software by Intelligen (McNulty 2018). SuperPro Designer facilitates modeling, evaluation, and optimization of integrated batch and continuous processes in a wide range of industries including food and pharmaceuticals (http://www.intelligen.com/superpro_overview.html). Shaklin et al. (2000) quantitatively evaluated Aspen Batch Plus and SuperPro software packages and found them well suited to perform basic material and energy balances, debottleneck, explore equipment/facility change outs, and perform economic analyses.4.Shelf life studiesWe will conduct shelf life studies on the AvoColor® product in its dry form stored in appropriate packaging at different temperatures. We will monitor its composition by HPLC, with attention directed to the appearance of any degradation or reaction products not present in the initial sample. Usually, a two-year study, the length of this Phase II project, is sufficient for this purpose (FDA 2009). We will also conduct stability studies in food matrices into which we propose to incorporate AvoColor®. The length and conditions of these tests will be determined by the matrices selected (Steele 2004). In these tests, we will evaluate the effect of other food ingredients, especially food acids and antioxidants.5.Exposure level estimationThe petition must include the amount of the color additive proposed for use and the color effect intended to be achieved, together with all directions, recommendations, and suggestions regarding the proposed use. We must establish the food products most suitable for incorporation of AvoColor® and determined appropriate use levels, after which we will follow FDA's Guidance for Industry: Estimating Dietary Intake of Substances in Food (https://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/ucm074725.htm). We have contracted with Intertek Scientific & Regulatory Consultancy Services for regulatory guidance and, as members, have assistance from the International Association of Color Manufacturers (IACM).6.NaOH stabilization studyThe color of AvoColor® is "stabilized" in a reaction with NaOH. This change is irreversible with re-acidification. The precise chemical changes occurring during this stabilization process are unknown. Given the chemical structure of the major colored compound found in AvoColor®, the major chemical changes expected to occur during the NaOH stabilization process are hydrolysis/de-esterification, polymerizations, and alterations in the affinity of cations that are affected by the pH values and are known to affect the color properties of colored molecules. Reaction monitoring and pre-post treatment comparisons will be performed using NMR spectroscopy. NMR spectroscopy is an excellent tool for real-time reaction kinetic monitoring and thus hydrolysis products will be identified by their characteristic signals at the 1H NMR spectrum. For studying potential polymerization we will use diffusion NMR as molecules with different degrees of polymerization will be well separated in a DOSY spectrum. In cases that monomers and dimer-polymers can form positive or negative ions, the NMR results will be further confirmed with high-resolution MS. The affinity of colorants with various cations will be studied using NMR since these changes will be reflected on the chemical shifts of nuclei that appear close to cations. Mass spectrometry will be also able to identify these changes as the bonding with different cations will result in different MWs. The cation profile of the mixture is important for drawing conclusions about how various metals/cations are bound to the colorant molecules and will be performed using ICP-MS. LC-MS, ICP-MS and NMR data collection will be outsourced but experiment design and data interpretation will be done in-house.7.SOPsThe petition must include a full description of the methods used in, and the facilities and controls used for, the production of the color additive. Following industry standards, we will write Standard Operating Procedures (SOPs), including production controls, necessary to maintain conformance with product specifications. Specifications for inputs e.g. seeds and reagents, and analytical procedures will be determined. Standard Sanitation Operating Procedures will be drafted.8.Methods developmentMethodologies to determine the identity and quantity of AvoColor® in any article of food and any substance formed in or on such article because of the use of AvoColor® will be based on those currently available for existing colorants (e.g. Yamjala et al. 2016). We have already developed such methodologies for quantifying AvoColor® in rodent food and feces (see above).9.Chemical specifications Generally, it consists of the following aspects of color additive composition (as appropriate):Volatile matter;Soluble, extractable or insoluble matter;Residual salts;Soluble impurity;Unreacted intermediates and related compounds;Subsidiary colors;Individual components that are included in the identity of the color additive;Pesticide residues;Solvent residues;Ash;Heavy metals;Total color content.

Progress 09/01/19 to 08/31/21

Outputs
Target Audience:As a small business, our target audience is potential customers, in this case, food color and consumer food product manufacturers. Food color additives require pre-market approval, so our audience also includes the Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A M.S. student in chemical engineering at UC-Davis was supported to conduct the techno-economic analyses. How have the results been disseminated to communities of interest?Results of this research have been disseminated at quarterly meetings of the International Association of Color Manufacturers (IACM); at the IFTNEXT Food Disruption Challenge™ competition (April 2020), at the Zoom conference of the Produce Marketing Association (July 2021), as well as, confidentially to potential customers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1:The qualitative and quantitative phytochemicalanalysis of multiple batches clearly illustrates the consistency in the composition of AvoColor®. Twenty-eight compounds were identified in AvoColor®. It contains classes of polyphenols including phenolic acids, flavonoids, and A- and B-type proanthocyanidins (PACs) in addition to perseorangin, the major colored compound. Perseitol, a 7-carbon sugar alcohol, was the most abundant component detected in AvoColor®. Persin, a polyhydroxylated lipid of reported toxicological concern found in avocado seeds, was not detected in AvoColor®. A number of phytochemicals found in AvoColor® are being actively investigated for their anti-inflammatory, cardioprotective, diabetes and cancer preventive potential (e.g. chlorogenic acid, epicatechin/catechin, and PACs). Objective 2: A computer model for process simulation and economic analysis was generated using Intelligen's software SuperPro Designer, Version 10. The model was used to determine equipment sizing, equipment process parameters, operations scheduling and raw material requirements. An economic analysis was performed, including the determination of the total capital expenditure (CAPEX), the total operating expenditure (OPEX) and the cost of goods sold (COGS). The breakdown of all costs (raw materials, consumables, utilities, labor, waste disposal, and various facility dependent costs) with the goal of identifying the materials and process steps which contribute most significantly to the total cost of production was performed. The sensitivity analysis was executed on several process parameters and design assumptions to assess their impact on the COGS and the optimal case scenario was proposed. For all scenarios analyzed, the base case for a green-field site, which does not include depreciation, local taxes, or insurance, gave a base COGS of $116.70 per kg of AvoColor®. Having to pay for the avocado seeds could increase the COGS. For example, if the price of the seeds is $0.41 per kg, the COGS will increase to $150 per kg. The proposed optimistic scenario resulted in a COGS of $74 per kg AvoColor®. The technoeconomic analysis stimulated a redesign of the process to eliminate several operations and dramatically reduce the consumption of ethanol as a solvent, further reducing the COGS to below $50. Objective 3:A long-term (2-year) stability study of the quality of AvoColor® under the influence of a variety of environmental factors such as temperature and light, was performed. An HPLC-based analytical method with an internal standard molecule (purpurogallin) was developed and used for the analysis of AvoColor® every 6 months. After 2 years of storage at 25°C or 50°C, AvoColor® showed minimal to no changes in its composition profile and its performance. The ideal condition of storage could be in air tight container (to avoid moisture) for a duration of up to 2 years at 25°C. Under extreme heat conditions, AvoColor® showed an increase in color intensity instead of the color bleaching. Potential color stabilizers were explored. Objective 4:A 90-day sub-chronic oral toxicity study in rats (males and females) was conducted to determine the repeated-dose oral toxicity and genetic toxicity of AvoColor® up to 5% in the diet. Body weights and clinical observations, ophthalmological examinations and neurotoxicity screening with motor activity assessment were performed. After euthanizing animals, the collected tissues were microscopically examined for signs of toxicity. The hematology, clinical chemistry, hormone measurements and genotoxicity were also assessed. Based on the results of this study, including evaluations of clinical observations, body weights, clinical chemistry, urine, micronucleus assay, comet assay, organ weights, functional observation battery, and histopathology, the administration of AvoColor® at doses of 0.1 to 5.0 % was unlikely either to present a neurotoxicity hazard, or to exhibit any signs of toxicity. The No-Observed-Adverse-Effect-Level (NOAEL) for AvoColor® was 2620.6 mg/kg/day for males and 3218.4 mg/kg/day females. The Acceptable Daily Intake (ADI) for humans was estimated at 26 mg of AvoColor®/kg/day. Based on the physical properties of AvoColor®, beverages are a suitable application model. The exposure level was estimated from the publicly available data of 2-day food consumption for the US population (2009-2012) and the intake of FD&C color tartrazine (yellow #5) and sunset yellow FCF (yellow #6) for different age groups of the same population. The cumulative maximum intake of AvoColor® ranged from 0.3 to 1.42 mg AvoColor®/kg bw/day (for all food categories); and the specific daily intake for drinks and beverages ranged from 29 to 292 μg AvoColor®/kg bw/day. These values represent a small fraction of the acceptable daily intake for humans. Objective 5:The chemical changes occurring during the optional alkaline treatment (NaOH stabilization step) were investigated using a previously described UHPLC -ESI-MS methodology. The NaOH stabilization process increased the specific concentration of perseorangin in AvoColor® and improved the purity by reducing the level of contaminant species. The effect of other alkaline compounds such as calcium hydroxide (Ca(OH)2) and potassium hydroxide (KOH) was explored and compared with the NaOH treated sample. There was no effect of other alkali (Ca(OH)2, KOH) on the color according to CIELAB values of finished products redissolved in water. Objective 6:The detailed description of the AvoColor® production process was written. Objective 7:The method, which consists of separating AvoColor® from the beverage matrix using a specific solid phase extraction (SPE) C-18 column (esprep, 6mL), and quantifying the recovered AvoColor® was developed and validated by evaluating the criteria of linearity, the repeatability, and the reproducibility. The range of the AvoColor® concentration was from 25 to 1000 µg/mL. The standard molecule successfully used was purpurogallin because of its physical and structural properties similar to AvoColor®. A relation between the concentrations of AvoColor® and purpurogallin was established. The variability within a day (repeatability precision) and between days (reproducibility precision) was assessed by estimating the coefficient of variation (CV). The coefficient of variation (CV) of the 3 different mixes measured on a single was 9.8%, 11.5% and 5.2%. The CV between days was 11%. A value of CV below 20% usually reflects a repeatable and reproducible method. Objective 8:The chemical specification of AvoColor® consisted of the evaluation of parameters such as: pesticide levels, heavy metals, and element analysis; the proximate analysis which consists of fat, volatile lipid, moisture, protein, sugar, calories, total carbohydrate, total phenolic and organic acid profile; the residual solvent and the microbiological profile, using standard methods of analysis. Up to 4 composited batches of AvoColor® were used for the tests and the parameters were expressed as mean + 2 standard deviation. The presence of 300 different pesticides were screened and their level was less than the limit of detection, 0.01 ppm. Heavy metal levels were well below the recommended FDA level in a food color additive. The minerals and residual ethanol were not at a concerning level, and the steam volatile oil was within the range for the color additive exempt from certification as defined by the FDA. The count for anaerobic, aerobic, coliforms, E. coli, mold and yeast was lower than the reporting limit (<10 CFU/g of AvoColor®). The cyanogenic and hydrocyanic acids were not detected in AvoColor® although tannic acid was reported at a level 0.24%.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Kemgang T, Dodds JW, Kennett MJ, Reidl KM, Hatzakis E, Ziegler GR, Lambert JD. A sub-acute toxicity study in mice of a potential food color additive derived from the seeds of the avocado (Persea americana, Lauraceae). Frontier in Nutrition 2022
  • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Hatzakis E, Mazzola EP, Shegog RM, Ziegler GR, Lambert JD. Perseorangin: A natural pigment from avocado (Persea americana) seed. Food Chemistry 2019, 293, 15-22, https://doi.org/10.1016/j.foodchem.2019.04.064
  • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Dabas D, Elias RJ, Ziegler GR, Lambert JD. In Vitro Antioxidant and Cancer Inhibitory Activity of a Colored Avocado Seed Extract. International J. Food Science 2019, Article ID 6509421, https://doi.org/10.1155/2019/6509421
  • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Dabas D, Ziegler GR, Lambert JD. Anti-Inflammatory Properties of a Colored Avocado Seed Extract. Adv. Food Technol. Nutr. Sci. 2019; 5(1): 8-12, https://doi.org/10.17140/AFTNSOJ-5-151
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Presentation to the Produce Marketing Association, July 14, 2021, via Zoom
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Participation to quarterly meetings of the International Association of Color Manufacturers (IACM) 2019-2020-2021
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Participation to IFT 2019 Annual Meeting and Food Expo, June 2-5 in New Orleans, LA
  • Type: Other Status: Other Year Published: 2019 Citation: video of the production process (confidential material)


Progress 09/01/19 to 08/31/20

Outputs
Target Audience:As a small business our target audience is potential customers, in this case food color and consumer food product manufacturers. Food color additives require pre-market approval, so our audience also includes the Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A M.S. student in chemical engineering at UC-Davis was supported to conduct the techno-economic analyses. An intern was supported to conduct starch extraction and isolation experiments. How have the results been disseminated to communities of interest?Results of this research have been disseminated at quarterly meetings of the International Association of Color Manufacturers (IACM); at the IFTNEXT Food Disruption Challenge™ competition (April 2020), at the 3rd Annual Delaware Sustainable Chemistry Alliance (DESCA) Investor Forum (November 2019), as well as, confidentially to potential customers. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, the quantitative analysis of the chemical composition of AvoColor® will be completed. The techno-economic analysis as well as the 2-year shelf life study will be finalized. The food applications and the methodology to accurately detect and quantify AvoColor® will be developed, and dietary exposure estimated. A meeting with FDA will be scheduled. The detailed standard operating procedure of the production process of AvoColor will be documented. The complete chemical specification of representative batches of AvoColor® will be proposed.

Impacts
What was accomplished under these goals? Objective 1: Composition analysis of AvoColor®. The biochemical composition of the extract has been completed and the molecules were identified by the UHPLC mass spectrometry. The qualitative analysis of AvoColor® showed the presence of 26 species with no history of toxicity concerns to humans. Objective 2: Techno-economic analysis. The techno-economic analysis was initiated with and contracted to The University of California Davis, Department of Chemical Engineering, under the direction of Professor Karen McDonald, to develop a computer model of the industrial scale production of AvoColor® in order to estimate total annual production costs and total capital investment for different production levels and process assumptions and to suggest operations to target for possible future process development. Objective 3: Shelf-life study. The shelf-life study showed AvoColor® powder performing well under tested storage condition (25 and 50 °C) for 13 months with very minimal color changes and no degradation products formed. Objective 4: Exposure level estimation. The safety assessment of AvoColor® through a 90-day sub chronic toxicity study with rats reveals no significant clinical, biological or behavioral signs of toxicity due to AvoColor® up to 5% concentration in the rat's diet. The No-Observed-Adverse-Effect-Level for AvoColor® was 2620.6 mg/kg/day for males and 3218.4 mg/kg/day females in rats. The Acceptable Daily Intake (ADI) for human was estimated at 26 mg of AvoColor®/kg/day. Objective 5: Alkaline "stabilization" process. The sodium hydroxide (NaOH) stabilization process increased the specific concentration of perseorangin in AvoColor® and improved the purity by reducing the level of contaminant species in the extract. Objective 6: Standard Operating procedure of AvoColor® production. Nothing to report. Objective 7: Method development for AvoColor® quantification. The detection and quantification method by HPLC using purpurogallin as internal standard is under development. Objective 8: Chemical specification of AvoColor®. The chemical specification of AvoColor® showed no concerning level of pesticides, heavy metals, residual solvent, salt, volatile oil, coliforms, or mold and yeast.

Publications

  • Type: Other Status: Other Year Published: 2020 Citation: Phase 3 Presentation to IFTNEXT Food Disruption Challengeâ¿¢ competition (April 2020)
  • Type: Other Status: Published Year Published: 2019 Citation: Publication in Centre Daily Time (https://www.centredaily.com/living/food-drink/article237832584.html) (Dec 2019)
  • Type: Other Status: Other Year Published: 2019 Citation: Presentation at the 3rd Annual Delaware Sustainable Chemistry Alliance (DESCA) Investor Forum on November 15, 2019 in Wilmington, Delaware