Progress 01/01/12 to 12/31/14
Outputs
Target Audience: Target audiences include agricultural specialists, specialty crop growers, food processing and engineering scientists,fellow research scientists, nutritional scientists, natural colors industries, regulatory agencies, the food industries thatuses natural color additives, and citizens thatconsumenatural colors in their diet. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? 1. We have been able to train 2 additional undergraduate students in 2014 on anthocyanin chemistry, simulated digestive models, and chemical stability. 2. The project has allowed for a PhD student to earn an internship in food chemistry at a major US food company, interested in natural colors. 3. The PhD student was awarded a fellowship following this project to continue writing publications from this work. The award included acceptance intoawriting-intensive workshop to help with manuscript preparation. 4. The PI and Co-PI have been able to use the basis fo this reseach to further develop models for human health benefits, with a focus on the stability of select polyphenolics as they traverse to the colon. Acyalted anthocyanins fit nicely into this category, and we hope to further this research into the stability and health benefits of those compounds that are not destroyed by the low-acid environemnt of the small intestine. How have the results been disseminated to communities of interest? Data presentations have been or will be madeyo the Institute of Food Technologists, American Chemical Society, and Experimental Biology.Meetings have taken place bi-annually between thePI's and the industrial collaborators, via conference calls and face-to-facemeetings. Publicity of the research was made via press releases,video, and print media through the American Chemistry Society. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The 2013 purple sweet potato crop had a greater success after a near complete crop failure of 2012 due to excessive rains in the North Carolina growing region. Published a paper in Food Chemistry on the identify of purple sweet potato anthocyanins, processing treatments that inhibited polyphenol oxidase, and found modifications to an aqueous extraction solution that would both inhibit oxidative enzymes and enhance recovery of pigments. 2. We discovered that the inhibition of oxidase enzymes, namely polyphenol oxidase, was the paramount processing issue facing the processing of purple sweet potatoes for pigment yield and stability, with rapid inhibition with a combination of heat, acid, and extraction salts/buffers being critical. We additionally evaluated the role of cell-wall active enzymes (pectinase and cellulose), protease, and amylase during processing and correlated a beneficial dose-response against pectinase and pigment extraction as the most critical enzyme to add following the inactivation of polyphenol oxidase from the potatoes. Pigment yields were increased by up to 25% more than with the combination of heat and solvent modifiers alone. This increase in yield and processing efficiency likely translates into a profitable processing scenario for the US pigment extraction industries. The last goal was to evaluate the bioactive properties of purple sweet potato pigment extracts and the role of acylatation on bioactive characteristics. We evaluated several factors related to bioactive properties including their chemical stability during digestion and the role of the low-acid environment of the gastrointestinal system on the hydrolysis and oxidation of anthocyanins. Anthocyanins were modeled by both alkali hydrolysis to remove the acyl moiety to create distinctive polyphenolic isolates and also hydrolyzed in non-human simulated digestive processes. Isolates of both pigments and non-pigmented polyphenolics were created. Their anti-inflammatory effects in CCD-18Co colon cells and selective cell proliferation in HT-29 cancer cells were evaluated. A 40% cell inhibition was observed for HT-29 cells for the anthocyanins up to a toxic-level dose. Purple sweet potato anthocyanins were found to be highly non-toxic to cells and therefore required a very high concentration (>500 mg/L) to reach cell toxicity. Anthocyanins with and without their acyl groups suppressed tumor necrosis factor alpha (TNF-α) and induced beneficial reactive oxygen species in cancer cells at 10 mg/L. Both anthocyanins and non-anthocyanin polyphenolic isolates served to down-regulate mRNA and protein expressions of inflammation using several common biomarkers. However, the results indicated the importance of non-pigmented polyphenolics that accompany the extraction of the pigmented anthocyanins in exerting maximal anti-inflammatory properties. Results also indicate that there is an anti-inflammatory benefit to the hydrolysis of acyl moieties during digestion, which provides valuable knowledge to role of acylated natural colors as healthy alternatives to synthetic food colors. Lastly, we further modeled the stability of acyalted purple sweet potato anthocyanins during simulated digestions against other acylated and non-acylated anthocyanin sources (ie. pomegranate, grape, raspberry, blackberry, black carrot, and red cabbage). Since the bioavailability of anthocyanins is largely believed to be low, at less than 1% of the ingested dose, our goal was to show that purple sweet potato and other acyalted sources may be more impactful for health in the colon, compared to the small intestine. We modeled this stability in non-human systems that simulate digestion based on pH changes from low in the stomach changing to a high pH in the intestines and evaluated them for differences in chemical structure, stability, and cancer cell proliferation. All were stable in the acidic gastric digestion conditions of pH 2.0 for 2 hours, but major changes to non-acyalted sources occurred in the simulated pH shift to the small intestine at pH 7 for up to 7 hours as a majority (up to 90%) of these anthocyanins was lost due to oxidation. By comparison, the acylated anthocyanins including purple sweet potato, were well maintained at this pH level at over 70% retention, allowing them to pass without significant chemical modification to the colon. However, for cancer cell viability it was revealed that non-acyalted anthocyanins were more effective in reducing cell counts at around 55% reduction at 50 μg/mL compared to only 30% for acylated anthocyanins. Results indicated a benefit to the non-acylated compounds in reducing cell viability, but in a matrix that rapidly destroys the compounds in the small intestine. Acylated sources, including purple sweet potato, may have had less impact on cell viability but the compounds were more stable and persisted in the digestion system longer and therefore exerted a greater overall benefit.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Cipriano, P.A., Ekici, L., Barnes, R.C., Gomes, C. and Talcott, S.T. Pre-Heating and Polyphenol Oxidase Inhibition Impact on Extraction of Purple Sweet Potato Anthocyanins. Accepted for publication, Food Chemistry, 2015.
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Progress 01/01/13 to 12/31/13
Outputs
Target Audience: Target audiences include fellow scientists, natural colors industries, government/regulatory agencies,any food industry that uses color additives, and any citizen that consumes natural colors in their diets. Changes/Problems: Our major problem was the poor crop performance infall of2012 due to excessive rainfall inNorth Carolina. We were able to obtain small amounts of research material, sufficent to contact the enzyme inhibition and extraction studies, however.However, this has hindered the application of post-harvest handling procedures and its impact on chemistry and stability. The 2013 crop looks to be an excellent growth year with good yield and crop quality. What opportunities for training and professional development has the project provided? 1. I was able to further train a post-doctoral research associate on anthocyanin extraction and phytochemistry. 2. I was able to train an undergraduate student in anthocyanin chemistry, for which he has been accepted into gradaute school under my direction. 3. I have trained two full-time PhD students as part of their educational experience. 4. The PI and Co-PI have been able to advance the commercialization of this research with our industrial partners. How have the results been disseminated to communities of interest? Data presentations were made to the Institute of Food Technologists, American Chemical Society, and Young Scientist Round Table in both oral and poster presentatins. Meetings have taken place every 4-5 months betweent the PI's and the industrial collaborators, via conference calls and on-site meetings. Publicity of the research was made via web, video,and print media through the American Chemistry Society and were publicized thorugh such media outlets asNational Public Radio and National Geographic. What do you plan to do during the next reporting period to accomplish the goals? We will complete the submission of our first 2 manuscipts for publication. The first on the chemistry, polyphenol oxidase inhibition, and extraction of purple sweet potato extracts and the second on the bioactive properties of acylated and non-acylated anthocyanin species from the potatoes. We will present research findings at both the Institute of Food Technologists and Experimental Biology meetings. We will continue to work withfood industry representatives to implement the findings into food manufacturing and quality control environments.
Impacts
What was accomplished under these goals?
After a near complete crop failure of 2012 due to excessive rains in the North Carolina growing region, we have been able to sufficiently advance the project goals by: 1. Identifying the phytochemistry of the purple sweet potatoes, both anthocyanins and non-anthocyanins. 2. Understand the role of polyphenol oxidase inhibition on polyphenolic recoveries using various processing techniques to inhibit the enzyme and optimize color recovery for commercial applications. 3. Understand the role of co-oxidation of pigments in the presence of polyphenol oxidase during purple sweet potato processing. 4. Evaluate the bioactivity of purple sweet potato anthochanins and non-anthocyanin polyphenolics and to generate strong hypotheses for 2014 with regard to the role of acylation on bioactivity in cell culture assays. Summary: Anthocyanins are pigments widely distributed in fruits, cereals, vegetables and are esponsible for the intense red, blue and purple colors found in nature. They are popular over synthetic dyes and have potent antioxidant properties and likely bioactive characteristics. Purple sweet potatoes (PSP) are becoming popular for use as natural colors, but processes to inactivate polyphenol oxidase and enable pigment recovery are challenging. Nine individual anthocyanins were tentatively identified by HLPC-MS from PSP using extracted ion chromatograms for each compound. PSP anthocyanins identified included cyanidin and peonidin based compounds with aglycons at m/z of 287 and 301, respectively with mono or diacylated moieties consisting of caffeic, ferulic, or p-hydroxybenzoic acids linked to sophorose at the 3-position of the anthocyanin and each with a 5-glucoside. Only one non-acylated anthocyanin was identified as cyanidin-3-sophoroside-5-glucoside at m/z at 773, producing fragment ions at m/z 611, 449 and 287. Mono acylated compounds were generally eluted first, as the high molecular weight compounds tended to have more affinity to the C18 column, except for the peonidin 3-(6”-caffeoyl sophoroside)-5-glucoside, which was co-eluted with cyanidin 3-(6”-caffeoyl-6”’-p-hydroxybenzoyl sophoroside)-5-glucoside. Diacylated compound seemed to be present in higher concentrations in all treatments, followed by cyanidin 3-(6" caffeoyl-6”’-feruloyl sophoroside)-5-glucoside and cyanidin 3-(6”-p-hydroxybenzoyl sophoroside)-5-glucoside. Extraction of PSP anthocyanins was conducted with and without a pre-heating step that was aimed at inhibiting polyphenol oxidase. Extraction solutions containing citric acid, oxalic acid, boric acid, and acidified water were also used that attempted to further inhibit co-oxidation reactions during processing. Pre-heated PSP treatments showed an anthocyanin concentration of 448, 630, 532, and 494 mg cyanidin/kg of PSP for the control, Oxalic Acid, Citric Acid and Sodium Borate treatments, respectively, while non-heated PSP showed concentrations of 410, 466, 478, and 511 mg cyanidin/kg of PSP for the same groups indicating benefits of a pre-heating step. Similar observations for the recovery of non-anthocyanin polyphenolics was observed with primary compounds, that can act as anthocyanin co-pigments, identified as chlorogenic acid, feruloylquinic acid, 4,5-di-caffeoyl quinic acid, 3,5-di-caffeoly quinic acid, and 3,4-di-caffeoyl quninic acid. Evaluations of bioactivity of concentrated PSP anthocyanins and polyphenolics followed, with cell viability decreasing in both normal colon cells (CCD-18Co) and carginogenic colon cells (HT-29). We are suspectingthat the extensive acylation of the anthocyanins in the high pH environment of the cell culture media is leading to the formation of pro-oxidative intermediates that are potentially harmful to the cells. These observations were found in both anthocyanin and non-anthocyanin extracts over a large dosing concentration.
Publications
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Through collaborative efforts with representatives of the processed vegetable and pigment industries, we have conducted a series of field plots of purple sweet potatoes in North Carolina whereby representative whole potatoes were evaluated for enhanced methods to extract, isolate, and prevent degradation of anthocyanins used as natural food colors. Where applicable, both technical and non-technical data was supplied back to these companies on improved processing methods and on-site consultations have progressed to share information obtained. An abstract of the work was prepared for public presentation of relevant data in the summer of 2013. These outcomes have allowed the supporting industries to further educate consumers on benefits and functions of natural colorants in their diet. PARTICIPANTS: Graduate students had the opportunity to train, gain practical experience, and publish their research on this project (Paula Cipriano, Enrique Carcia, and Kimberly Krenek) as well as a post-doctoral research associate (Dr. Lutfiya Ekici), and undergraduate research scholars (Ryan Barnes and Gustavo Reyes) . Collaborators included Dr. Susanne Talcott and industrial collaborators at Avoca Inc., Sensient Technologies, and Yakima Fruit Works. TARGET AUDIENCES: Natural colors industries, fruit and vegetable industries, beverage industry, functional foods industry, ingredient industries, consumers. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Purple sweet potato (PSP) anthocyanins were found to be highly acylated natural colors that give excellent stability in foods, making them good substitutes for synthetic food colors. During whole PSP evaluations, we discovered that native polyphenoloxidase was easily activated and resulted in co-oxidative reactions that destroyed the targeted anthocyanins. Studies were designed to inhibit or destroy the oxidative enzymes through the use of pre-heating treatments to whole or cubed PSP and anti-browning agents or enzyme inhibitors added to evaluate their role on anthocyanin stability. Experimentally, PSP were heated for 10 min at 90C and then extracted with a 2% citric solution for 1 hr at 70C and compared to a non-heated PSP samples. Anti-browning agents added to the extraction solution included 1% sodium tetraborate, oxalic acid, and citric acid each added to the 2% citric acid solution and likewise PSP samples were extracted for anthocyanins. After extraction using commercially viable methods for anthocyanin recovery, each extract was subjected to color stability trials by holding at 80C for up to 6 hrs. When compared to the control groups, the addition of anti-browning agents led to higher pigment yields ranging from 29 to 74% when coupled with a pre-heating step, compared to increases of only 9 to 28% in non-heated samples. The synergistic effect of pre-heating with the anti-browning agents served to inactivate polyphenol oxidase while simultaneously aiding in the extraction and recovery of the valuable pigments. The stability of the extracted anthocyanins followed first-order degradation kinetics. The half-life of the non-heated PSP anthocyanin extracts (9.0 to 16.4 hrs) was slightly longer than the pre-heated samples (8.4 to 10.9 hrs) among the anti-browning treatments, potentially indicating thermally-induced oxidation products that impacted pigment stability. However, results indicate that the use of anti-browning agents associated with a mild thermal inactivation of polyphenol oxidase is a viable processing method to increase overall pigment yield from PSP. Additionally, PSP isolates are being evaluated in cell culture models for potential health benefits related to inflammatory pathways. Using a bio-guided fractionation technique, the anti-inflammatory properties of isolated anthocyanins and non-anthocyanin phenolic acids were compared to whole, commercial PSP concentrates. Results indicated that both anthocyanins and non-anthocyanins are major contributors to the reduction in inflammation responses in vitro. These results are valuable in selecting PSP varieties, extraction, and concentration methods on an industrial scale for further advancing not only the quality of the pigments but also for enhanced health benefits of food products.
Publications
- No publications reported this period
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