COLORED POTATO BIOACTIVE COMPOUNDS AS ANTI-OBESITY AGENTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0222463
• Project No.: COL00621
• Project Start Date: Jul 1, 2010
• Project End Date: Jun 30, 2012

Project Director
Vanamala, J.

Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523

Performing Department
Food Science & Human Nutrition

Non Technical Summary
An effective strategy to combat the growing threat of childhood obesity includes a combination of dietary, lifestyle, educational, environmental, behavioral and policy changes. Emerging evidence suggests that diet plays an important role in pathophysiology of childhood obesity. Functional foods with putative anti-obesity properties marketed today are expensive, thus, it is imperative to develop evidence-based functional foods that are affordable to low income consumers susceptible to obesity. The potato (Solanum tuberosum L.) is the world's 3rd-largest food crop and the leading vegetable crop in the United States, with per capita consumption of about 126 lbs annually. In recent years, demand for specialty potatoes has risen sharply in developed countries and the United States, in particular. Due to their health benefiting properties, specialty and/or colored potatoes represent one mechanism for enhancing human nutrition through popular and palatable means. Colored potatoes are rich in anti-oxidant and anti-obesity bioactive plant compounds, such as carotenoids, and anthocyanins. However, potatoes are stored and processed before consumption and the effect of storage and processing on the health benefitting properties of specialty potatoes is not known. For producers and consumers to fully benefit from these properties, it is imperative to document the specific mechanisms of action of the protective foods and to understand the effects of post-harvest storage and processing methods on the biological activity of the functional compounds found in specialty potatoes. To address the above need for research, we propose to determine the effect of post-harvest treatments (fresh vs. stored potato) and processing methods (baking vs. frying) on the total phenolic and acrylamide content and antioxidant capacity. We will also determine the extent to which raw and processed potato carotenoids and anthocyanins differ in their alteration of Bacteroidetes-rich "lean microbiota" or Firmicutes-rich "obese microbiota" in the gut of obese pig. Obesity is associated with higher proportion Firmicutes, and lower proportion of Bacteroidetes, and reduced diversity. To answer these research questions we will utilize samples collected from pigs provided with high-fat diet for two months followed by high-fat diets containing either fresh, baked or fried potatoes for 6 weeks. Research findings will be communicated through a variety of mechanisms that results in greater technology and knowledge transfer to stakeholders including consumers, potato producers and processors.

Animal Health Component

20%

Research Effort Categories

Basic

60%

Applied

20%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
204	1310	1010	10%
701	1310	1010	20%
701	1310	1040	20%
701	1310	1090	10%
702	4010	1010	20%
702	4010	1040	10%
702	4010	1090	10%

Knowledge Area
204 - Plant Product Quality and Utility (Preharvest); 701 - Nutrient Composition of Food; 702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
1310 - Potato; 4010 - Bacteria;

Field Of Science
1010 - Nutrition and metabolism; 1090 - Immunology; 1040 - Molecular biology;

Keywords

obesity

colored potatoes

processing

bioactive compounds

gut microbiota

pig

dissemination

extension

antioxidant activity

phenolics

Goals / Objectives
The overall goal of this integrated project is to identify consumer-preferred colored potato varieties with high content of putative anti-obesity bioactive compounds, and appropriate storage and processing methods that have minimal effects on the content, composition, bioavailability and "lean gut microbiota" promoting ability in pigs (an agriculturally important animals)consuming a high-fat diet, and to communicate research finding through a mechanism that results in technology and knowledge transfer to appropriate stakeholders. Project Objectives: 1. Identify elite colored/specialty potato cultivars with desirable agronomic parameters and health benefiting bioactive compounds: Six specialty potato cultivars (three yellow and three purple) will be analyzed for drought, pest and disease resistance, length of growing season, specific gravity, storability, tuber size, and yield, and the content of bioactive compounds as compared to the commercial white potato variety Atlantic. 2. Determine the effect of storage and processing (baking and chipping) on antioxidant capacity, phenolic and acrylamide content and sensory properties of colored potato cultivars. Fresh and processed (baked vs. chipped) tubers will be analyzed for antioxidant capacity, phenolic and acrylamide (a potential human carcinogen at higher doses) content. Baked and chipped samples will be analyzed for appearance, color, taste, crispiness and overall acceptability, as compared to the commercial white variety Atlantic using sensory analysis. 3. Determine the extent to which raw vs. processed potato bioactive compounds differ in their alteration of Bacteroidetes-rich "lean microbiota" or Firmicutes-rich "obese microbiota" in the gut of obese pig. One of the current funded grants (USDA NRI Integrated Grant, 2009-55200-05197) provides the fecal samples from obese pigs fed with processed purple and yellow potatoes. These fecal samples will be analyzed for lean (Bifidobacterium and Lactobacillus) and obese (Staphylococci and Clostridia) microbiota using media exclusion and fluorescence in situ hybridization (FISH) techniques. Ratio of lean vs. obese gut microbiota will be correlated with back fat thickness, lipogenesis and lipolysis measured in the obese pig as part of the funded USDA grant. 4. (a) Translate the knowledge obtained from agronomic practices, food processing operations, phytonutrient analyses, and functional studies (lean vs. obese microbiota in relation to lipolysis vs. lipogenesis) into an Extension program to educate producers,consumers and food industry partners in Colorado. Information will be shared with Colorado producers at field days, consumers and industry partners via workshops, and CSU Extension website to increase the knowledge on colored potato selections, appropriate processing methods and health benefits, and food safety issues related to acrylamide. (b) Survey the effectiveness of various dissemination methods and the impact of the research on the horticultural, post-harvest and processing practices and methods employed towards utilization of specialty potatoes.

Project Methods
While the overarching goal of this research is to understand the role of post-harvest and processing effects on bioavailability and "lean gut microbiota" promoting ability, we also aim to communicate research results to appropriate stakeholders so that they may incorporate findings into their production practices and marketing plans. The research component of the proposal provides the first insight into postharvest handling and processing (baking and frying) effects on the phenolic, acrylamide content and antioxidant capacity using spectrophotometric and HPLC One of the current funded grants (USDA NRI Integrated Grant, 2009-55200-05197) provide the fecal samples from obese pigs fed with processed purple and yellow potatoes. In brief, the experimental design of funded USDA grant is; seventy two castrated male pigs will be housed individually and fed a standard (control) diet (10 pigs) or high-fat diet (62 pigs) for 8 wk. At the end of the 8 wk, 56 pigs from high-fat diet group and eight pigs from standard diet group will be selected for the experimental diet intervention. Eight pigs on standard diet continue on standard diet for an additional 9 weeks. Fifty six pigs on high-fat diet will be assigned to one of seven dietary treatment groups (n = 8): (1) chow diet; (2) yellow potato at y % that is (a) baked and (b) raw (freeze-dried); (3) purple potato chip at p % that is (a) fried and (b) raw; (4) white raw potato at y % or p % (whichever is higher); and (5) y % yellow baked + p % purple fried potato. The percentage dietary concentration of potato (y % and p %) will be decided based on the results of the dose-response study (conducted in summer 2010). Feed allowance will be 90% of voluntary feed intake observed from the dose-response study to ensure all pigs consume similar amounts of feed during the 9wk experimental period. Fresh urine and fecal samples will be collected after experimental diet intervention at wk 0, 2, 4, 6, 7, 8 and 9 of the study. Fresh fecal samples will be plated in triplicate on a variety of differentially-selective media (e.g. genus-specific) and incubated for detection of lean vs. obese bacteria. For confirmation, a representative samples will be subjected to fluorescence in situ hybridization (FISH) followed by flow cytometry detection using 16S rRNA-targeted oligonucleotide probes. Consumer preference of the colored vs. traditional (white) potatoes will be measured using sensory analysis. Researchers will utilize the following methods/tools to disseminate the findings: presentations at grower association meetings, horticulture-field days, development of an educational web-site (http://potato.colostate.edu/) that may be linked to industry organizations and extension sites, additional workshops and publications in popular and scientific newsletters and/or journals.

Progress 07/01/10 to 06/30/12

Outputs
OUTPUTS: Color-fleshed potatoes are a rich source of anthocyanins, which may contribute to the protection of high-fat diet induced inflammation and obesity. However, Color-fleshed potatoes can undergo 3-6 months of storage before processing/consumption and the effect of storage and processing on their bioactive anthocyanins remains unknown. We hypothesized that Color-fleshed potatoes retain polyphenolic content (TP), antioxidant activity (AA), anthocyanins and sensory attributes with minimal changes in acrylamide levels even after 3 months of storage and processing (baking and chipping). We further hypothesized that consumption of color-fleshed potatoes will suppress weight gain and inflammatory markers, and will concomitantly elevate gut bacterial diversity in pigs fed a high-fat diet. To test these hypotheses, we utilized: i) white-, yellow- and purple-fleshed potato clones ii) 40 pigs on high-fat diet (HFD) for 12 wk provided with one of the five diets: HFD, white-fleshed potato (WP) or PP chips at 10/20% for 5 wk. Purple-fleshed potatoes had greater phenolic content (TP) and antioxidant activity (AA) as compared to traditional white- and yellow-fleshed genotypes. The AA of all clones increased with storage; however, an increase in TP was observed only in purple-fleshed clones. Baking caused minimal losses while chipping reduced the phenolic and anthocyanin content, and AA of the potatoes. With storage, total phenolic and anthocyanin content, and AA increased in baked samples while in the chipped samples, they remained constant. Principal component analysis of approximately 1600 metabolite fragments obtained by UPLC-MS revealed that storage and processing (chipping vs. uncooked) caused a shift in the metabolite profiles of potato clones. However, baking retained similar metabolite profile as that of uncooked potato. Consumers were willing to pay a premium for Color-flesh potatoes if they were educated on their potential health benefits. TP and AA of purple-fleshed potatoes, after baking, were comparable with those of anthocyanin-rich berries. In the pig study, serum, distal colon mucosa/digesta and fecal samples were utilized to analyze oxidative stress (MDA; colorimetric assay) and inflammatory (TNFα; ELISA and prostaglandins; LC-MS/MS) markers and gut bacteria (pyrosequencing). Potato diets, during short feeding period (5 wks), had no effect on weight gain, feed intake or back-fat thickness. However, all potato diets, except WP (20%), suppressed (p = 0.04) systemic levels of TNFα compared to HFD. However, only purple-fleshed potatoes suppressed systemic MDA and colonic PGE2 levels (p ≤ 0.05). Only purple-fleshed potatoes increased distal colon bacteroidetes to firmicutes ratio (BFR) compared to HFD. This is particularly important as BFR is reduced during obesity. Indeed, MDA and TNFα elevated during obesity positively correlated with firmicutes (0.518, p = 0.002; 0.380, p = 0.024, respectively), and MDA negatively correlated with BFR (-0.413, p = 0.036). In summary, these results suggest that the potato diets, particularly purple-fleshed potato, suppress inflammation by modulating gut bacterial signature. PARTICIPANTS: Project team remains the same. In addition, Dr. Holm helped to grow and procure potato cultivars. Dr. Knight from University of Colorado, Boulder helped with establishing gut microbiota analysis. TARGET AUDIENCES: 1) Potato breeders/industry 2) Food industry 3) Food and nutrition researchers/students 4) Potato researchers 5) Obesity and cancer researchers PROJECT MODIFICATIONS: Instead of using FISH for limited number of bacteria identification, we used more advanced pyrosequencing.

Impacts
Purple-fleshed (Purple Majesty and CO97227-2P/PW) varieties/advanced selection were identified as rich sources of bioactive compounds and anti-oxidant capacity compared to all other varieties tested. Demonstrated that: 1) Even after 3 months of storage followed by processing (baking), CO97227-2P/PW retained the anti-oxidant capacity and anti- colon cancer properties (obesity and/or type 2 diabetes promotes colon cancer); 2) baking not only retains metabolite profile similar to that of uncooked but also retains biological activity against human colon cancer cell lines - suggesting baking is a preferred method of processing color-fleshed potatoes compared to chipping; 3) consumers prefer tastier evidence-based health food products whereas potato producer's primary preference is the ability to sell the crop - thus, it is critical to develop farm to fork operations that optimizes the health-benefits without losing the sensory attributes; 4) Results strongly suggest the concept that purple-fleshed potatoes suppress the high-fat diet induced inflammation via modulating gut bacterial signature; 5) Provided preliminary data for two USDA grant proposals - one received high priority and the other medium priority during the proposal period; one resubmitted and the other will be resubmitted next month; 6) provided transdisciplinary training to two graduate students on the proper methodology and considerations required for conducting analytical and in vitro studies using plant products; 7) one graduate student was also trained on using pigs, an agriculturally important animal model for assessing anti-inflammatory/anti-obesity properties of whole food products; and 8) Graduate students received several national awards for this novel research - ex. Institute of Food Technologists Graduate Student Poster Competition (2nd place), Potato Association of America Graduate Students Oral Presentation (1st place), New York Academy of Science Graduate/Post-doc Student Poster Competition (1st place).

Publications

• Amer, F., G. Madiwale, L. Reddivari, M. Stone, D. Holm and J. Vanamala. 2012. Storage effect on glycoalkaloid and acrylamide content in potato chips is genotype dependent and these toxicants negatively correlate with consumer preference. Potato Association of America - PAA 2012, Aug 13, 2012, Denver, CO.
• Vanamala, J., L. Reddivari, S. Radhakrishnan, R. Knight, S. W. Kim and D. Holm. 2012. Color-Fleshed Potato Phytochemicals: Colonic-Systemic Oxidative Stress and Colon Cancer. University of Colorado Cancer Center Conference, Oct 30, 2012, Aurora, CO
• Gaurav, M., L. Reddivari, D. Holm and J. Vanamala. 2012. Combined effects of storage and processing on the bioactive compounds and pro-apoptotic properties of color-fleshed potatoes in human colon cancer cells. Journal of Agriculture and Food Chemistry, 60(44):11088-96.
• Vanamala, J., S. Radhakrishnan, L. Reddivari, and A. Massey. 2012. Anthocyanins as Apoptotic Regulators. In George G. Chen and Paul B. S. Lai (Eds.), Novel Apoptotic Regulators in Carcinogenesis (1st edition, pp. 93-122). Springer.
• Radhakrishnan, S., L. Reddivari, S. W. Kim and J. Vanamala. 2012. Uncooked and baked purple potatoes suppress oxidative stress and inflammatory markers in high-fat diet consuming pig. Potato Association of America - PAA 2012. Aug 13, 2012, Denver, CO.
• Reddivari, L., G. P. Madiwale, D. G. Holm and J. Vanamala. 2012. Purple-fleshed potatoes retain anti-cancer properties in vitro even after prolonged storage and processing. Potato Association of America - PAA 2012, Aug 13, 2012, Denver, CO.
• Radhakrishnan, S., L. Reddivari, S. B. Smith, S. W. Kim and J. Vanamala. 2012. Purple potato, even after processing, suppresses oxidative stress and inflammatory markers in high-fat diet consuming pigs. FASEB J, March 29, 2012; 26:823.5.

Progress 01/02/11 to 12/31/11

Outputs
OUTPUTS: Colored potatoes, rich in anthocyanins and carotenoids, are becoming popular for their putative health benefits. Recent evidence suggests that plant bioactive compounds such as anthocyanins alter the gut bacterial profile. Gut bacterial is connected to a variety of chronic diseases including obesity, type 2 diabetes etc. However, it is not known extent to which the colored potato bioactive compounds alter the gut microbial profiles, particularly after storage and/or processing. We established all the analytical techniques to assess the effect of genotype and storage and/or processing on: 1) Bioactive polyphenols/anthocyanis; 2) acrylamide; and 3) glycoalkaloids. We also established the protocols to assess the changes in microbial fingerprint in the gut digesta and fecal material. This year we focused on: 1) Selection and procurement of ten potato cultivars with desirable agronomic characters such as drought, pest and disease resistance, specific gravity, storability, tuber size, and yield among selected cultivars; 2) determination of the effect of genotype, storage and/or processing on health benefiting and toxic compounds; 3) determination of gut microbiome (pyrosequencing) of digesta (distal ileum and proximal, and distal colon) and fecal samples from pigs consuming high-fat diet for 12 weeks followed by supplementation with white or purple potato chips (10 or 20%); 4) collection of gut digesta and fecal samples from pigs consuming lean diet, high-fat diet and high-fat diet supplemented with white or purple potato (raw, baked and chipped) for 15 weeks; and 5) dissemination of results through conference presentations, field days and seminars. The outcomes from the above studies: 1) Riogrande Russet, Russet Burbank and Atlantic - most popular white varieties; Purple Majesty, All blue and Mountain Rose - most popular specialty cultivars; and ACC 99-375-1, CO97227-2P/PW - advanced selections were selected for the study; 2) advanced selection CO97227-2P/PW showed desirable agronomic traits and the highest content of bioactive compounds compared to the other cultivars, and the amount of toxic compounds were comparable to other commercial varieties. Storage increased the acrylamide and GA content, and the percent increase was cultivar dependent. Baking led to no or minimal losses while chipping significantly reduced the phenolic and anthocyanin content, and antioxidant activity of the potatoes compared to the uncooked potatoes; 3) gut microbial signature of the distal digesta samples correlated with the fecal samples. Gut microbiota signature was distinct among distal ileum and proximal and distal colon digesta. 4) analysis samples collected from pigs consuming white or purple potatoes (raw, baked or chipped) will aid us to determine the effect of processing and long-term feeding on gut microbial signature; and 5) results were disseminated through San Luis Valley Experiment Station Field days, IFT, PAA and Potato Expo and University of Colorado Cancer Center (UCCC) Retreat to educate growers, industry, students and researchers, and medical professional on health-benefiting properties of color-fleshed potatoes. PARTICIPANTS: Project team remains the same. In addition, Dr. Holm helped to grow and procure potato cultivars. Dr. Knight from University of Colorado, Boulder helped with establishing gut microbiota analysis. TARGET AUDIENCES: 1) Potato breeders/industry 2) Food industry 3) Food and nutrition researchers/students 4) Potato researchers 5) Obesity and cancer researchers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Identified purple-fleshed CO97227-2P/PW as an advanced selection with greater levels of bioactive compounds, anti-oxidant capacity and anti-colon cancer activity compared to all other varieties tested. Demonstrated that: 1) Even after 3 months of storage followed by processing (baking), CO97227-2P/PW retained the anti-oxidant capacity and anti- colon cancer properties (obesity and/or type 2 diabetes promotes colon cancer); 2) dietary component effects on pig distal colon microbiome could be assessed non-invasively using fecal microbiome; 3) consumers prefer tastier evidence-based health food products whereas potato producer's primary preference is the ability to sell the crop; 5) provided transdisciplinary training to two graduate students on the proper methodology and considerations required for conducting analytical and in vitro studies using plant products; and 6) one graduate student was also trained on using pigs, an agriculturally important animal model for assessing anti-inflammatory/anti-obesity properties of whole food products.

Publications

• Reddivari, L., J. Vanamala, S. Radhakrishnan, P. Yang, S. W. Kim, R. Knight. 2011. Purple potatoes alter the gut microbiota and inflammatory eicosanoids in obese pigs consuming high-fat diet. Potato Association of America - PAA 2011, 08/15/2011, Wilmington, NC.
• Madiwale, G., L. Reddivari, M. Stone, D. Holm, J. Vanamala. 2011. Effect of storage and processing on the composition, anticancer properties and sensory attributes of colored-flesh potatoes. New York Academy of Sciences Research Competition (Food Science Division), 12/12/2011.
• Madiwale, G.P., L. Reddivari, D. G. Holm and J. Vanamala. 2011. Fresh vs. stored colored potatoes: Bioactive compounds and anti-colon cancer properties. Public Health Symposium: Food Systems abstracts, 04/04/2011, Fort Collins, CO.
• Radhakrishnan. S., L. Reddivari, G. Madiwale, S. W. Kim and J. Vanamala. 2011. Purple potato anthocyanins exert anti-cancer properties and suppress oxidative stress even after processing. Potato Association of America - PAA 2011, 08/15/2011, Wilmington, NC.
• Bond, J., S. Kroll, C. Bond, J. Vanamala, L. Reddivari, D. Iverson, E. Larson, C. Nicoletti, N. Janusch. 2011. Consumer Demand for Value-Added Potatoes: An Experimentation and Sensory Analysis with Discussion of Supply-Side Production Motivations and Market Outlook. Potato Expo 2011, Las Vegas, Nevada.