The Impact of Cocoa Processing on Flavanol Content Absorption and Health Effects

Lead Research Organisation: University of Reading
Department Name: Food and Nutritional Sciences

Abstract

Representing one of the most important lifestyle factors, diet can strongly influence the incidence of cardiovascular disease and thus a healthy diet is crucial for healthy ageing. Recent dietary intervention studies, in particular those using wine, tea and cocoa, have demonstrated beneficial effects on reducing cardiovascular disease risk, including an ability to lower blood pressure and to prevent blood vessel ageing. While such foods and beverages differ greatly in chemical composition, nutrient content and calories per serving, they have in common that they are amongst the major dietary sources of a group of plant compounds known as flavanols. The beneficial effects of flavanols have been attributed to their potential to be absorbed into the blood and to exert direct actions on blood vessels. Whilst flavanol-rich foods can be regarded as being protective against cardiovascular disease, the content of active flavanols is significantly reduced during industrial processing. This is because during heating and storage, the flavanols react with to varying degrees with sugars to form new products known as flavanol-Maillard conjugates. At present, information regarding the absorption of these new conjugates and whether they possess similar beneficial properties to native flavanols is lacking. This proposal is designed to address these questions and to unravel the significance of industrial food processing on their inherent health properties. On completion of the proposal, we will be in an excellent position to advise manufacturers of flavanol-rich foods on the best processing conditions required to produce foods with optimum beneficial cardiovascular effects. Building on existing human work conducted in our laboratory, this multidisciplinary study is designed to: 1) investigate the formation of flavanol-Maillard products during the processing of a common flavanol-rich food, namely cocoa; 2) determine the fate of these compounds in the human stomach, small intestine and large intestine; and 3) assess their ability to exert beneficial effects human cells. The first objective will inform us of the major flavanol-Maillard conjugates formed in the flavanol-rich foods during the processing, in this case heating of the cocoa beans during roasting. Although we will study cocoa, heat processing is relevant to many other flavanol rich-foods and therefore will have wider relevance. Secondly, we will examine the absorption of these conjugates, along with native flavanols by feeding processed cocoa to human and measuring them in blood. This will tell us whether heat processing reduces the absorption of native flavanols and also whether the flavanol-Maillard conjugates are absorbed by humans. We will also investigate their metabolism in the large intestine and whether they have a beneficial effect on the balance of the gut microflora, akin to changes seen with pre-biotic functional foods. Lastly, we will examine the beneficial effects of the conjugates in cellular models of human colon cancer and cardiovascular disease using state-of-the-art molecular techniques. We predict that this proposal will help determine the optimum industrial processing conditions required to generate flavanol-rich foods capable of exerting the strongest cardiovascular protection. It will inform both industry and the consumer and will help us develop future strategies to maximise flavanols in our foods. The proposal will broaden understanding of the role that diet plays in the prevention of cardiovascular disease and will help provide evidence for new and promising dietary strategies for tackling cardiovascular disease. The results of this study therefore have important implications for an ageing population where an improvement in healthy ageing is greatly desired. Moreover, the potential benefits in terms of quality of life are relevant to the population as a whole, as are the potential savings in health care costs.

Technical Summary

Dietary derived flavanols have been shown to reduce cardiovascular risk. These beneficial effects have been attributed to their absorption and actions on the vascular endothelium. However, food processing alters the flavanol content of foods due to interactions with Maillard reaction products, formed primarily during heating. This proposal is designed to unravel the significance of these reactions for the beneficial vascular effects of flavanol-rich foods. We will determine which flavanol-Maillard conjugates are generated from native flavanols and carbohydrate precursors under heat processing [150C; 30min or 120C; 5min], using state-of-the-art analytical analysis to characterise products, including specially developed HPLC protocols and LC-MS/MS. Major flavanol-Maillard derivatives identified will be synthesised. To assess human absorption and vascular function we will perform randomised, double blind, triple cross-over human intervention studies. These will use analytically well characterised foods: 1) standard high-flavanol, 2) heat processed high-flavanol; and 3) a low-flavanol. Blood samples will be collected at 0, 0.5, 1, 2 and 4 hr and flavanols and/or flavanol-Maillard conjugates will be measured by HPLC. Vascular function will be measured using state-of-the-art Laser Doppler imaging with iontophoresis. We will also investigate the absorption of individual flavanol-Maillard conjugates in cellular models of the human small intestine. The extent to which flavanol-Maillard conjugates are metabolised in the large intestine and their effects on the microflora will be performed using HPLC and fluorescent in situ hybridisation (FISH) with molecular probes targeting 16S rRNA, respectively. Finally the cellular activity of flavanol-Maillard conjugates will be assessed (using Western immunoblotting), in terms of their ability to: 1) inhibit the growth of colon adenocarcinoma cells and 2) to increase endothelial nitric oxide production by eNOS activation.

Publications

10 25 50

 
Description Dietary flavanols are well reported to induce beneficial effects on the human vascular system and thus reduce the risk of cardiovascular disease. As such, there is interest in developing food products that are rich in these natural food components. Cocoa is an excellent source of flavanols and improves blood perfusion and lowers blood pressure on consumption. However, during industrial processing the levels of flavanols in cocoa are reduced, in part due to their reaction with carbohydrates present in the cocoa to form flavanol-Maillard conjugates. At present information regarding these processes and their effects on the beneficial properties of cocoa are unknown. Our proposal was designed to unravel the significance of industrial food processing on the inherent health properties of cocoa, in particular the roasting and alkalisation steps where the greatest degree of flavanols are thought to be lost.



The major findings of our work were as follows:



1. Initially we studied the reactions of flavanols with cocoa carbohydrates under typical roasting and alkalizing conditions. Here, cocoa flavanols were found to react with sugar derivatives generated as a result of the Maillard reaction to form flavanol-Maillard complexes. In addition, flavanols were observed to yield novel flavanol-C-glycosides. High levels of flavanol-C-glycosides and lower amounts of flavanol-Maillard complexes were identified in commercial cocoa powders.



2. A randomized, controlled, human intervention trial was run in order to assess the impact of cocoa alkalisation on its inherent beneficial cardiovascular effects. Three cocoa powders were used which had been subjected to varying degrees of alkalization: 1) natural or non-alkalized cocoa (1740 mg flavanols); 2) medium alkalised cocoa (403 mg flavanols); and 3) heavily alkalized cocoa (1.3 mg flavanols). The impact of these cocoa powders on the vascular system was assessed in 10 healthy men by measuring physiologically significant increases in blood flow/perfusion (using Flow-Mediated Dilatation, a technique used by cardiologists to assess the global health of the vascular system). Our data indicated for the first time that both non-alkalised and medium alkalised cocoas induce a similar extent of vascular benefits at 2 and 4 h post ingestion. In contrast the heavily alkalized cocoa had no beneficial effects.



3. The 3 alkalised cocoas were also found to exert a positive effect on the growth of beneficial bacterial species, Bifidobacterium spp and Eubacterium rectal-C. coccoides. Furthermore, they inhibited of the growth of the pathogenic C. histolyticum group, which has been shown to contribute to the progression of colonic cancer and the onset of inflammatory bowel disease. The degree of cocoa processing did not alter these beneficial effects. These findings were supported by observations that cocoa roasting also had not effect on its positive influence on the gut microbiota.



4. Alkalised cocoa was observed to reduce the growth of human large intestinal adenocarcinoma cells, although to a lesser extent then that of the non-alkalised equivalent. These differences were found to be dependent on the ability of native flavanols to inhibit the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, which plays critical role in cancer cell proliferation.



Our data provide information that will allow industry to optimise levels of these beneficial micronutrients in foods. Overall, the data broaden our understanding of the role that food processing plays in inherent health benefits of foods containing flavanols and other phytochemicals. Indeed, although our data was collected using cocoa as a model food, the data have relevance to other food and beverages, including tea, coffee, fruit juices/smoothies and a variety of foods containing fruit and vegetables. Our data will help to deliver healthier foods to the consumer thus reducing disease risk and health care costs at the population level.
Exploitation Route By using only metabolic forms of polyphenols in mechanistic assessment
Sectors Agriculture, Food and Drink,Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description BBSRC Bioscience for Health Strategy Committee
Geographic Reach National 
Policy Influence Type Membership of a guidance committee
 
Description BBSRC research grant 2014
Amount £534,820 (GBP)
Funding ID BB/M002802/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2015 
End 12/2017
 
Description EUropean Union 7th Framework research grant
Amount £320,000 (GBP)
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 06/2010 
End 05/2012
 
Description Mars Nutrition Research Council funding
Amount £86,000 (GBP)
Organisation Mars Nutrition Research Council, Mars Incorporated 
Sector Private
Country United States
Start 08/2008 
End 06/2011
 
Description Mars Symboscience 
Organisation Mars Incorporated UK
Department Mars Symboscience
Country United States 
Sector Academic/University 
PI Contribution R&D of new products based on the research
Collaborator Contribution Help with the synthesis of physiologically relevant metabolites of flavanols, through the use of their facilities.
Impact Manuscript and addtional funding
 
Description The effects of flavanols on brain and vascular health 
Organisation Mars Incorporated UK
Country United Kingdom 
Sector Private 
PI Contribution Wrote successful grant application and hosted PhD student
Collaborator Contribution Supply of research intervention diets for human clinical trial (£60,000). Fully funded PhD studenship (stipend + fees + runing costs). Enhances my groups work in the flavonoid vascular area.
Impact Greatly expands the flavonoid brain and vascular research programme.
Start Year 2007
 
Description 3rd International Conference on Polyphenols and Health 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact International conference; 40 min oral presentation

Press release and round table discussions.
Year(s) Of Engagement Activity 2009
 
Description 3rd International Congress on Wine and Health 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact International conference; 30 min oral presentation

Press release
Year(s) Of Engagement Activity 2008
 
Description 4th Internation Conference on Polyphenols and Health 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 45 min Plenary lecture with questions and discussion afterwards, around 20 min

Requests for publications and further work with Industry stimulated.
Year(s) Of Engagement Activity 2011
 
Description 5th Internation Conference on Polyphenols and Health 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 1h Plenary session with round table discussion (1h). Discussion was wide ranging and set targets for research over the next 2-5 years

requests for Industry projects and manuscript requests
Year(s) Of Engagement Activity 2013
 
Description Diet and Brain Health Film 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Recorded and film for the University of Reading regarding the research area. ~15 min and published on the Universities website

Increased recruitment. Increased Industrial funding.
Year(s) Of Engagement Activity 2010
 
Description Dissemination conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Public engagement event run by the Department of Food and Nutritional Sciences targeted primarily at the public but with literature reaching a wider audience.

After session I received 18 emails requesting further information, publications and advice
Year(s) Of Engagement Activity 2013,2014
 
Description Fourth European Nutrigenomics Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact European Conference; 20 min oral presentation

Press release for conference and media interest
Year(s) Of Engagement Activity 2008
 
Description Mars Nutrition Research Council 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Industrial Conferenece; 40 min oral presentation

Funding directed towards this research in 2007, 2008 and 2009.
Year(s) Of Engagement Activity 2007
 
Description Oxygen Club California 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact International conference; 45 min oral presentation

Press conference and discussion regarding future research focus in the area.
Year(s) Of Engagement Activity 2009
 
Description Oxygen Club of California 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact International conference; 30 min oral presentation.

media interest in the USA
Year(s) Of Engagement Activity 2007
 
Description Royal Pharmaceutical Society 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact International conference; 45 min oral presentation

Press release and media interviews
Year(s) Of Engagement Activity 2009
 
Description Sixth Form Summer School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact organisation and running of the laboratory sessions as part of the School of Food Biosciences 'Sixth Form Open Day'. This course gives A-level students a chance to experience the academic research environment. Experiments related to this MRC grant were used in order to show how certain nutrients may be beneficial to human brain health. June 2005, 2006, 2007 and 2008

Increased recruitment of students.
Year(s) Of Engagement Activity
 
Description Society for Free Radical Research-International 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact International conference; 30 min oral presentation

Media interest
Year(s) Of Engagement Activity 2007
 
Description TV Programme 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Part of a TV programme 'The Food Hospital". Filmed a piece relating to the MRC funded research.

A number of schools have requested lay talks to pupils.
Year(s) Of Engagement Activity 2014
 
Description The Nutrition Society 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact International conference; 30 min oral presentation

Press release and significant media interest. Collaborations made and grants applied for.
Year(s) Of Engagement Activity 2008
 
Description The Times, The Observer, The Daily Mail and The Wine Spectator 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Edited Interviews for publication, mainly 2007 and 2008

Interest from the general public and NHS
Year(s) Of Engagement Activity 2009
 
Description The Wye Valley School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk to 6th-form students about University level science and research.

Increased recruitment and interest in science in general.
Year(s) Of Engagement Activity 2012
 
Description Third European Nutrigenomics Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact European conference; 20 min oral presentation

Conference press release and media interest
Year(s) Of Engagement Activity 2007
 
Description Website articles - Various 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Usually telephone interviews followed by publication of edited interview on public websites.

Interest from the general public usually by email
Year(s) Of Engagement Activity