International Journal of Nutraceuticals, Functional Foods and Healthy Ingredients
ISSN 1722-6996 AIHTEI
Agro FOOD Industry Hi Tech - vol. 26(5) September/October 2015 Published by Tekno Scienze Srl Poste Italiane Spa - Spedizione in Abbonamento Postale D.L. 353/2003 (conv. L. 27/02/2004 n. 46) art. 1 comma DCB M ilano The intelligent way to make foods functional.
Indena may be a new name in the functional foods market. But we weren’t born yesterday. Far from it. For more than 90 years, we at Indena have been researching, developing and producing plant derived active principles. Natural solutions, whose safety and efficacy are guaranteed also by intensive research and rigorous testing. For many years, Indena products have been widely used and appreciated in the pharmaceutical, cosmetic and health food industries. We are already marketing a number of functional food ingredients. And more are on the way. To know more, indena.com. Agro FOOD Industry Hi Tech is a peer-reviewed journal CONTENTS devoted to functional food and nutraceuticals addressed to a readership belonging to the industry. The 2014 impact factor is 0.225, according to the Thomson Reuters Journal www.algea.com Citation Reports. Agro FOOD Industry Hi Tech vol. 26(5) September/October 2015 TEKNO SCIENZE SRL Viale Brianza, 22, 20127 Milano (Italy) Tel +39-02-26809375/28381260 COVER STORY Fax +39-02-2847226 2 A powerful marine ingredient: application as a e-mail: [email protected] valuable salt replacer in industrial products web: www.teknoscienze.com Algea Food
Editor in Chief EDITORIAL 4 Trans fats: towards a global ban on using partially hydrogenated oils Carla Scesa - [email protected] I. Pravst Editorial Director Silvana Maini - [email protected] 6 NEWS FROM EFSA Associate Editors Gayle De Maria - [email protected] NUTRIGENOMICS 8 Nutritional genomics and disease prevention: a case for colorectal cancer Florian Weighardt - [email protected] B. E. Abernathy, E. Chung, C. C. Muscoplat Marketing & Sales Giulio Fezzardini - [email protected] 14 NEWS ON STEVIA Silvia Baldina - [email protected] Simona Rivarollo - [email protected] DIETARY SUPPLEMENTS Marketing & Events 16 The effects of bilberries, blackcurrants and their constituent anthocyanins on heart health in humans Paola Passadore - [email protected] R. A. Koning, I. Grayson, C. Weckbecker Production Manager Elisa Novaresi - [email protected] 22 Vitamin K2 for heart health. Important for preventing calcification of heart and Production Assistants & IT department blood vessels Luis A. Alburqueque Diaz - [email protected] M. E. Møller Matteo Cattane - [email protected] 28 The vitamin hype includes some hazards. The history of vitamin use and Administrative & Account abuse Alba Aprea - [email protected] S. E. Grayson, C. C. Muscoplat
PRINTING OFFICE: PRODUCT FOCUS Arti Grafiche Colombo (Muggiò - MI, Italia) 32 Importance of protein quality for the ageing population FrieslandCampina DMV Authorization from Tribunale di Milano n. 514 of July 27th 2006 (Autorizzazione del Tribunale di 35 NEWS FROM FIE Milano n. 514 del 27 Luglio 2006). 39 NEWS FROM SCIENCE SCIENTIFIC ADVISORY BOARD: Anton Alldrick (CCFRA) NUTRACEUTICALS 42 Evaluation of nutraceuticals effect from in vitro assays Sybille Buchwald-Werner (Vital Solutions GmbH) E. Vezzoli, V. Di Pietro, R. Calò, D. Marchesi, E. De Vecchi, D. Bijno, L. Marabini, Undurti N Das (UND Life Sciences) S. Rodighiero, M. Francolini Inga Koehler (Pfizer Consumer Health Care) Ray A. Matulka (Burdock Group) SENIOR NUTRITION Vicente Micol (University Miguel Hernández) 46 Ageing population trends and the flourishing nutraceuticals market Arthur Ouwehand (Danisco Health & Nutrition) C. Stott, C. Stirling Demosthenes B. Panagiotakos (Harokopio University) WOMEN'S HEALTH Denis Poncelet (ONIRIS and Capsulae sarl) 49 Eve and the apple: the origin of original sin. Apple comsumption and female Igor Pravst (Nutrition Institute – Slovenia) sexual quality of life: an intriguing relationship Joe Romano (Waters) T. Cai Yang Zhu (Eurasia Agrofood Biotech) Ralf Zink (DMK - Deutsches Milchkontor) FUNCTIONAL FOOD 52 Sodium / potassium relationship in biofortified cucumber S. Montoya, A. C. Salgado, M. L. Lorenzo, E. Ortega
PLANT INGREDIENTS 56 Plant protein ingredients for future foods N. Sozer, K. Poutanen
FOOD ANALYSIS 60 Sensory and physico-chemical evaluation of commercial coffees consumed in All rights reserved to Tekno Scienze. No part of this Banja Luka (Bosnia and Herzegovina) – Part 2 publication may be reproduced, stored in a B. Odžaković, N. Džinić, S. Grujić, S. Kravić, M. Jokanović retrieval system or transmitted in any form or by any means: electronic, mechanical, photocopy- 64 Salvia jurisicii Košanin and Salvia amplexicaulis Lam. Chemical composition of the essential oils ing, recording or otherwise without the prior per- A. Alimpić, D. Pljevljakušić, K. Šavikin, V. Matevski, P. D. Marin, S. Duletić-Laušević mission of the publisher Tekno Scienze Srl. ANTIOXIDANTS Associated to USPI - Unione della Stampa 68 Antioxidant activity and phenolic contents. Pomegranate vinegar Periodica Italiana. N. H. Budak
Agro FOOD Industry Hi Tech - vol 26(5) - September/October 2015 1 COVER STORY Agro FOOD Industry Hi Tech customer's publication
Salt is an important nutrient if used in small doses: probably you are eating more sodium than your necessity. Moreover, sodium consumption is related with increase of blood pressure and cardiovascular diseases (CVD’S). Cardiovascular diseases (CVD’s) are a global problem that cause the death of over 17 million people only in 2008. More of 3 million of these deaths can be prevented and occurs in people aged less than 60 (1). Scientific evidence shows that simply reducing the population-wide sodium intake by 15% could prevent 8.5 million cardiovascular-related deaths worldwide over 10 years (2). More recently, the case for decreasing salt intake has been reinforced by the European Food Safety Authority’s (EFSA) positive opinion, which concluded that there is a cause and effect relationship between high dietary sodium intake and increased blood pressure, and that a reduction in dietary sodium intake helps maintain normal blood pressure (ID 336) (3).
The WHO recommends a population salt intake of less than 5 grams per person per day to help prevent CVD. It is estimated that the current global levels are of 9–12 grams per day: for example Americans get most of their daily sodium -more than 75%- from processed and restaurant foods (4). To benefit the population and the economy, governments are pushing for population wide sodium reduction.
Salt is largely used because does not change the organoleptic characteristics of a food while is a taste enhancer, a preservation agent and is useful for food processing. These functions influence how, and if, sodium levels can be reduced in food (5). It may be a valuable solution to replace the common salt that can be found in processed foods with natural ingredients, such as seaweed for example. Ascophyllum nodosum can be a valuable ingredient to substitute part of the added sodium in processed food, poor in salt but having a natural richness of the flavour.
Therefore, in order to offer complete solutions we industrially tested AlgeaFood Phyto in different types of processed products like:
SAUCE: Mayonnaise with a little bit of mustard, reduction of salt 60% FOODS for VEGETARIANS: Tofu with spinach and seaweed, salt reduction around 50% READY FOODS: Tomato sauce with grouper, salt reduction 50% PROCESSED MEAT: Patè of meat with Arctic seaweed, salt reduction 40%
No changes in the industrial parameters by using the Arctic phytocomplex, for sensorial evaluation all of these products were tested in a panel test with 106 panellists; all of them representative of general consumers’ population (age 25-55, women (55%), men (45%)) were individually interviewed for 30 minutes.
2 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 MIRIAM PASCIUTI Contact us to have more information: Algea AS, [email protected] P.O.B 2143 Løkkemyra, N-6502 Kristiansund, Norway www.algea.com
A powerful marine ingredient: application as a valuable salt replacer in industrial products
The test performed, in cooperation with a reliable partner, was with four tastings: - Blind taste compared with the white for 2 products Explain to panellists the differences between the two products: seaweed to reduce salt and only salt: - Informed taste of a product compared with the white - Placebo taste of product with a wrong information regarding the presence of seaweeds or only salt
Here is an example, for tomato sauce with grouper for each taste: placebo effect works, because the “wrong” taste (the product that consumer believed with salt) achieves lower vote in all of three taste types.
Finally, there are a growing number of publications in scientific journals attempting to findnew ways to replace sodium chloride without losing food flavour, and several of them are focusing on brown seaweeds. To mention just a few of them, it has been shown that brown seaweeds can also be successfully used in low-salt meat products (6) and in low salt seasonings (7), and the benefits of their use are not only reduced amounts of sodium used.
Algea, The Arctic Company In Norway, beyond the Arctic Circle, in one of the purest habitats in the world, we have been harvesting and processing Ascophyllum nodosum with great passion for over 75 years. Because this seaweed undergoes strong climate stress due to being sometimes exposed to the sun and bad weather and other times immersed into freezing Arctic waters it developed active components of the highest quality and purity. We harvest Ascophyllum nodosum following a strict process to preserve its quality, with tools and technologies that are advanced in order to preserve the environment. Processing takes place near harvesting areas to keep all the properties intact. It is how AlgeaFood Phyto is produced: a marine phytocomplex made with brown Arctic seaweed, rich in high quality natural active compounds than can help against fat accumulation and sodium intake. The low content of sodium and a balanced intake of algal carotenoids, alginates, vitamins and minerals make AlgeaFood Phyto a great alternative to kitchen salt, helping the body better process fats and facilitating weight control and the achievement of natural well-being.
References 1. Mendis S. et al., “Global Atlas on cardiovascular disease prevention and control”, World Health Organization, 2011 2. Keast, R. SJ, Hayes J. E., “Successful Sodium Reduction”, The World of Food Ingredients, September 2011 3. EFSA Journal 2011 at: http://www.efsa.europa.eu/en/ efsa journal/doc/2237.pdf 4. Mattes RD, Donnelly D. Relative contributions of dietary sodium sources. J Am Coll Nutr. 1991;10:383–93 5. Lim, M., Wing, P. L., Salt Solutions, International Food Ingredients, (2) 2011Jimenez-Colmenero F., Meat Science, 2010, 84(3):356-63. Sanchez- Machado DI, Biomed. Chromatogr.,2004, 18(3):183- 190. Lopez-Lopez I., Meat Science, 2009, Epub ahead of print 6. Ahern DA, J. Am. Diet Assoc., 1989, 89(7):935-8
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 3 EDITORIAL
IGOR PRAVST Nutrition Institute, Tržaška cesta 40, 1000 Ljubljana, Slovenia Member of the Agro FOOD Industry Hi Tech Scientific Advisory Board Trans fats: towards a global ban on using partially Igor Pravst hydrogenated oils
Very recently, Golomb and coworkers published a paper with the meaningful title “A Fat to Forget” in which they show evidence that the consumption of dietary trans fatty acids (TFAs) predicted declines in memory, and that memory became worse with increased intake of trans fats (1). This is only one of the recent additions to the list of evidence of the harmful effects of TFAs on human health. However, while for years TFAs have been a well-recognised risk factor for the development of cardiovascular diseases and associated with all-cause mortality (2), products with high TFA levels can still be found in some markets. An extensive market basket investigation of biscuits in 20 European countries performed by Stender and co-workers showed that TFA levels in foods in Eastern Europe are much higher than in Western Europe (3). These results suggest that some population groups might still be consuming TFAs in amounts that increase their health risks.
TFAs are isomers of unsaturated fatty acids which are not synthesised by the human body and not required in the diet. Their biggest source in the diet is processed foods which contain partially hydrogenated oils (PHOs). These are used to produce semi-liquid and solid fats that can be included when producing foods such as margarine, shortenings and biscuits with a long shelf life. Such products can constitute up to 60% of TFAs (3). PHOs can also withstand repeated heating and are therefore used for frying. For these reasons, as well as consumers’ positive perception of vegetable fats (compared to animal fats), PHOs became very popular in the middle of last century.
The first safety concerns with TFAs present in PHOs emerged in the 1970s and suggested an increased risk of coronary heart disease. Based on strong scientific evidence, it was concluded that TFA intake should be as low as possible within the context of a nutritionally adequate diet (4). Responsible food manufacturers reformulated food products and stopped using PHOs, while policymakers started to regulate this area. Various food policies are being used in different countries to limit dietary intake of TFAs, for example mandatory labelling of the presence of PHOs (currently in use in the EU), labelling of the content of TFAs, and the most restrictive policy – regulatory limits on TFA content in foods.
In Canada and the USA, compulsory labelling of the amount of TFAs in foods was introduced in 2005 and 2006, respectively. In 2013, the US FDA published a preliminary report stating that PHOs were no longer Generally Regarded as Safe (GRAS) and, very recently, in June 2015, it was decided that PHOs will no longer be allowed in foods (5). The food industry has a three-year compliance period to phase out industrial TFAs.
In the EU, mandatory labelling of PHOs on pre-packed food products was introduced in the 1990s. Currently, PHOs must be labelled on ingredient lists as “partially hydrogenated fat/oil”, but there are no general EU limits on TFA levels in foods. Most members currently rely on food producers to voluntarily reduce TFAs. Denmark was the first EU country to introduce legislative limits on TFAs in 2004, prohibiting the sale of foods containing more than 2 g of industrial TFAs per 100 g of fats. Similar rules were introduced later in Austria (2009) and in Hungary (2014).
The acceptance of Regulation (EU) No 1169/2011 on the provision of food information to consumers (6) provided an opportunity to harmonise this issue on the EU level. According to this regulation, the EC shall prepare a report on the presence of TFAs in foods and in the overall diet of the EU population, including a proposal to provide information about TFAs to consumers or restrictions on their use. The EC shall accompany this report with a legislative proposal, if appropriate (6). While such a report should have been published before December 2014 it has been delayed and its publication is expected these days. The EC has been under strong public pressure to resolve this issue as soon as possible. A number of organisations, including the European Society of Cardiology, have called on European policymakers to ban the use of industrial TFAs. It seems that member states are also in favour of such a decision, and there are no opponents publicly defending the use of PHOs in foods. It seems like an excellent opportunity to permanently forget PHOs as a food ingredient also in the EU. While this would be a significantly delayed decision, it would be a very important one. Because several countries are looking closely at food policy decisions in the EU, such a decision would also have a strong global message.
REFERENCES
1. Golomb BA et al. 2015. PLoS ONE, 10(6): e0128129. 2. Kiage JN et al. 2013. Am J Clin Nutr, 97: 1121–8. 3. Stender S et al. 2014. BMJ Open 2014 20;4(5):e005218. 4. EFSA. 2004. EFSA Journal, 81: 1-49 5. US FDA. 2015. Final Determination Regarding Partially Hydrogenated Oils (Removing Trans Fat) 6. Regulation (EU) No 1169/2011 on the provision of food information to consumers
4 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 Superior minerals for infant formula
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Highly bioavailable citrates Strictly controlled safety and purity Advantageous for wet and dry mixes Improved taste of final product
www.jungbunzlauer.com The environmental risk of insect farming human consumption and is processed is also expected to be comparable to hygienically. The food enzyme is used other animal production systems. Existing together with added fibrinogen; the waste management strategies should thrombin catalyses the transformation be applicable for disposing of waste of fibrinogen to fibrin, which interacts from insect production. with collagen, enabling the binding EFSA’s opinion is based on data from of meat/fish pieces resulting in meat INSECTS AS FOOD AND FEED: WHAT ARE peer-reviewed scientific literature, preparations, meat products and fishery THE RISKS? assessments performed by Member products. States and information provided by The typical use and the recommended Interest is growing in the potential relevant stakeholders. maximum use levels of the food enzyme benefits of using insects in food and NEWS have been provided. animal feed, but what would be the Background Dietary exposure to thrombin from its use risks from production, processing and Insects represent a niche food market as a food enzyme was estimated using consumption of this alternative source of in the EU, with several Member States the EFSA Comprehensive European protein? reporting occasional Food Consumption Database. The EFSA has addressed human consumption. estimated mean and 95th percentile this question with a risk Nonetheless, the use exposure across five population groups profile that identifies the of insects as a source ranged from 3 to 12 µg TOS/kg body potential biological and of food and feed weight/day and from 6 to 24 µg TOS /kg chemical hazards as potentially hasNEWS important bw/day, respectively. well as allergenicity and environmental, economic The food enzyme has been environmental hazards and food security characterised by determining the associated with the use benefits. The insect temperature and pH optima and of farmed insects as food species reported to have the thermostability. Its composition is and feed. The Scientific the greatest potentialNEWS characterised by measuring the enzyme Opinion also compares for use as food and/or activity, content of protein and total these potential hazards feed in the EU include organic solids. with those associated houseflies, mealworms, As the food enzyme is derived from with mainstream sources crickets and silkworms. edible parts of animals, no toxicological of animal protein. A number of tests are required. The possible presence of organisations – including Considering the origin of the food biological and chemical the Food and Agricultural enzyme, the CEF Panel considers that hazards in food and feed Organisation of the the likelihood of a food allergic reaction products derived from insects would United Nations (FAO) – have studied to this thrombin is low and, therefore, it depend on the production methods, the possibility of using insects forNEWS food does not give rise to safety concerns. what the insects are fed on (substrate), and feed, and three EU Member States the lifecycle stage at which the insects – Belgium, France and the Netherlands – are harvested, the insect species, as have performed risk assessments related well as the methods used for further to insects as food or feed. processing, EFSA’s scientific experts say. The European Commission is currently EFSA concludes that when non- co-financing a research project to processed insects are fed with currently explore the feasibility of using insect permitted feed materials, the potential protein for feed. The Commission is also occurrence of microbiological hazards considering how to develop policy in the is expected to be similar to that areas of novel foods and animal feed associated with other non-processed to reflect the potential use of insects as sources of protein. There are limited NEWSfood and feed. EFSA’s Scientific Opinion data available on the transfer of was requested to support this work. chemical contaminants from different Based on the origin of the food types of substrate to the insects enzyme from edible parts of animals, themselves. SCIENTIFIC OPINION ON THROMBIN the manufacturing process, and the The occurrence of prions – abnormalNEWS FROM CATTLE AND PIG’S BLOOD compositional and biochemical data proteins that can cause diseases such provided, the Panel concluded that this as Bovine Spongiform Encephalopathy Following a request from the European food enzyme does not give rise to safety (BSE) in cattle and Creutzfeldt-Jakob Commission, the EFSA Panel on Food concerns under the intended conditions Disease in humans – is expected to be Contact Materials, Enzymes, Flavourings of use. equal or lower if the substrate does not and Processing Aids (CEF Panel) was include protein derived from humans asked to deliver a scientific opinion on (manure) or ruminants. the food enzyme thrombin (EC 3.4.21.5) News edited by The Scientific Opinion also considers obtained from blood plasma of cattle GAYLE DE MARIA the possible hazards associated with and pigs. NEWS Agro FOOD Industry Hi Tech other types of substrate, such as kitchen The food enzyme is obtained from [email protected] waste, and animal manure. cattle and pig’s blood that is fit for NEWS
6 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015
NUTRIGENOMICS
BREANN E. ABERNATHY1, ERICA CHUNG2, CHARLES C. MUSCOPLAT3* *Corresponding author 1. Department of Food Science and Nutrition University of Minnesota, Twin Cities 65 Food Science and Nutrition, 1334 Eckles Ave, St. Paul, MN 55108, USA 2. Department of Food Science and Nutrition, University of Minnesota, Twin Cities, 225 Food Science and Nutrition, 1334 Eckles Ave, St. Paul, MN 55108, USA 3. University of Minnesota, Twin Cities, 717 Delaware St. SE, Room 478, Mail code: 1932, Minneapolis, MN 55414, USA Charles C. Muscoplat Nutritional genomics and disease prevention: a case for colorectal cancer
KEYWORDS: Nutrigenomics, nutrigenetics, epigenetics, colorectal cancer, cancer prevention, nutrition.
Within a regular diet, there are many bioactive compounds that have been shown to exhibit Abstract chemopreventative properties. In many cases, this is due to their interactions with the genome, or, moreover, the epigenome. Nutritional genomics is the field of study that aims to elucidate the mechanisms and downstream effects that said bioactive compounds employ with respect to disease. Of the chronic ailments that can be modulated by the diet in such a way, cancer prevention is the most heavily studied, particularly colorectal cancer (CRC). This interest is due to the increased exposure and susceptibility for the colonic microenvironment to be modulated by small molecules in the diet. Understanding these mechanisms and implications in cancer prevention is vital for the future of dietetics and personalized medicine. *The following case study is entirely theoretical. Its purpose is to illustrate the potential understanding of how dietary constituents can impact the onset and progression of cancer. All patient information in the case study is fictional. Further research is needed for such medical and dietetic advances.
that allow them to modulate the molecular environment of CASE STUDY the cells (3). Prevention is largely achieved by controlling Patient 1 is a fifty-year-old male presented with blood in unwanted DNA adduct formation and altered epigenetic stool and abdominal pain. A colonoscopy showed an changes within the microenvironment of the colon. ulcerative non-metastatic tumor. He subsequently underwent surgical extirpation of the tumor. The patient With global CRC rates on the rise, researchers are looking for exhibits central obesity with elevated cholesterol, new forms of prevention or treatment that are less expensive hypertension, heart disease, and diabetes with and more readily available through the diet (1). The neuropathy. He has a history of alcohol consumption and regulation of gene expression by small molecules in the diet smoking. The family history revealed that both his father has recently attracted a great deal of research. This field of and uncle died of colorectal cancer and his mother was study is termed nutritional genomics. Nutritional genomics obese and possessed type II diabetes. aims to understand the association between the genome and bioactive compounds in the diet, and their impact on health and disease. INTRODUCTION
Colorectal cancer (CRC) is the third most common cancer in NUTRITIONAL GENOMICS, MECHANISMS men and second in women worldwide (1). Recently, Dr. Bert Vogelstein published data that showed a linear correlation Nutritional genomics is defined as the study by which dietary with the number of cell divisions and cancer incidence constituents can modulate gene expression and how within specific tissues (2). As colorectal cells have one of the genetic variation can influence an individual’s response highest turnover rates, incidence of CRC is also high (2). This to dietary constituents (4). In most applications, the is due to the increased rate of mutations that occur in highly downstream impact is the prevention and control of disease, proliferating tissues (2). Additionally, colorectal tissues are while in some cases the opposite is possible. highly susceptible to environmental exposure. Over the past decade, this field of study has gained interest In addition to being one of the most common cancers, it is among researchers, namely those interested in CRC (5). also one of the most easily preventable (3). A large part of this A few of the mechanisms and dietary interactions related prevention hypothesis is due to the exposure of the colon to in genetic variation and gene expression are discussed small molecules from the diet, many of which have properties following.
8 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 Folate One example of a dietary constituent that plays multiple to increase CRC progression in a few studies (9,10). As roles in the realm of nutritional genomics is folate. Folate is patient 1 likely possessed a preneoplastic lesion, this widely known as the B vitamin involved in proper neural tube supraphysiological dose of folate could have promoted development, and is now added to processed cereal grains tumorigenesis. for fortification in many countries (6). Additionally, it is naturally found in green leafy vegetables, such as spinach. However, Methylation the over supplementation of folate is beginning to gain Methyl groups are single carbon units that can be attention as serum folate concentrations have increased transferred to small molecules, proteins, and DNA via a over 100% since mandatory fortification began in the United variety of mechanisms, primarily by DNMTs. The methylation States (7). Notably, the developmental genes that are of DNA occurs in cytosine rich regions, called CpG islands. involved in normative neural tube development (and others) When CpG islands are in abundance in the promoter regions can also act as oncogenes when activated at improper of DNA, and can therefore become methylated, the gene time points within an individual’s lifecycle. This makes the role in question is usually silenced (5,12). Hypermethylation of folate in the regulation of these genes extra important in can result in the aberrant silencing of tumor suppressor cancer progression. genes, where hypomethylation can result in the activation of oncogenes. Both of these scenarios are undesirable. Once in the body, dietary folate equivalents are converted to However, the opposite can also be true, leading to a more 5-methylenetetrahydrofolate (MTHF), which is involved in the positive outcome. conversion of homocysteine to methionine. The methyl groups that are donated from methionine and choline are then used Caffeic acid phenethyl ester to produce S-adenosylmethionine (SAM). SAM is considered Caffeic acid, a natural polyphenol found in coffee, is to be the universal methyl donor, and is involved in the action recognized for its antioxidative and anti-inflammatory of DNA methyltransferases (DNMTs) (8). DNMTs transfer methyl properties. Recent studies have shown that caffeic acid groups from SAM to cytosine residues in CpG rich promoter inhibits the proliferation of cancer cells and may be an regions of DNA. Hypermethylation can cause genomic anti-cancer agent itself (13). Caffeic acid phenethyl ester silencing, which is key for tumor suppressor genes in cancers. (CAPE), a derivative of caffeic acid, has also been shown The involvement of folate in this pathway is important, as to have anticancer properties. Two proteins involved in supraphysiologic concentrations of folate could lead to tumorigenesis, PSAT1 and PSMA1, were both down-regulated increased aberrant methylation patterns, and therefore after CAPE treatment (14). Several studies have indicated increased susceptibility to cancers, such as CRC. that CAPE induces apoptosis in liver, oral, and colon cancer cells, in many models. It has also been shown to inhibit the Additionally, folate is involved in thymidylate synthesis, which development of azoxymethane-induced aberrant crypts in is the rate-limiting step in DNA synthesis (6). Impairment of the colon of rats (14). These findings show increasing promise thymidylate synthase, the enzyme that catalyzes the synthesis that caffeic acid may be used as a chemopreventive agent of deoxythymidine monophosphate (dTMP) from deoxyuridine against certain forms of cancer, including CRC. monophosphate (dUMP), can lead to the misincorporation of uracil into DNA (9,10). This misincorporation can cause point Epigallocatechin-3-gallate mutations in the DNA, and, with increasing driver mutations, A major phytochemical in green tea, epigallocatechin- susceptibility to carcinogenesis increases. This is important in 3-gallate (EGCG), has long been known for its health determining the proper dose of folate for chemoprevention, benefits (15). This is especially true in cancer prevention, as too much folate leads to increased DNA synthesis and through various mechanisms including decreasing global therefore increased cellular proliferation (9,10). Importantly, DNA methylation levels, reactivating estrogen receptor- it has been shown that high folate intake is protective in alpha expression in breast cancer, or the demethylation most individuals, the exception being those with preexisting of the Wnt oncogene promoter in lung cells (4). CRC is neoplastic lesions (9, 10). a recent addition to the list of cancers that EGCGs may have protective effects on. A study conducted by Chen Methyl-tetrahydrafolate reductase (MTHFR) is another et al. indicated that after human CRC cells were treated important enzyme involved in folate metabolism, and is with EGCGs for 36 hours, EGCGs caused mitochondrial key in DNA synthesis and methylation (6, 8, 10). A common damage and induced apoptosis triggered by oxidative mutation, C677T, results in reduced activity of MTHFR (30% stress (16). Another study demonstrated that EGCGs from reduction in heterozygotes, 65% reduction in TT homozygotes) green tea have the ability to inhibit DNMT activity and (10, 11). The homozygous TT genotype is associated with reactivate methylation-silenced genes in cancer cells (17). increased CRC incidence and global DNA hypomethylation This reactivation of methylation-silenced genes through (6, 10, 11). Realistically, serum folate concentrations require the usage of EGCGs was demonstrated in human CRC, careful monitoring in at risk individuals, in order to offset the esophageal cancer, and prostate cancer cell lines. progression of CRC. EGCG have also been shown to have anticancer activity CASE STUDY by inducing cellular apoptosis, specifically for patients with Impact on Patient 1: Prior to diagnosis, patient one had CRC. When sodium butyrate (NaB), a naturally occurring been taking 1 mg of folic acid equivalents in supplemental non-toxic compound produced in the colon, is combined form daily. This dose is 1.5 times greater than the with EGCG for treatment, apoptosis and cell cycle arrest recommended daily allowance of folate, and has shown in various CRC cell lines occurs. In addition, it was found that survivin, an antiapoptotic protein that is usually over-
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 9 expressed in CRC, was down-regulated in the CRC cells oversupplementing by a factor of 10, in addition to when exposed to both NaB and EGCG (18). This combined consuming high quantities of vitamin A (23). This treatment provides a novel approach to combating oversupplementaion of antioxidants prevents the CRC with naturally occurring compounds at lower than apoptotic effects of ROS and RNS on cancer cells and conventional doses. may have lead to proliferation of CRC cells in Patient 1.
CASE STUDY While the case study highlights that antioxidants can Impact on Patient 1: Patient 1 recently disclosed his be detrimental to a person when oversupplemented, TAS2R38 genotype, which revealed that he is considered antioxidants also have the ability to enhance cancer- to be a “supertaster” (19). This means that patient 1 is fighting mechanisms. ROS and RNS can also inhibit cancer- highly sensitive to bitter compounds, which are present in fighting mechanisms in the body, and antioxidants are able high concentrations in coffee, tea, and certain to extinguish these free radicals, allowing the cancer-fighting vegetables. Consequently, patient 1 typically turns to soda mechanisms to continue (24). For this reason, a balance instead of coffee or tea for caffeine. This choice provides between antioxidants and free radicals (such as ROS and patient 1 with minimal CAPE and EGCG, both of which RNS) is necessary for proper body maintenance. Without this have shown to be beneficial in CRC prevention. Had balance, undesirable outcomes can accrue. patient 1 ingested such compounds, apoptosis of aberrant cells could have been encouraged, therefore leading to Histone coding decreased carcinogenesis. A more specific form of post-translational modification is through histone coding. Histones are small proteins that octamerize and are then wound by DNA. These complexes POST-TRANSLATIONAL MODIFICATIONS form nucleosomes, which are the basic components of chromatin. The “openness” of the chromatin determines the Post-translational modifications regulate the balance activity of transcription of the genes present in that area, between the survival and death of cells, and can be with more accessible chromatin being considered “open” modulated through the diet. Reactive oxygen species (25,26). The state of accessibility is determined by the small (ROS) and reactive nitrogen species (RNS) induced post- functional groups that are attached to lysine residues on the translational modifications have been recognized to play histone tails. Acetylation, methylation, phosphorylation, and a role in triggering apoptosis and may be a promising ubiquitination are a few examples of the types of reactions method of targeting cancer cells (20–22). Autophagy, carried out that add the respective functional groups to which is a cellular process of turnover for useless proteins histones, the most predominantly studied being acetylation and organelles, can be considered a temporary and (27,28). These reactions are catalyzed by histone acetylases reversible path to cell death. It has been shown that RNS (HATs) and histone deacetylases (HDACs), which add and induces autophagy through suppression of the mTORC1 remove acetyl groups, respectively. Increased acetylation pathway, decreasing cell viability in MCF-7 breast leads to more accessible chromatin, and therefore cancer cell line (20). Recent studies have indicated increased transcription of the genes in those regions (29). that many cancers, including breast and colorectal cancers, are initiated and maintained by cancer stem Sulforaphane cells (CSC), which can initiate tumorigenesis. CSC may Sulforaphane is a bioactive metabolite of glucoraphanin, also contribute to tumor recurrence, metastasis, and a glucosinolate found in cruciferous vegetables such as treatment resistance in some cancers. CSC can be broccoli and Brussels sprouts. It is well understood that counteracted through aberrant N-linked glycosylation glucoraphanin induces the activity of phase II detoxification of cell surface receptors. This action has been widely enzymes, and the metabolite then modulates HDAC activity implicated in regulating tumorigenicity and tumor and histone coding, primarily by inhibition (27). HDAC progression, affecting CSC self-renewal (21). N-linked inhibitors, such as sulforaphane, cause increased acetylation glycosylation of cell surface receptors can be altered of histones, which opens up the chromatin in those regions through changes in expression of glycan-modifying such that those genes are more accessible to transcriptional enzymes (21). Altered expression of glycan-modifying machinery (28). Increased acetylation allows for the enzymes is a result of aberrant expression of sialic acids activation of epigenetically silenced genes, which, in cancer that cap these glycan chains. An increased intake of the are commonly tumor suppressors and other regulators of the non-human Sia N-glycolylneuraminic acid (Neu5Gc), a cell cycle (28). natural component found in milk and red meat, results in metabolic-glycoengineering of human carcinomas (22). In an in vitro study by Rajendran et al., colon cancer cells were treated with sulforaphane and analyzed via a number of methods (27). They found that while the vehicle treated CASE STUDY control cells had a dramatic increase in cell viability and cell Impact on Patient 1: ROS and RNS produce free radicals in count, the sulforaphane treated cells showed no change in the body, and therefore are regulated by the antioxidant viability and the cell count remained constant. Additionally, defense system, two of which are vitamins A and E. Patient HDAC activity in the sulforaphane treated cells did not 1 disclosed that he takes 400 mg of vitamin E daily and change, compared to the control cells where HDAC activity consumes large quantities of sweet potatoes, which are was increased. Moreover, a significant reduction in class I high in vitamin A. While 40 mg of vitamin E is HDAC protein expression was found in the cells treated with recommended for CRC protection, patient 1 is sulforaphane, especially in HDAC3. When sulforaphane
10 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 was removed from the treatment, there was a reversal in provides a survival advantage for those with CRC (32). The HDAC protein expression and general increase in HDAC downregulated miRNAs, including miR-135b and miR-21, activity (27). The downstream effect is that histones remain are responsible for the regulation of tumor and metastasis acetylated, and therefore the genes in such regions can suppressing genes (31–33). Decreased expression of be epigenetically reactivated (27,28). This is important these miRNAs results in increased tumor and metastasis in cancer, as many tumor suppressors are silenced in suppression (33). cancer cells. Activating these genes allows for increased transcription, and therefore induction of apoptosis and cell CASE STUDY differentiation, and the reduction of proliferation by cell Impact on Patient 1: Patient 1’s most recent food cycle regulation (25–29). frequency questionnaire exposes his high consumption of grapes and red wine. Therefore, it can be hypothesized CASE STUDY that patient 1’s tumor suppressing miRNAs are upregulated, Impact on Patient 1: As previously while his oncogenic miRNA levels are downregulated. This discussed, patient 1 is considered a could contribute to delayed onset and decreased supertaster. As bitter compounds, such as progression of carcinogenesis. glucosinolates, are particularly potent to patient 1, he avoids cruciferous vegetables. This choice minimizes his exposure to sulforaphane, the active metabolite of a glucosinolate. This avoidance of cruciferous vegetables may have diminished patient 1’s susceptibility to CRC prevention. microRNA Many genes and pathways in the human genome are regulated by small, noncoding RNAs, dubbed microRNAs (miRNA). The primary mechanism by which miRNAs are able to silence genes is by their incorporation into RNA-induced silencing complexes (RISC), which then unwind the associated miRNA for activation (30). Upon activation, the complex is then able to bind to and inhibit the translation of its target mRNAs, leading to the silencing of those genes (30). While the mechanisms of how miRNAs are involved in the regulatory pathways of cancer is yet to be completely understood, some researchers have made large strides in their findings.
Grape seed extract Recently, the impact of grape seed extract (GSE) on miRNA expression and its downstream effects on signaling pathways in CRC has been under investigation. In a novel study by Derry et al., the chemopreventive Table 1. Dietary bioactive compounds, their genomic mechanisms, and downstream effects of GSE regulated miRNAs were effects (4). studied in a mouse model (31). Mice were injected with a chemical carcinogen, azoxymethane (AOM), in order to induce CRC, and fed OTHER CHRONIC AILMENTS IMPACT CARCINOGENESIS diets containing various amounts of GSE, in hopes of establishing a dose-response relationship. The five groups In addition to the previously discussed mechanisms, were later evaluated for miRNA and cytokine expression. targeting the upstream predispositions for CRC are also They found that GSE altered expression of cytokines/ important. Obesity and diabetes compose two of the most interleukins involved in proliferation and apoptosis (31). important extraneous factors that are often attributed to Additionally, they found that the AOM + 0.5% GSE group CRC (50). had very large changes in miRNA expression for multiple miRNAs when compared to the AOM only group (31). Obesity Importantly, the upregulated miRNAs, including miR- Obesity and its many comorbidities have become let7a and miR-20a, are regulators of cell cycle regulatory highly prevalent in the past 4 decades, affecting both proteins (31–33). The increased expression of these miRNAs young and old (51). Research suggests that obesity is
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 11 a transgenerational disease, and that treatment must You have seen now how our patient may have received encompass strategies for mitigating obesity in future many of his health problems from his parents, but it has also generations. Both maternal and paternal obesity are been been shown that some or many of these problems can be linked with body weight of future generations. For mothers, ameliorated or eliminated with serious changes to the diet. diets high in fat during gestation selects for an obesogenic phenotype in fetuses thereby increasing the chances of Abbreviations obesity and metabolic syndrome in offspring (52). In a CRC colorectal cancer study conducted by Soubry et al. paternal obesity was DNMTs DNA methyltransferases found to be associated with the Insulin-like Growth Factor 2 CAPE caffeic acid phenethyl esters (IGF2) gene hypomethylation in newborns, indicating that EGCG epigallocatechin gallate paternal weight does affect the health of future offspring ROS reactive oxygen species (53). RNS reactive nitrogen species HDACs histone deacetylases Diabetes AOM azoxymethane Obesity is a strong predictor of diabetes in a person. More than 90% of diabetes cases are accounted for by Type II Diabetes (T2DM), which is largely modulated by lifestyle REFERENCES choices. A study by Reyman et al. indicated that severe vitamin D deficiency in childhood obesity is associated with 1. American Cancer Society. Global Cancer Facts & Figures.; lower insulin sensitivity and higher levels of inflammatory 2015. doi:10.1002/ijc.27711. mediators (54). However, another study revealed that 2. Tomasetti C, Vogelstein B. Variation in cancer risk among tissues can be explained by the number of stem cell when this deficiency is corrected, in some cases, so was divisions. Science (80- ). 2015;347(6217):78–81. doi:10.1126/ the patient’s insulin sensitivity problems (55). The above science.1260825. example indicates that it is not impossible to counteract 3. Weinberg R. The Biology of Cancer, 2nd.; 2013. Available at: predisposed maternal and paternal traits through dietary http://garlandscience.com/product/isbn/9780815342205. choices. Accessed June 3, 2015. 4. Hardy TM, Tollefsbol TO. Epigenetic diet: impact on the epigenome and cancer. Epigenomics. 2011;3(4):503–18. CONCLUSION doi:10.2217/epi.11.71. 5. Kussmann M, Krause L, Siffert W. Nutrigenomics: where are There are several important factors that one should we with genetic and epigenetic markers for disposition and susceptibility? Nutr Rev. 2010;68 Suppl 1:S38–S47. doi:10.1111/ understand before diving head first into the field of j.1753-4887.2010.00326.x. nutritional genomics. It is a new and emerging field, and 6. Liu J, Ward RL. Folate and One-Carbon Metabolism and has been discussed here as a small piece of a larger Its Impact on Aberrant DNA Methylation in Cancer. 1st ed. picture. Patient 1 may have benefited from increased Elsevier Inc.; 2010. doi:10.1016/B978-0-12-380864-6.00004-3. caffeic acid, sulforaphane, and vitamin D intake to 7. Mason JB, Dickstein A, Jacques PF, et al. A temporal ameliorate some of his symptoms and delay cancer association between folic acid fortification and an increase progression, but this treatment plan does not apply to in colorectal cancer rates may be illuminating important everyone. It is imperative that we understand that an biological principles: a hypothesis. Cancer Epidemiol individual’s genome and epigenome is unique. While Biomarkers Prev. 2007;16(7):1325–9. doi:10.1158/1055-9965.EPI- there are some similarities, if we choose to use food as a 07-0329. 8. Duthie SJ. Folate and cancer: How DNA damage, repair and form of preventative medicine, ultimately, every patient methylation impact on colon carcinogenesis. J Inherit Metab will have a treatment plan specifically tailored to them. Dis. 2011;34(1):101–109. doi:10.1007/s10545-010-9128-0. The field of nutritional genomics is rapidly expanding, as 9. Protiva P, Mason JB, Liu Z, et al. Altered folate availability mechanisms are being elucidated at an increasing rate. modifies the molecular environment of the human colorectum: In order for medicine and dietetics practice to continue Implications for colorectal carcinogenesis. Cancer Prev Res. advancing, changes in scientific knowledge need to be 2011;4(4):530–543. doi:10.1158/1940-6207.CAPR-10-0143. embraced. We have now seen that epigenetics may 10. Van den Donk M, Pellis L, Crott JW, et al. Folic acid and vitamin have profound effects on diet, lifestyle, and overall health. B-12 supplementation does not favorably influence uracil What an individual eats is driven by many factors; such as incorporation and promoter methylation in rectal mucosa culture, taste and smell, affordability, and psychological or DNA of subjects with previous colorectal adenomas. J Nutr. 2007;137(9):2114–2120. emotional factors. We know that taste can be genetically 11. Hanks J, Ayed I, Kukreja N, et al. The association between determined and therefore contributes to an individual’s mthfr 677C>T genotype and folate status and genomic diet and health. Will there be a difference in cancer onset and gene-Specific dna methylation in the colon of and progression in someone who is sensitive to beneficial, individuals without colorectal neoplasia1-4. Am J Clin Nutr. bitter compounds, such as those in cruciferous vegetables, 2013;98(6):1564–1574. doi:10.3945/ajcn.113.061432. in comparison to someone who is not? 12. Kussmann M, Fay LB. Nutrigenomics and personalized nutrition: science and concept. Per Med. 2008;5(5):447–455. Epigenetics has shown us that an individual’s overall health doi:10.2217/17410541.5.5.447. may not just be due to our choices anymore – but may also 13. Chiang E-PI, Tsai S-Y, Kuo Y-H, et al. Caffeic acid derivatives be influenced by the epigenomes of previous generations. inhibit the growth of colon cancer: involvement of the PI3-K/Akt and AMPK signaling pathways. PLoS One. Despite patient 1’s father and uncle both dying of 2014;9(6):e99631. doi:10.1371/journal.pone.0099631. colorectal cancer, with serious changes to the diet, he 14. He Y-J, Li W-L, Liu B-H, Dong H, Mou Z-R, Wu Y-Z. Identification can start preventing further complications that may arise. of differential proteins in colorectal cancer cells treated
12 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 with caffeic acid phenethyl ester. World J Gastroenterol. 2014;20(33):11840–9. doi:10.3748/wjg.v20.i33.11840. 15. Lee WJ, Shim J-Y, Zhu BT. Mechanisms for the inhibition of DNA methyltransferases by tea catechins and bioflavonoids. Mol CREATING TOMORROW’S SOLUTIONS Pharmacol. 2005;68(4):1018–30. doi:10.1124/mol.104.008367. 16. Chen H, Landen CN, Li Y, Alvarez RD, Tollefsbol TO. Epigallocatechin gallate and sulforaphane combination treatment induce apoptosis in paclitaxel-resistant ovarian cancer cells through hTERT and Bcl-2 down-regulation. Exp DO YOU WANT AN Cell Res. 2013;319(5):697–706. doi:10.1016/j.yexcr.2012.12.026. 17. Fang MZ, Wang Y, Ai N, et al. Tea Polyphenol ( − ) EMULSIFYING AND -Epigallocatechin-3-Gallate Inhibits DNA Methyltransferase and Reactivates Methylation-Silenced Genes in Cancer Cell STRUCTURE- Lines Reactivates Methylation-Silenced Genes in Cancer Cell Lines. 2003:7563–7570. BUILDING FIBER? 18. Saldanha SN, Kala R, Tollefsbol TO. Molecular mechanisms for inhibition of colon cancer cells by combined epigenetic- modulating epigallocatechin gallate and sodium butyrate. Exp Cell Res. 2014;324(1):40–53. doi:10.1016/j. yexcr.2014.01.024. 19. GeneCards. TAS2R38 Gene. GeneCards. 2014. Available at: http://www.genecards.org/cgi-bin/carddisp. pl?gene=TAS2R38. Accessed May 27, 2015. 20. De Sanctis F, Sandri S, Ferrarini G, et al. The emerging immunological role of post-translational modifications by reactive nitrogen species in cancer microenvironment. Front Immunol. 2014;5:69. doi:10.3389/fimmu.2014.00069. 21. Guo H, Nagy T, Pierce M. Post-translational Glycoprotein Modifications Regulate Colon Cancer Stem Cells and Colon Adenoma Progression in Apcmin/+ Mice through Altered Wnt Receptor Signaling. J Biol Chem. 2014;289(45):31534–31549. doi:10.1074/jbc.M114.602680. 22. Padler-Karavani V. Aiming at the sweet side of cancer: aberrant glycosylation as possible target for personalized- medicine. Cancer Lett. 2014;352(1):102–12. doi:10.1016/j. canlet.2013.10.005. 23. Mayo Clinic. Vitamin E Dosing. Mayo Clin. 2013. Available at: http://www.mayoclinic.org/drugs-supplements/vitamin-e/ dosing/hrb-20060476. Accessed June 4, 2015. 24. Milkovic L, Siems W, Siems R, Zarkovic N. Oxidative Stress and Antioxidants in Carcinogenesis and Integrative Therapy of Cancer. Curr Pharm Des. 2014;20(42):6529–6542. doi:10.2174/1 381612820666140826152822. 25. Ho E, Clarke JD, Dashwood RH. Dietary Sulforaphane , a Histone Deacetylase Inhibitor for Cancer Prevention 1 , 2. Prostate, The. 2009:2393–2396. doi:10.3945/jn.109.113332.2393. 26. Traka M, Gasper A V, Smith JA, Hawkey CJ, Bao Y, Mithen RF. Transcriptome analysis of human colon caco-2 cells exposed to sulforaphane. J Nutr. 2005;(May):1865–1872. 27. Rajendran P, Delage B, Dashwood WM, et al. Histone deacetylase turnover and recovery in sulforaphane-treated colon cancer cells: competing actions of 14-3-3 and Pin1 in HDAC3/SMRT corepressor complex dissociation/reassembly. Mol Cancer. 2011;10(1):68. doi:10.1186/1476-4598-10-68. 28. Rajendran P, Ho E, Williams DE, Dashwood RH. Dietary phytochemicals, HDAC inhibition, and DNA damage/ repair defects in cancer cells. Clin Epigenetics. 2011;3(1):4. Our naturally occurring, starch-derived carbohy- doi:10.1186/1868-7083-3-4. drate CAVAMAX® W6 offers you new possibilities 29. Rajendran P, Kidane AI, Yu TW, et al. HDAC turnover, CtIP for novel food formulations: acetylation and dysregulated DNA damage signaling in colon • Heat-stable, oil-based icings and frostings cancer cells treated with sulforaphane and related dietary • Whipping without fat isothiocyanates. Epigenetics. 2013;8(6):612–623. doi:10.4161/ • Highly efficient oil-in-water emulsions, for salad epi.24710. dressings and egg-free mayonnaise type sauces, 30. Kong ANT, Zhang C, Su ZY. Targeting epigenetics for cancer even for meat applications or meat replacements prevention by dietary cancer preventive compounds- the case of miRNA. Cancer Prev Res. 2013;6(7):622–624. CAVAMAX® W6 is plant-based and vegan, doi:10.1158/1940-6207.CAPR-13-0202. approved as a soluble dietary fiber, and has convincing advantages: Readers interested in a full list of references are invited to visit our • Tasteless website at www.teknoscienze.com • Stable even at low pH • Free of all main allergens
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Scientific opinion on the safety of the extension of use of steviol glycosides (E 960) as a food additive
KEYWORDS: food additive, steviol glycosides, E 960, sweetener, extension of use.
Following a request from the European Commission to take The present exposure estimates of steviol glycosides (E 960) are into account an extension of use of steviol glycosides (E 960) based on the MPLs of use currently authorised in Annex II to as food sweeteners, a revised exposure assessment for five Regulation (EC) No 1333/2008 and take into consideration the population groups was carried out based on the maximum maximum levels as proposed by the applicant for an extension permitted levels (MPLs) authorised in Annex II to Regulation of use in tea, coffee, herbal infusion beverages, instant coffee (EC) No 1333/2008 and the extension of use at the levels and instant cappuccino products up to 29 mg/L of steviol proposed by the applicant for the food category 14.1.5 equivalents, rather than 10 mg/L, as assessed in the previous Coffee, tea, herbal and fruit infusions, chicory; tea, herbal and 2014 EFSA opinion (EFSA ANS Panel, 2014). fruit infusions and chicory extracts; tea, plant fruit and cereal preparations for infusions, as well as mixes and instant mixes of The individual raw consumption data for five population these products (sub-category 14.1.5.2 Other). groups (toddlers, children, adolescents, adults and the elderly) Steviol glycosides (E 960) are natural sweet-tasting constituents from the EFSA Comprehensive European Food Consumption and their use as food sweeteners is regulated under the Database were used for the calculation of the revised European Parliament and Council Regulation (EC) No exposure assessment of steviol glycosides (E 960). 1333/2008 on food additives. Stevioside was previously evaluated by the Scientific Committee on Food (SCF) in 1984, According to this requested extension of use, the impact on 1988 and 1999 (SCF, 1984, 1989, 1999). The Joint FAO/WHO dietary intake appears to be negligible in comparison with Expert Committee on Food Additives (JECFA) reviewed the the revised estimate of 2014. The Panel noted that, overall, safety of steviol glycosides (E 960) in 2000, 2005, 2006, 2007 and the mean exposure estimates remain below the ADI of 4 mg/ 2009 (JECFA, 2000, 2005, 2006, 2007, 2009). The EFSA Panel on kg bw per day for all population groups, with the exception of Food Additives and Nutrient Sources added to Food (ANS) toddlers (in one country) at the upper range of the high-level evaluated the safety of steviol glycosides (E 960) used as food exposure estimates (95th percentile: 4.3 mg/kg bw per day), additives in 2010 and established an Acceptable Daily Intake which remains above the ADI. (ADI) of 4 mg/kg body weight (bw) per day (EFSA ANS Panel, The Panel concluded that dietary exposure to steviol 2010). Refinement of the estimated intake was performed by glycosides (E 960) is similar to the exposure estimated in 2014 EFSA in 2011 and in a requested extension of use in 2014 (EFSA and therefore does not change the outcome of the safety ANS Panel, 2011a, 2014). assessment (EFSA ANS Panel, 2010, 2014).
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ROSELLA AMANDA KONING*, IAN GRAYSON, CHRISTOPH WECKBECKER *Corresponding author Evonik Nutrition & Care GmbH Rodenbacher Chaussee 4, 63457 Hanau, Germany
Rosella Amanda Koning The effects of bilberries, blackcurrants and their constituent anthocyanins on heart health in humans
KEYWORDS: Berries, anthocyanins, polyphenols, supplementation, cardiovascular disease, atherosclerosis.
Berries have a long history of use in humans, as they have been shown to have numerous positive health Abstract effects. In this review the focus is on the effects of bilberries and blackcurrants and their purified extracts containing higher levels of anthocyanins on human heart health. Anthocyanins have an effect on several different areas of cardiovascular health, including improvement of endothelial function, anti-oxidative effects, anti-inflammatory effects and the normalization of HDL and LDL lipoproteins in the blood. Purified anthocyanin supplements derived from bilberry and blackcurrant show positive effects against all these cardiovascular disease targets.
INTRODUCTION anthocyanidins found in nature are pelargonidin, cyanidin, delphinidin, peonidin, petunidin and malvidin, with cyanidin Berries have been consumed for many centuries because as the most widely occurring (Figure 1). of their beneficial effects for vitality, prevention of viral infections, eye health, bladder and kidney health, and gastro-intestinal health (1). The interest in dietary supplementation with berries has increased in recent times because of the recognition of their beneficial effects on cardiovascular disease, cancer prevention, neurodegenerative diseases, aging skin protection, inflammation-related diseases, type 2 diabetes and metabolic syndrome (2, 3). Bilberries and blackcurrants are an important source of micro- and macronutrients. Their beneficial health effects have been associated with the high levels of bioactive substances they contain, such as phenolic acids, flavonoids and especially anthocyanins. Anthocyanins are the largest group of water-soluble pigments in the plant kingdom and are responsible for the red, blue and purple colors of many fruits and vegetables. Products with high anthocyanin content include berries, red grapes, red cabbage, eggplant and the black variants of soybean and rice. Figure 1. Main anthocyanidin-glycosides occurring in nature. Anthocyanins are classed as flavonoids, part of the (poly)phenolic group of natural products, and are usually found in the form of flavylium cations. Anthocyanins The average anthocyanin content of bilberry is around 430 are classified according to the number and location of mg and that of blackcurrant is around 270 mg per 100 g of the hydroxy and methoxy groups attached to the flavan edible fruit (6). Bilberries contain a wide variety of at least nucleus, and by the nature of the attached mono-, di- or 16 different anthocyanins; the main ones are delphinidin- tri-saccharides, which can contain glucose, galactose, 3-glucoside (D3G), delphinidin-3-galactoside (D3Gal), arabinose, rhamnose and xylose groups (4,5). The six main cyanidin-3-glucoside (C3G), cyanidin-3-galactoside
16 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 (C3Gal) and petunidin-3-glucoside (P3G). Blackcurrant Endothelial dysfunction has been recognized as an early contains only 4 anthocyanins: C3G, cyanidin-3-rutinoside marker for atherosclerosis (11). Several risk factors (C3R), D3G and delphinidin-3-rutinoside (D3R). including hypertension, smoking, chronic inflammation and oxidative stress (reactive oxygen species or ROS In recent years there has been increased scientific support formation) worsen endothelial function. Biomarkers used to for the positive effects of anthocyanins in the area of heart measure endothelial function in humans are Flow health. Epidemiological studies have shown positive Mediated Dilation (FMD), plasma nitrite and nitrate, and correlations between regular flavonoid intake and a cellular adhesion markers as vascular cell adhesion reduced risk of cardiovascular disease (CVD), CVD molecule-1 (VCAM-1) and intercellular adhesion molecule 1 mortality and coronary heart disease (CHD) mortality (7,8). (ICAM-1) (12). A recent epidemiological study showed that a high anthocyanin intake reduced the risk of myocardial Several randomized placebo-controlled trials have been infarction (9). performed to determine the effect of blackcurrant juice and purified anthocyanins from bilberry and blackcurrant CVD covers a variety of heart and circulatory pathologies on endothelial function. They show that a short-term including coronary heart disease, congenital heart disease, blackcurrant juice consumption in healthy individuals stroke, angina and myocardial infarction. All have different (single consumption of 250 mL 20% blackcurrant juice, pathology profiles, however a common factor is an providing 50.5 mg total cyanidin and delphindin) does not impaired endothelial function. Oxidative stress can worsen have a significant effect on endothelial dependent or the situation by oxidizing lipoproteins which can be more endothelial independent vascular reactivity, and on easily taken up by macrophages. Fat-laden macrophages biomarkers of endothelial function (plasma nitrite, nitrate, can become foam cells leading to inflammation of the ICAM-1 and VCAM-1) (13). A blackcurrant juice blood vessel wall. This then expands, leading to supplementation study in healthy volunteers with a habitual atherosclerosis and restriction of blood flow (10). consumption of less than 2 portions of fruit and vegetables per day (4 x 250 mL blackcurrant juice providing 143 mg The objective of this review is to provide an overview of anthocyanins per day for 6 weeks) found a significant human studies performed with bilberry, blackcurrant and increase in FMD. These effects are attributed to the their constituent anthocyanins and their effects on heart improved endothelial function, as the endothelium- health, especially in relation to endothelial function, anti- independent glyceryl trinitrate-induced dilation was not oxidative effects, inflammation and atherosclerosis. affected. Oxidative stress, measured as F2-isoprostanes, was significantly lowered after supplementation (14). As the long-term blackcurrant supplementation study does show ENDOTHELIAL FUNCTION an improvement in endothelial function, the lack of effect in the earlier trial is most likely due to low amount of active Endothelial cells line the blood vessels and play an constituents consumed. important role in vascular health. Impaired endothelial function leads to a lower ability of the blood vessel to dilate It is not only blackcurrant juice which shows positive because of the lower availability of nitric oxide (NO) (a effects on endothelial function. Supplementation studies vasodilator), and other vasoactive molecules such as using purified anthocyanins from bilberry and blackcurrant endothelin-1 and prostacyclin. Nitric oxide synthase (NOS) is in hypercholesterolemic subjects (320 mg anthocyanins present in the endothelial cell, and converts arginine into per day for 12 weeks) showed significantly increased FMD NO. This diffuses into the vascular smooth muscle cells and and cGMP levels. The cGMP levels and FMD were found to activates guanylyl cyclase (GC), leading to the production be positively correlated. These effects can be attributed of cGMP. This second messenger activates a chemical to an improved endothelial function, as the endothelium- cascade leading to vasodilation (see Figure 2). independent vasodilation (not NO dependent) was not influenced by anthocyanin supplementation. Also on exposure to the NO-cGMP inhibitor L-N-monomethyl arginine (L-NMMA), the beneficial effects on FMD were abolished. This has been confirmed in a rat aortic ring model in the same study. The levels of VCAM-1 were also significantly decreased (15). A similar study using the same anthocyanins, the same dosage and type of subjects over 24 weeks also found that the endothelial adhesion factor VCAM-1 was significantly decreased in plasma. It was confirmed in a porcine arterial endothelial cell line that the anthocyanins C3G and D3G are most likely to be responsible for the significant reduction of VCAM-1 (12).
It is therefore likely that the positive effects of anthocyanins, or anthocyanin-rich products, on endothelial function in humans is mediated via the activation of NO-cGMP signaling and reducing the Figure 2. Nitric oxide mediated endothelial cell relaxation. plasma levels of cellular adhesion molecules, thereby lowering the progression towards atherosclerosis.
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 17 ANTI-OXIDATIVE EFFECTS Activation of NF-KB leads to activation of target genes, and pro-inflammatory mediators are secreted (21). Oxidative damage plays a significant role in the progression of CVD (16). As described above, the presence of NO is Supplementation with bilberry juice in subjects with at needed to maintain normal endothelial function. However, least one risk factor for CVD (330 mL juice per day for 4 reactive oxygen species can interact with NO and reduce its weeks) resulted in a significant decrease in the plasma bioavailability (17). Anthocyanins are powerful anti-oxidative concentrations of inflammatory mediators CRP, IL-6, IL-15 compounds, which have the ability to scavenge radicals and and MIG. All of these are target genes activated by chelate metal ions preventing the cells from oxidative NF-KB (21). damage (18). Several randomized placebo-controlled trials Blackcurrant juice was assessed for its anti-oxidative potential. supplementing purified anthocyanins from bilberry and Blackcurrant juice supplementation in healthy subjects (250 blackcurrant showed positive effects on inflammatory mL juice per day for 1 week) showed significantly increased mediators. Supplementation in healthy subjects (300 mg serum anti-oxidative status (serum SH groups and PON1 anthocyanins per day for 3 weeks) resulted in a significant lactonase activity) (19). A recent human intervention study decrease in NF-KB controlled pro-inflammatory mediators. with bilberry pomace extract demonstrated the anti-oxidative IL-4, IL-13, IL-8, ‘regulated upon activation, normal T cell effects at the molecular level. It was found that the expressed and secreted’ (RANTES), and IFN-α were metabolite phloroglucinol aldehyde (PGA), formed after significantly decreased after anthocyanin supplementation. digestion of the anthocyanin-rich extract activates the NRF2/ No change was found in TNF-α, CRP and IL-1β. IL-4, IL-8 and ARE stress-response mechanism. The transcription factor IL-13 are pro-inflammatory cytokines that induce NF-KB, and nuclear factor E2 (Nrf) is activated by anthocyanins. It moves IFN-α also induces NF-KB. Within the same study monocytes to the nucleus and activates the antioxidant response were exposed to the same purified anthocyanins and a element (ARE) sequence. Upon activation several genes LPS-induced activation of NF-KB was significantly coding for anti-oxidative and anti-inflammatory factors are suppressed. This shows that the anthocyanins are most likely expressed (20). to inhibit the NF-KB activation, and decrease the amount of pro-inflammatory mediators (22). A similar study in subjects with hypercholesterolemia (320 mg anthocyanins per day INFLAMMATION for 24 weeks) also found anti-inflammatory effects; decreased levels of CRP and IL-1β, cytokines which were Inflammation in CVD plays an important role in the not found to be affected in healthy individuals (15, 22). The progression of atherosclerosis. During oxidative stress exposure and target group could explain this difference. In lipoproteins in the blood are oxidized, and can be taken up a HepG2 cell line assay the single anthocyanins C3G and more rapidly by macrophages in the blood. Oxidized D3G inhibited the IL-6 and IL-1β induced CRP production in lipoproteins can harm vascular endothelial cells as the loaded a dose dependent manner. The anti-inflammatory effects macrophages can turn into foam cells (13). These foam cells found with simultaneous exposure of C3G and D3G were attach to the endothelial wall and become fatty streaks. stronger compared with the effects of exposure to the More inflammatory mediators will be attracted to this place, single anthocyanins, indicating a synergistic effect (15). the fatty streak will increase, and will be strengthened with fibrin to form a plaque. The inflammatory response of the Not all the studies done with purified anthocyanins gave body involves macrophages, which play a central role in the positive effects on inflammatory markers. Another regulation of NO, prostaglandins and cytokines. Transcription randomized, placebo-controlled, double-blind trial factor NF-KB plays a central role in the inflammatory response. performed in pre-hypertensive subjects (640 mg purified anthocyanins from bilberry and blackcurrant per day for 4 weeks) showed no significant difference in anti- inflammatory markers (23). The lack of effect can be due to the small sample size, low power of the study, target group and/or time window.
It is most likely that the anti-inflammatory effects of anthocyanins, or anthocyanin-rich products are mediated either via the direct inhibition of NF-KB or via the inhibition of its activators, preventing the activation of pro- inflammatory reactions in the body.
ATHEROSCLEROSIS
Atherosclerosis is characterized by thickened artery walls, narrowed by plaques consisting of white blood cells, dead cells, triglycerides and cholesterol, which impedes the blood flow. The process starts with oxLDL uptake by macrophages. This can be caused by either an increased uptake of LDL cholesterol, or a reduced presence of HDL.
18 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 HDL facilitates the reverse cholesterol transport, meaning randomized, placebo-controlled, double-blind trial in pre- that it promotes the efflux of cholesterol from hypertensive subjects (640 mg anthocyanins per day for 4 macrophages, foam cells and atherosclerotic plaques, so weeks) showed a significant increase in HDL cholesterol, but that it can be transported to the liver and excreted as bile no change in LDL cholesterol. The lack of effect on LDL can (24). This is shown in Figure 3. be due to the small sample size, low power of the study, initial health status of the target group or time window (23).
The improvements in serum lipid profiles after anthocyanin consumption contribute to a lower risk of atherosclerotic plaque formation. The anthocyanins promote cholesterol efflux to the serum and HDL cholesterol, removing the excess of cholesterol from the blood vessels, while simultaneously CETP and LDL cholesterol are limited, reducing the availability of cholesterol for incorporation into the plaques.
CONCLUSION
Anthocyanins have been revealed to be powerful compounds in the support of heart health because of their action on multiple targets. In the pathology of cardiovascular disease they improve endothelial function, anti-oxidative Figure 3. Atherosclerotic plaque formation. markers, anti-inflammatory effects, and also improve the lipoprotein profile. These targets cannot be seen in isolation, as for instance an improvement found in the lipoprotein Short-term supplementation with blackcurrant juice in healthy profile is also found to be correlated to endothelial function subjects (single dose of 250 mL 20% juice) does not show a (12). By influencing these targets, anthocyanins contribute to significant effect on plasma triacylglycerides and non- the maintenance and improvement of heart health. Whether esterified fatty acid concentration levels (13). A similar study the effects observed are directly caused by the anthocyanins with blackcurrant juice in healthy subjects (250 ml 100% juice themselves, or their metabolites is a current focus of research. for 1 week) showed a significant decrease in macrophage The bioavailability is found to be extremely low in humans; cholesterol content; however the cholesterol efflux rate from <0.1% of the intact anthocyanins are excreted in urine. This the cells was not affected. As the efflux rate has not indicates that after ingestion anthocyanins undergo an changed, it is most likely that the uptake of cholesterol has extensive first-pass metabolism, and enter the systemic been inhibited (19). The difference between the two results circulation as metabolites. The concentration of the phenolic could be due to dose, exposure time and the subjects on acid metabolites is found to be significantly higher in the which the study was performed. blood plasma than the concentration of the parent compound. It is therefore likely that not only the parent Supplementation with purified anthocyanins from a mixture of compounds, but also the metabolites are bioactive, and thus bilberry and blackcurrant in dyslipidemic subjects (320 mg per likely to be responsible for the beneficial health effects (25, day for 12 weeks) showed significant increases in HDL 26). cholesterol, decreases in LDL cholesterol, and increased cellular cholesterol efflux to serum. The mass and the activity Although bilberries and blackcurrants are important natural of plasma cholesteryl ester transfer protein (CETP) was sources of anthocyanins and other phenolic compounds, significantly decreased and its change was correlated with they require a relatively long exposure time before a positive the change in LDL cholesterol, while the change in HDL effect on heart health is observed. Short term studies gave cholesterol was found to be correlated to the cellular weak or modest results at best (19), whereas studies cholesterol efflux to serum (24). A similar study with the same performed with purified anthocyanins (320 mg to 640 mg/day, anthocyanins (320 mg per day for 12 weeks) in over several weeks) showed positive effects on several hypercholesterolemic subjects showed significant decrease in aspects of cardiovascular health. This is most likely due to the LDL cholesterol, and an increase in HDL cholesterol. The higher dosages of anthocyanins found in supplements. An change in cGMP (marker of endothelial function) and HDL edible 100 gram portion of bilberry contains ca. 430 mg and were positively related to FMD, therefore the improvement in blackcurrant ca. 270 mg total anthocyanins (6). Consumption serum lipid profile could have contributed to the of an effective anthocyanin dose over the long term only improvement in endothelial function (15). A study using through berries would require a large daily intake, which may anthocyanin supplementation in hypercholesterolemic stress the digestive system, especially with tannin-rich berries. subjects for 24 weeks showed a significant decrease in LDL The use of supplementation with purified anthocyanins, or cholesterol, and an increase in HDL cholesterol. No significant extracts containing high concentrations of anthocyanins, changes in the levels of triacylglycerides and total cholesterol should be explored in more detail to determine their effects were observed. The decrease in cholesterol was positively on long term supplementation in large, well-designed correlated to the change in CRP. CRP has the ability to bind placebo-controlled randomized design trials with surrogate LDL or oxLDL and promote the uptake via macrophages. Thus, markers or clear clinical end points of CVD, while the change in serum lipid profiles could be mediated via the simultaneously monitoring the absorption and metabolism anti-inflammatory effects of anthocyanins (15). Another (26-29). The current available studies are encouraging and
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 19 have shown that supplementation using purified anthocyanins activation in hypercholesterolemic individuals”, Clinical Chemistry, can lead to effective dietary levels of anthocyanins, which 57, 11, 1524-1533 (2011) have been found to have positive effects on biomarkers of 16. Pashkow, F.J. “Oxidative stress and inflammation in heart disease: human heart health. do antioxidants have a role in treatment and/or prevention?”, International Journal of Inflammation, ID 514623 (2011) 17. Rocha, M., Apostolova, N., Hernandez-Mijares, A., et al. “Oxidative stress and endothelial dysfunction in cardiovascular disease: REFERENCES AND NOTES mitochondria-targeted therapeutics”, Current Medical Chemistry, 17 (32), 3827-3841 (2010) 1. Edwards, S.E., da Costa Rocha, I., Williamson, E.M., et al. 18. Kong, J.M., Chia, L.S., Goh, N.K., et al., “Analysis and biological Phytopharmacy: an Evidence-Based Guide to Herbal Medicinal activities of anthocyanins”, Phytochemistry, 64, 923-933 (2003) Products, Chichester, Wiley-Blackwell (2015) 19. Rosenblat, M., Volkova, N., Attias, J., et al. “Consumption of 2. Chu, K-W., Cheung, C.M., Lau, R.A.W., et al. in Herbal Medicine: polyphenolic-rich beverages (mostly pomegranate and black Biomolecular and Clinical Aspects, Benzie, I.F.F., Wachtel-Galor, S., currant juices) by healthy subjects for a short term increased Eds., 2nd Edition, Boca Raton, CRC Press, 55-72 (2011) serum antioxidant status, and the serum’s ability to attenuate 3. Wallace, T.C., Giusti, M.M. Anthocyanins in Health and Disease, Boca macrophage cholesterol accumulation”, Food and Function, 1, Raton, CRC Press (2013) 99-109 (2010) 4. Mazza, G.J. “Anthocyanins and Heart Health”, Ann Ist Super Sanità, 20. Kropat, C., Müller, D., Böttler, U., et al. “Modulation of Nrf2- 43 (4), 369-374 (2007) dependent gene transcription by bilberry anthocyanins in vivo“, 5. Bei, R., Masuelli, L., Turriziani, M., et al. “Impaired expression and Molecular Nutrition and Food Research, 57, 545-550 (2013) function of signaling pathway enzymes by anthocyanins: role on 21. Karlsen, A., Paur, I., Bøhn, S.K et al. “Bilberry juice modulates plasma cancer prevention and progression”, Current Enzyme Inhibition, 5, concentration of NF-kappaB related inflammatory markers in 184-197 (2009) subjects at increased risk of CVD”, European Journal of Nutrition, 49 6. Basu, A., Rhone, M., Lyons, T.J. “Berries: emerging impact on (6), 345-355 (2010) cardiovascular health”, Nutrition Reviews, 68 (3), 168-177 (2010) 22. Karlsen, A., Retterstøl, L., Laake, P., et al. “Anthocyanins inhibit 7. Mink, P.J., Scrafford, C.G., Barraj, L.M., et al. “Flavonoid intake and Nuclear Factor-κB activation in monocytes and reduce plasma cardiovascular disease mortality: a prospective study in concentrations of pro-inflammatory mediators in healthy adults”, postmenopausal women”, The American Journal of Clinical Nutrition, The Journal of Nutrition, 137, 1951-1954 (2007) 85, 895-909 (2007) 23. Hassellund, S.S., Flaa, A., Kjeldsen, S.E., et al. “Effects of 8. Wallace, T.C “Anthocyanins in Cardiovascular Disease”, Advances anthocyanins on cardiovascular risk factors and inflammation in Nutrition, 2, 1-7 (2011) in pre-hypertensive men: a double-blind randomized placebo- 9. Cassidy, A., Mukamal, K.J., Liu, L., et al. “High anthocyanins intake is controlled crossover study”, Journal of Human Hypertension, 27, associated with a reduced risk of myocardial infarction in young and 100-106 (2013) middle-aged women”, Circulation, 127, 188-196 (2013) 24. Qin, Y., Xia, M., Ma, J., et al. “Anthocyanin supplementation 10. Dimmeler, S. “Cardiovascular disease review series”, EMBO improves serum LDL- and HDL-cholesterol concentrations Molecular Medicine, 3 (2), 697 (2011) associated with the inhibition of cholesteryl ester transfer protein in 11. Bell, D.R., Gochenaur, K., “Direct vasoactive and vasoprotective dyslipidemic subjects”, The American Journal of Clinical Nutrition, properties of anthocyanins-rich extracts“, Journal of Applied 90, 485-492 (2009) Physiology, 100, 1164-1170 (2006) 25. Fang, J. “Bioavailability of anthocyanins”, Drug Metabolism 12. Zhu, Y., Ling, W., Guo, H., et al. “Anti-inflammatory effect of purified Reviews, 46 (4), 508-520 (2014) dietary anthocyanin in adults with hypercholesterolemia: a 26. Rodriguez-Mateos, A., Heiss, C., Borges, G., et al. “Berry (poly) randomized controlled trial”, Nutrition, Metabolism & Cardiovascular phenols and cardiovascular health”, Journal of Agricultural and Diseases, 23, 843-849 (2013) Food Chemistry, 62, 3842-3851 (2014) 13. Jin, Y., Alimbetov, D., George, T., et al. “A randomized trial to 27. Del Rio, D., Rodriguez-Mateos, R., Spencer, J.P.E., et al. investigate the effects of acute consumption of a blackcurrant “Dietary (poly)phenolics in human health: structures, juice drink on markers of vascular reactivity and bioavailability of bioavailability, and evidence of the protective effects against anthocyanins in human subjects”, European Journal of Clinical chronic diseases”, Antioxidants & Redox Signalling, 18 (14), Nutrition, 65, 849-856 (2011) 1818-1892 (2012) 14. Khan, F., Ray, S., Craigie, A.M., et al. “Lowering of oxidative stress 28. Tangney, C., Rasmussen, H.E. “Polyphenols, inflammation and improves endothelial function in healthy subjects with habitually cardiovascular disease”, Current Atherosclerosis Reports, 15 low intake of fruit and vegetables: a randomized controlled trial of (5), 324-339 (2013) antioxidant- and polyphenol-rich blackcurrant juice”, Free Radical 29. Habauzit, V., Morand, C. “Evidence for a protective effect Biology and Medicine, 72, 232-237 (2014) of polyphenols-containing foods on cardiovascular health: 15. Zhu, Y., Xia, M., Yang, Y., et al. “Purified anthocyanin an update for clinicians”, Therapeutic Advances in Chronic supplementation improves endothelial function via NO-cGMP Disease, 3 (2), 87-106 (2012)
20 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 Healthberry™ 865 Pulling together the power of bilberries and blackcurrants
Boost the health-promoting potential of food and nutraceutical formulations with a powerful mix of bioactives. Healthberry™ 865 combines anthocyanins from bilberries and blackcurrants in an effective way with proven health benefits.
www.evonik.com / healthberry [email protected] DIETARY SUPPLEMENTS
MONA E. MØLLER Kappa Bioscience AS, Gaustadalléen 21, 0349 Oslo, Norway
Mona E. Møller Vitamin K2 for heart health Important for preventing calcification of heart and blood vessels
KEYWORDS: Vitamin K2, heart health, cardiovascular disease, vascular calcification, calcium.
The strong link between the silent arterial calcification and increased risk for cardiovascular events Abstract suggests needs for preventive actions. Research points to a pivotal role for the Matrix Gla protein (MGP), one of the strongest natural inhibitors of vascular calcification, in preventing calcification. Vitamin K2 (menaquinone-7, MK-7) is a key factor for MGP activation. However, vitamin K2 deficiencies are quite common in the general population. Newer research suggests that intake of vitamin K2 in dietary supplement doses may increase MGP activation and contribute to prevention of calcification of heart and blood vessels in healthy people.
INTRODUCTION attacks and strokes are usually acute events and are mainly caused by a blockage that prevents blood from flowing to Cardiovascular disease (CVD) - large challenges to the the heart or brain. The most common reason for this is a build- society up of fatty and calcium rich deposits on and in the inner walls Cardiovascular disease (CVD) is the number one cause of of the blood vessels that supply the heart or brain - known death globally; more people die annually from CVD than as atherosclerosis.There are two major types of calcium from any other cause. CVD is closely related to life-style mineralization; calcification associated with atherosclerotic factors. Heart disease and stroke can to a large extent be plaque (calcification of the tunica intima), and medial arterial prevented through a healthy diet, regular physical activity calcification (in vascular smooth muscle cells, VSMC, in the and avoiding tobacco smoke. vessel wall), Figure 1 (for a review on calcification of the arteries, see (5)). Arterial calcification was initially referred to An estimated 17.3 million people died from CVD in 2008, as Mönckeberg’s sclerosis and both the medial and internal representing 30% of all global deaths. Of these were an elastic lamina (layer of elastic tissue layer between the intima estimated 7.3 million due to coronary heart disease and and media tunica) are involved (6).Vascular calcification 6.2 million due to stroke (1). CVD causes 47% of all deaths reduces arterial elasticity and result in stiffening of the vessels. in Europe, and is the main cause of death for both women and men in most countries. Overall CVD is estimated to cost Figure 1. Cross the EU economy nearly € 196 billion annually (2). In the US it section through has been estimated that in the next twenty years the cost of the artery wall; medical care for heart disease will rise from $273 billion (2010) Atherosclerotic plaques containing to $818 billion (2030). In addition, heart disease will result in lipids and calcium cost related to productivity; $172 billion in 2010 increasing to (hydroxyapatite) $276 billion in 2030 (3). Due to the aging US population it is result in lumen deforming. Medial estimated that by 2030, almost 4 % of US adults will have had calcification a stroke during their life time (4). (within the vessel wall) containing hydroxyapatite Cardiovascular calcification –a risk factor for CVD result in loss of CVD is caused by disorders of the heart and blood vessels. elasticity and stiff vessels. CVD includes coronary heart disease (heart attacks), cerebrovascular disease (stroke), raised blood pressure (hypertension), peripheral artery disease, rheumatic heart Calcification of the arteries is closely linked to increased disease, congenital heart disease and heart failure. Heart risk of cardiovascular disease (7-11). In patients, an inverse
22 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 relationship between calcification of vessels and survival is circulating de-phosphorylated, under-carboxylated MGP (dp- demonstrated (12). Vascular calcification (VC) is frequent in ucMGP, inactive) correlates well with vascular mineralization the general population and is increasing with age. Recent and a low vitamin K status (19). In accordance with this, studies points to a key role of Matrix Gla protein (MGP) in activated MGP (in the presence of vitamin K2) prevented prevention of VC (13-15), MGP is dependent on vitamin K, in and reversed calcification of vessels in animal models and particularly vitamin K2 for its function (13-15). This paper will human cells in vitro (15, 19, 25-26). Furthermore, Gla-peptide discuss the role of MGP and vitamin K2 for prevention of VC. fragments of MGP inhibited formation of hydroxyapatite crystals in vitro (27). An inverse relationship between dp-ucMGP (inactive) and MATRIX GLA-PROTEIN, MGP, A STRONG INHIBITOR OF VESSEL survival in cardiovascular patients indicates an important CALCIFICATION role of activated MGP in preventing vessel calcification (28). Theuwissen and co-workers summarizes studies showing a One of the strongest inhibitors of vessel calcification is the close correlation between high circulating levels of dp- vitamin K-dependent protein MGP (16, 17). The precise ucMGP and calcification (29). A recent publication supports mechanism of MGP is not known, but a major role is inhibition these findings; in a study in 577 community-dwelling older of soft-tissue calcification. MGP is expressed in bone, lung, people with a follow up of 5.6 years, high plasma dp-ucMGP kidney, arteries and cartilage (18, 19). Just like osteocalcin was associated with increased risk of cardiovascular disease in bone, MGP is dependent on vitamin K for its function; independent of classical risk factors and vitamin D status. The vitamin K is a co-factor for the enzyme γ-glutamyl-carboxylase effect was attributed to low vitamin K status (30). The same which activates “so-called” Gla-proteins by introducing result were found in hemodialysis patients; a 4.5 fold higher carboxyl-groups in the proteins. In the activated form, the serum levels of dp-ucMGP and a 8.4 fold higher under- Gla-proteins can bind calcium and contributes to control the carboxylated osteocalcin (ucOC) was found compared to calcium distribution in the body. Vitamin K is necessary for the controls (31). The studies confirm that both in the general activation to take place and is crucial for the body’s ability to population as well as in hemodialysis patients vitamin K regulate calcium. Activated MGP binds calcium, preventing deficiency is common and may lead to vascular calcification. calcium deposits in arteries (Figure 2).
VITAMIN K2- A KEY PLAYER FOR OPTIMAL CARDIOVASCULAR HEALTH
Vitamin K comprises a number of structurally related compounds including phylloquinone (vitamin K1) and menaquinones (vitamin K2s); menaquinone-4 (MK-4) and menaquinone-7 (MK-7) being the most important. Figure 3 shows the molecular structure of all-trans MK-7, the active form of MK-7 (32).
Figure 3. 3D model of all-trans MK-7. Atoms: black = oxygen, grey = carbon, white = hydrogen.Vitamin K may have different three-dimensional configuration; cis or trans forms. Only Figure 2. Left: Intake of vitamin K2: Healthy vessel with active the all-trans form is MGP, cMGP. Right: No intake of vitamin K2: calcified vessels in the biological active. presence of inactive MGP, dp-ucMGP.
The major differences in effect between vitamin K1, MK-4 MGP was first described by Price (20), and its presence and MK-7 are due to differences in absorption, lipid transport in arteries and in atherosclerotic plaques has later been in blood and tissue distribution (33-35). The half-life for MK-7 demonstrated (21-23). Lou and coworkers showed that is substantially longer than for vitamin K1 and MK-4; 72 hours mice lacking MGP developed to term, but died within two vs. 1-2 hours, resulting in higher blood and tissue levels. As a months as a result of arterial calcification which lead to result, MK-7 has better ability to activate Gla-proteins in extra blood-vessel rupture (24). In accordance with this, Price was hepatic tissue (i.e. bone and vessels) even in microgram able to demonstrate that in warfarin treated rats, inhibiting doses (34). vitamin K dependent MGP activation, the animals developed calcification of the elastic lamellae in arteries and heart valves (21). PREVENTING SOFT TISSUE CALCIFICATION – MAY SUFFICIENT MGP is carboxylated in five residues and may also be INTAKE OF VITAMIN K2 MAKE A DIFFERENCE TO PUBLIC HEALTH? phosphorylated at serine-residues (19). The function of the phosphorylation is not exactly known but is believed to Epidemiological studies have shown a relationship between be connected to protein secretion. The plasma level of intake of vitamin K2 and positive effects on cardiovascular
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 23 health. In a study in the Netherlands (The Rotterdam Study tissue, respectively. As warfarin inhibits the vitamin K-cycle, – 2004), involving 4807 Dutch men and women followed for the inhibition takes place not only in the liver. This implies 8-11 years, it was demonstrated that intake of food with high that warfarin may lead to vitamin K deficiency in peripheral content of vitamin K2 (menaquinones 4-10) dramatically tissue, potentially giving serious side-effects. The side-effects reduced the risk of cardiovascular diseases and mortality of warfarin treatment on Gla-proteins in extra-hepatic tissue (Figure 4) (36). Vitamin K1 had no effects on outcome in have been reviewed by Danziger (49). this large study. In a follow-up study in 564 postmenopausal women using food-frequency questionnaire to estimate In a cross sectional study in middle aged long term intake of vitamin K, it was demonstrated that high intake coumarin users and a matched control group, it was of menaquinones, but not vitamin K1 was associated with demonstrated that chronic coumarin therapy is associated reduced coronary calcification (37). with enhanced vascular calcification (47). It was suggested that the effects were related to the inhibition of MGP activation, probably due to vitamin K2 deficiency. In a recent study, use of anti-coagulants was associated with negative effects on cardiovascular health (48). The authors suggest that chronic use of vitamin K antagonist may enhance potentially harmful coronary calcification in elderly low-risk atrial fibrillation patients. The possible link between VC and anti-coagulant treatment is discussed by Chatrau and co-workers (50).
VITAMIN K IN FOOD – IS THERE A DEFICIENCY IN THE GENERAL POPULATION?
Vitamin K1 found in greens is considered as the major dietary source of vitamin K, accounting for approximately 90% of total vitamin K intake (50). Vitamin Figure 4. The Rotterdam study: Effect of vitamin K2 intake on heart K2, menaquinones, is found in animal products and and cardiovascular health (modified from Geleijnse et al. 2004). fermented food products such as cheese. The content of menaquinones in the daily food intake has decreased over the past hundred years. In the previous century it was A similar study in 16057 women showed that there was common to eat fermented products like “sauerkraut”, cured an inverse correlation between intake of vitamin K2 fish and aged cheeses. Today the most efficacious vitamin (menaquinones MK7, MK-8 and MK-9) and the risk of K, MK-7, is only found in high concentration in the natto coronary heart disease (CHD). Vitamin K1 intake was not dish (fermented soybean) in Japan. However, the content associated with reduction of CHD (38). These data supports vitamin K2 in Western diet is low and it has been suggested earlier findings that dietary vitamin K1 had little or no effect that there is a vitamin K2 deficiency in the population in on coronary calcification (39). most countries in Europe and in the US (51).
The importance of vitamin K2 for cardiovascular health was The intake of K vitamins is sufficient for 100% activation of the recently documented in a double-blind placebo controlled clotting factors in the liver in the healthy population (33, 29). study in 244 healthy postmenopausal women. After daily In contrast to the coagulation factors, several studies intake of 180 µg MK-7 for three years the treatment group have demonstrated that both OC and MGP in serum are showed improved arterial stiffness compared to placebo. not fully activated in the general population (only 10- The improvement was highest in women having high arterial 40% activated) and that supplementation increases the stiffness at start of the study (40). degree of activation (29, 31, 53-56). Potentially, extra- hepatic carboxylation of Gla-proteins contribute to better health (29). As mentioned, elderly community-dwelling WHAT HAPPENS IN PEOPLE ON ANTI-COAGULANT TREATMENT people showing vitamin K-deficiency (high dp-ucMGP) (SEVERE VITAMIN K DEFICIENCY)? followed for 5.6 years showed increased risk of incidence of CVD (30). Theuwissen and coworkers (29) showed that Oral anti-coagulants or vitamin K-antagonists like the coumarins intake of MK-7 in supplement doses (i.e. 90 µg and above) (warfarin) are used globally for primary and secondary resulted in significantly increase in carboxylated OC and prevention of both arterial and venous thrombosis (41). MGP in circulation. Coumarins interfere with the activation of vitamin K-dependent coagulation factors, thereby lowering the ability for blood Very few studies have focused on the vitamin K status in clotting. While warfarin may prevent stroke and pulmonary children. In a recent study (57) it was found that children embolism, it may possibly contribute to complications have a low level of dp-ucMGP which increased progressively associated with low vitamin K activity, such as osteoporosis, with age. After supplementation of children (45µg/day bone fractures, and calcification of arteries (42-48). for seven weeks, 6-10 years) or adults (90µg/day for seven weeks, 20-40 years) a significant decrease in dp-ucMGP was The coagulation factors are activated in the liver, while found both for both groups compared to placebo groups. MGP and OC are activated in the vessel wall and bone The authors conclude that children and adults above 40
24 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 years showed the largest tissue-specific conclude that calcium supplementation vitamin K2 deficiency and may benefit from for prevention of osteoporosis may actually supplementation (57). increase the risk of CVD (72), pointing to the importance of optimal calcium distribution.
THE CALCIUM PARADOX – THE LINK BETWEEN The skeleton is built up by bone BONE- AND CARDIOVASCULAR HEALTH (hydroxyapatite). Build-up cells (osteoblasts) and break-down cells (osteoclasts) regulate As pointed out, vascular calcification is the strength of bones. Bone remodeling an independent risk factor for CVD and (or metabolism) is a lifelong process where mortality. Interestingly, osteoporosis shows mature bone tissue is removed and new a co-occurrence with CVD and vascular tissue is formed. These are slow processes calcification. Several recent publications and in adults, only 10% is remodeled every demonstrate a link between CVD and year. osteoporosis (53, 60-63). The link is mainly Osteoporosis occurs when the activity based on epidemiological studies showing of bone break-down cells (osteoclast) that people with arterial calcification also have increased dominate over the activity of the bone building-cells bone loss and vice versa (64-68). The diseases share (osteoblast) resulting in loss of calcium and fragile bones. common risk factors such as old age and inactivity and In healthy bones, osteoblasts produce osteocalcin which the relationship has been regarded as general effect of binds calcium into bone (hydroxyapatite). Osteocalcin is aging. Recent reviews suggest that CVD and osteoporosis dependent on vitamin K to be active. Thus, not surprisingly, share the same pathophysiological mechanisms (67, 68). vitamin K deficiency, resulting in low bone building activity is Why is there a connection between these apparently common in osteoporotic patients. different diseases? Both processes are complex and regulated by a.o. vitamins, hormones, lipids and signal Vascular calcification is an active process, resulting in proteins, but mineralization is a common denominator. calcification within the cells of the vessel walls (as discussed Calcification of soft tissue such as blood vessels is a process above). During this process vascular smooth muscle cells resembling the bone formation process and several (VSMC) are developing into osteoblast-like cells and start research groups have outlined a common mechanism (10, a mineralization process. What is triggering this process? 61, 69). The mineral composition of bone (hydroxyapatite) is Studies show that vitamin K2 deficiency (MK-7) results in high chemically very similar to that observed in calcified deposits concentration of inactive MGP, which contributes to VC. in atherosclerotic arteries (70, 71). A recent meta-analysis (55, 73, 74). Development of osteoporosis and CV disease are due to Metab. 286(5):E686-96 (2004). complex pathophysiological mechanisms and many factors 11. Truong J.T., Booth S.L., Emerging Issues in Vitamin K Research, are involved. However, one common denominator stands out; Journal of Evidence-Based Complementary & Alternative vitamin K2 deficiency. Vitamin K2 acts a cofactor for activating Medicine, 16(1) 73-79 (2011). 12. Rosenhek R., Binder T., Porenta G., et al. Predictors of outcome osteocalcin associated with building calcium into bone and for in severe, asymptomatic aortic stenosis; N Engl J Med. activating MGP, inhibiting calcium accumulation in the vessel 343(9):611-7 (2000). walls (67, 68, 73); i.e. this vitamin is a pre-requisite for optimal 13. Schurgers L.J., Spronk H.M., Skepper J.N. et al.: Post-translational calcium distribution). Vitamin K2 deficiency may be responsible modifications regulate matrix Gla protein function: importance for the so-called “calcium paradox”; the lack of calcium in the for inhibition of vascular smooth muscle cell calcification,J bone and its accumulation in the vessel wall (74). More studies Thromb Haemost. 5(12):2503-11 (2007). are urgently warranted to evaluate the importance of vitamin 14. Dalmeijer G.W., van der Schouw Y.T., Magdeleyns E.J., K2 in calcium regulation. Vermeer C., Elias S.G., Velthuis B.K., de Jong P.A., Beulens J.W.: Circulating species of matrix Gla protein and the risk of vascular calcification in healthy women,Int J Cardiol. Oct 15;168(6):e168-70 (2013). CONCLUDING REMARK 15. Schurgers L.J., Spronk H.M., Soute B.A.,et al.; Regression of warfarin-induced medial elastocalcinosis by high intake of Vascular calcification of hearth and vessels is to a great vitamin K in rats, Blood. 109:2823-31 (2007). extent asymptomatic and leads to CVD and mortality. 16. Shearer M.J., Newman P.: Metabolism and cell biology of Today, no treatment for cardiovascular calcification vitamin K. Throm Haemost. 100:530-47 (2008). exists. Recent studies suggest that VC is an active 17. Schurgers L.J., Uitto J., Reutelingsperger C.P.; Vitamin process where vitamin K2 plays a key role in prevention K-dependent carboxylation of matrix Gla-protein: a and reversal of the process. It has been suggested that crucial switch to control ectopic mineralization; Trends Mol circulating level of dp-ucMGP (and ucOC) can be used Med.;19(4):217-26 (2013). as a biomarker for vitamin K status; a high concentration 18. Hale J.E., Fraser J.D., Price P.A.; The Identification of Matrix Gla Protein in Cartilage, J Am Soc Biochem & Molecul Biol, 263, of dp-ucMGP (low vitamin K status) may indicate a risk 5820-24 (1988). of calcification of soft tissue and a risk of development 19. Schurgers L.J., Cranenburg E.C., Vermeer C.; Matrix Gla-protein: of CVD (74). Recommendation of vitamin K2 (MK- the calcification inhibitor in need of vitamin K,Thromb Haemost. 7) supplementation to the general population could 100:593-603 (2008). contribute to prevent or reduce risk of CV and risk of 20. Price P.A., Faus S.A., Williamson M.K.; Warfarin causes rapid bone diseases. Several studies are ongoing for further calcification of the elastic lamellae in rat arteries and heart documentation of the beneficial effects of vitamin K2 to valves; Arterioscler Thromb Vasc Biol. 18(9):1400-7 (1998). prevent VC and will hopefully confirm the importance of 21. Price P.A., Urist M.R., Otawara Y.; Matrix Gla protein, a new vitamin K2 for prevention of VC (75). gamma-carboxyglutamic acid-containing protein which is associated with the organic matrix of bone; Biochem Biophys Res Commun. 117(3):765-71(1983). 22. Shanahan C.M., Cary N.R., Metcalfe J.C., et al.; High expression REFERENCES of genes for calcification-regulating proteins in human atherosclerotic plaques; J Clin Invest. 93(6):2393-402 (1994). 1. Nichols M., Townsend N., Luengo-Fernandez R., et al. “European 23. Fraser J.D., Price P.A.; Lung, heart, and kidney express high Cardiovascular Disease Statistics 2012. European Heart Network, levels of mRNA for the vitamin K-dependent matrix Gla protein. Brussels, European Society of Cardiology, Sophia Antipolis Implications for the possible functions of matrix Gla protein (2012). and for the tissue distribution of the gamma-carboxylase J Biol 2. WHO Cardiovascular diseases (CVDs), Fact sheet No 317, Chem. 263(23):11033-6 (1988). (2013). 24. Luo G., Ducy P., McKee M.D., et al.; Spontaneous calcification 3. Heidenreich P.A., Trogdon,J.G., Khavjou, O.A. et al. Forecasting of arteries and cartilage in mice lacking matrix GLA protein. the furture of cardiovascular disease in the United States: Nature. Mar 6;386(6620):78-81(1997). A policy Statement from the American Heart Association, 25. Proudfoot D., Shanahan C.M. Molecular mechanisms mediating Circulation, 123, 933-44 (2011). vascular calcification: role of matrix Gla protein.Nephrology 4. Ovibiagele B., Goldstein L.B., Higashida R.T. et al., Forecasting (Carlton).;11:455-61 (2006). the future of stroke in the United States: A policy statement 26. Wallin R., Schurgers L., Wajih N.; Effects of the blood coagulation from American Heart Association and the American Stroke vitamin K as an inhibitor of arterial calcification,Thromb Association, Stroke, 44, 2361-75 (2013). Res122(3):411-7 (2008). 5. Doherty T.M., Fitzpatrick L.A., Inoue D., et al. Molecular, 27. Goiko M., Dierolf J., Gleberzon J.S., et al.; PLoS One. Nov endocrine, and genetic mechanisms of arterial calcification; 12;8(11):e80344 (2013) Endocr Rev. 25(4):629-72 (2004).. 28. Ueland T., Gullestad L., Dahl C.P., et al. Undercarboxylated 6. Micheletti RG., Fishbein G.A., Currier J.S., et al.;Mönckeberg matrix Gla protein is associated with indices of heart failure and sclerosis revisited: a clarification of the histologic definition of mortality in symptomatic aortic stenosis, J Intern Med. 268:483- Mönckeberg sclerosis; Arch Pathol Lab Med.132(1):43-7 (2008). 92 (2010). 7. Chapman I. Anatomic and clinical significance of calcification 29. Theuwissen E., Magdeleyns E.J., Braam L.A., et al.; Vitamin K of the aortic knob visualized radiographically. Am J Cardiol 6: status in healthy volunteers; Food Funct.;5:229-34 (2014). 281-286 (1960). 30. van den Heuvel E.G., van Schoor N.M., Lips P., et al.; Circulating 8. Giachelli C.M.; Vascular calcification mechanisms; J Am Soc uncarboxylated matrix Gla protein, a marker of vitamin K Nephrol. 15:2959-2964 (2004). status, as a risk factor of cardiovascular disease; Maturitas. 9. Johnson R.C.; Leopold,J.A. Loscalzo,J.: Vascular calcification: Feb;77(2):137-41(2014). Pathobiological mechanisms and clinical implications. Circ. Res.;99:1044-1059 (2006). 10. Vattikuti R., Towler D.A.; Osteogenic regulation of vascular Readers interested in a full list of references are invited to visit our calcification: an early perspective;Am J Physiol Endocrinol website at www.teknoscienze.com.
26 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015
DIETARY SUPPLEMENTS
SHIRA E. GRAYSON1, CHARLES C. MUSCOPLAT1,2* *Corresponding author 1. Department of Food Science and Nutrition, College of Food, Agricultural and Natural Resource Sciences, University of Minnesota, USA 2. University of Minnesota, Twin Cities, 717 Delaware St. SE, Room 478, Mail code: 1932, Minneapolis, MN 55414, USA
Charles C. Muscoplat The vitamin hype includes some hazards The history of vitamin use and abuse
KEYWORDS: Vitamins, vitamin supplements, dietary supplements, nutrition, health.
The discovery of vitamins in the last century has helped to eliminate many nutrient deficiency diseases Abstract and diet-related disorders that have plagued humankind since the dawn of civilization. This understanding of the interactions between vitamins and human health has launched the fortified food industry that has led to the addition of vitamins to many of the food products we eat on a regular basis such as milk, bread, juice, and the majority of processed snacks and cereals. Today we also see vitamins and related supplements sold in mega-doses, marketed by the media as the universal cures or preventions for nearly every conceivable ailment such as enhancing immunity, increasing, lifespan, boosting sexual drive, improving exercise capabilities, or preventing diseases such as cancer or Alzheimer’s disease. This review will briefly summarize the role of vitamin supplements and examine the exaggerated claims and possible dangers of unrestrained vitamin use from a scientific perspective.
THE SUPPLEMENT HYPE about their effectiveness and their safety and ultimately contribute greatly to their consumption patterns (11,12). Throughout the past decades, the use of dietary supplements has increased significantly alongside the growth of the An excellent portrayal of this in effect is with the cover story of multi-billion dollar dietary supplement industry (1-3). Over half the April 6th 1992 issue of Time magazine, entitled “The New of the U.S population uses dietary supplements defined as Scoop on Vitamins”. The article explained that the traditional vitamins and minerals and thousands of claimed beneficial medical views of vitamins and minerals were too limited natural or synthetic products of diverse origins (3-5). It is clear and that vitamins in doses much higher than those usually from these purchasing habits that consumers have placed a recommended may protect against a range of illnesses (13). considerable amount of value on these dietary supplements, The scientific sources used in the article to support these claims but what is not so clear, and perhaps, what is puzzling, is to were sparse and included no results from any well-designed try to decipher why and upon what evidence. Why are we longitudinal clinical trials. Yet, the article became the top buying these products so readily and incorporating them selling issue of the year and was well received by numerous into our every-day routines and health practices and in such pharmaceutical companies and lobbying groups such as the massive quantities? What has led us to believe that these National Nutritional Foods Association (NNFA). concentrated antioxidants, vitamins, natural products, and minerals are the key to reaching optimal health? The scope of this paper is too narrow to cover all types of dietary supplements such as all dietary antioxidants, nutrients, Anthropological studies of supplement practices have and micronutrients beyond vitamins. Examples of these dietary uncovered that the reasons for use are complex and rooted agents may include green tea, red wine, flowers, grains, or any in the current culture that incentivizes proactively taking dietary antioxidants from plant oils or natural products. It may be responsibility for one’s own health (6-8). While the desire to be a that these dietary supplements eventually prove to have health smart consumer committed to improving personal health and benefits, or at least not add to our risks. However, these dietary prevent illness is impressive, what is troubling is that much of the agents have not been adequately tested through actual studies information about health and science comes from popular to be discussed here in this report. press and aggressive marketing strategies, not from legitimate scientific research (9-11). Thus, the way in which dietary Instead, the scope of this article will specifically cover the supplements are framed by the popular press, advertisements, history and science of vitamin supplements and how they or other private companies can create societal assumptions have come to play such a major role in the consumer health
28 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 industry. Additionally, this article will focus on why, based on Up until the mid-1930’s, vitamins were obtained through food existing evidence, there is little justification for the general and intake alone and levels were regulated by changes made widespread use of high dose vitamin supplements. to the diet. However, since the mid 20th century, vitamins have been produced as commodity chemicals made widely We need adequate amounts of all thirteen vitamins to survive available as inexpensive semi-synthetic and synthetic sources and to thrive, and the discovery and isolation of vitamins has of multivitamin dietary supplements and as additives in fortified led to the prevention of vitamin deficiency conditions as well as foods (24). This transition rapidly shifted the focus from adequate the eradication of several devastating nutrient deficiency-linked vitamin intake to high dose or “mega dose” vitamin intake in diseases (14-16). order to maximize health outcomes. The words “vitamins” and “antioxidants” have become synonymous to “health” and However, the health-conscious American society today has “fitness” and the adage that “the more vitamins we consume, succeeded in eradicating and treating nutrient deficiency the better” has been made independent from modern associated conditions and diseases to the point where we now scientific understanding and solid clinical data. live in an excess of vitamin availability. In addition to the vitamins and minerals that occur naturally in the foods we eat, we are Many of the results showing harmful long-term health also consuming combinations of vitamins and minerals added effects of seemingly safe vitamins or supplements are met to ‘enhance’ juices and sports drinks, in fortified or enriched with strenuous disbelief. Despite these findings, the dietary products, and concentrated in supplemental pill forms (17). supplemental industry continues to grow with the creation Nevertheless, there is little to no conclusive long-term research to and sales of new pills, serums, drinks and powders containing support that the consumption of vitamins at doses higher than vitamin concentrations way beyond the Recommended Daily recommended is beneficial to our health in any way, let alone a Allowances (RDAs) established by the Food and Nutrition Board safe decision. of the National Academy of Sciences (25-26).
Advocates will say that all of the 65,000 dietary supplements, THE HISTORY AND SCIENCE BEHIND VITAMIN USE including vitamin products that are currently on the market, represent a vast amount of new storehouse of health waiting The belief that the diet is a powerful tool for improving health to be tapped by the consumer. Empowered consumers and preventing illness dates back centuries before there was a can read up on boastful new claims, but they may also be real knowledge of nutrition. The Ancient Egyptians, for example, unproven and anecdotal reports regarding anything from the ate liver to cure night blindness long before it was known that prevention of cancer with daily multivitamin use, increased night blindness was associated with vitamin A deficiency, fitness performance with antioxidant supplements, increased and sailors on long-distance voyages throughout the 17th immune function with vitamin C pills to the latest herb cure for and 18th centuries ate citrus foods to avoid scurvy prior to the depression. understanding that the disease was caused by low levels of vitamin C (18). There is no contest that these vitamins in their elemental or natural form may be associated with positive health effects The term “Vitamine” was officially coined in 1912, by a Polish such as the prevention of diseases, increased fitness ability, and biochemist named Casimir Funk who was one of many scientists immune function. However, countless studies have shown that to contribute to the discovery of vitamins throughout the early vitamins in supplement form may not have identical health 19th and mid 20th century (19). The discovery of vitamins effects as vitamins consumed through their natural food sources helped clinicians recognize that diseases like scurvy, beriberi, (27, 28). rickets, pellagra, and xerophthalmia were caused by specific vitamin deficiencies as opposed to infections or toxins (20, 21). While not vitamins, glucosinolates, an anticancer compound, It was through clinical observations of vitamin deficiencies that provide a valuable example of how beneficial chemicals in chemists were able to isolate and characterize the individual foods can be absorbed differently by the body when eaten as vitamin structures. real foods in comparison to when they are consumed in store bought supplements. To illustrate, a study in 2011 found that Vitamins are defined today as essential nutrients that subject given fresh broccoli florets absorbed and metabolized are needed in small amounts for the normal growth and seven times more glucosinolates than the subjects given development of organisms (21). There are thirteen universally isolated glucosinolates in capsule form (27). recognized vitamins that must be obtained through the diet because they cannot be synthesized in sufficient quantities Additionally, while several studies have shown antioxidants by the body; vitamins include vitamin A, C, D, E, K, B1-B3, B5- may protect against free radicals involved in oxidative B7, B9 and B12. Vitamin D is an exception since its precursor stress associated with many diseases, cognitive impairment, cholecalciferol, can be synthesized by the skin with adequate dementia, and byproducts of high intensity exercises (29-31), sunlight and can be converted in the kidney to vitamin D. additional studies have shown the opposite effect to be true Vitamins are further categorized into fat-soluble vitamins with antioxidants in high-dose supplement form. Specifically, (vitamins A, D, E, and K), which are stored in the liver and high doses of Vitamin C has been shown to diminish muscle adipose tissue, and water-soluble vitamins (vitamins B and growth with strength training (32,33) and antioxidant C), which are readily excreted from the body. For fat soluble supplement combinations have been shown to blunt the vitamins, the vitamin can become sequestered in fatty tissues, or signaling of molecular pathways and mitochondria creation, the opposite insofar as deficiencies might take a very long time necessary for exercise training adaptations (34, 35). Additionally, to appear since the vitamin stores are accessed over a very high doses of vitamin C and calcium have been shown to be long time period (21-23). associated with an increased risk of cancer (36, 37).
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 29 One explanation is that these inconsistencies are most likely due One example is The Physician’s Health study that followed to other chemicals naturally found in food products that may a total of 14,641 US male physicians above the age of 50 improve nutrient potency and absorption by the body. There receiving different vitamin supplement combinations from 1997 is another explanation, specifically in the case of antioxidants until 2007 with follow up studies lasting until 2011. Results from discussed above, that has been hypothesized by a number of this longitudinal study found that there were no immediate or scientists referred to as the “antioxidant paradox”. It explains long-term effects on the risk of total cancers with vitamin E or that while we associate free radicals with destructive activity C supplementation. Also, there was no difference in cognitive like damaging DNA and interfering with cellular membranes, performance or verbal memory from the individuals that free radicals also have some vital qualities like killing bacteria, took daily vitamins versus the individuals that took placebos eliminating new cancer cells, and acting as messenger throughout this study (49). initiators for many necessary biochemical processes (38). Thus, as antioxidants are known to absorb and deactivate these In addition to the null results of vitamin supplement use, free radicals, most likely, when people are taking antioxidants several large clinical trials such as the Alpha Tocopherol Beta- such as vitamin C and E in large concentrations they disrupt Carotene Cancer Prevention Trial, The Beta-Carotene and the balance between free radicals and antioxidants to the Retinol Efficacy Trial, The Women’s Health Initiate, and The point that critically necessary biochemical processes such as Iowa Women’s Health Study have found that the regular use immune function, cell communication, and muscle growth are of vitamin and mineral supplements including multivitamins, interrupted (39). vitamin B6, vitamin E, folic acid, supplemental iron, magnesium, zinc and copper, resulted in increased incidences of mortality A way to better demonstrate the difference in antioxidant and diseases including several types of cancers (48, 50-52). doses naturally found in foods versus in supplemental form is to compare the concentrations of vitamin C. For a reference, There have also been numerous studies that show that calcium the RDA for vitamin C for a non-smoking adult is 60 milligrams supplementation with or without vitamin D increases the risk per day. The amount of vitamin C in one orange is between of cardiovascular events, specifically, myocardial infarctions 50-75 milligrams and in one cup of orange juice is about 75- (53-55). 90 milligrams. In contrast, a typical vitamin C tablet contains 1,000 milligrams accounting for over 1,000% of the RDA, and An important thing to consider, that is often overlooked, is some labels advise taking two tablets per day (40). Marketers that individual factors such as age, gender, weight, total encourage consumers to continue high dose antioxidants by vitamin intake, medication use, predisposition for disease, claiming these products contain higher antioxidant scores, exercise frequency, and other environmental exposures such better solubility profiles or even stronger or longer biological as cigarette smoke can greatly affect the way in which each activity. However, the basis of this logic is flawed because many individual tolerates vitamin intake (56). In other words, vitamin claims of antioxidant success have based only in vitro studies, use in combination with predisposition for cancer or tobacco which cannot observe long-term side effects or toxicities in vivo. use for example have been shown to increase the likelihood for possible harmful health effects (57). Also, of great interest, is the data from the National Health WHY VITAMIN SUPPLEMENTS RAISE SOME CONCERN and Nutrition Examination Survey (NHANES) of 2003-2006 that showed that individuals who use dietary supplements Countless studies have shown that people who eat containing vitamins and minerals tend to have higher intakes more vitamin rich fruits and vegetables have a of fortified foods and vitamin-rich diets from natural food lower incidence of cancer, heart disease and live sources than non-vitamin users to begin with (1, 58). Thus, longer (41,42). Like earlier discussed, clinical trials it is those individuals who are already getting adequate in the past several years, however, have vitamin intake who are consuming the extra, unnecessary failed to find beneficial qualities for mega-doses of vitamins in supplement form. This combination the prevention of chronic diseases can be dangerous as users who reach levels above the with regular use of vitamins in recommended Upper Limits (UL) of vitamin intake for nutrients
supplement form, and many such as folic acid, vitamins A, B6, C, calcium, iron, zinc and have found harmful health magnesium increase the potential for nutrient toxicities and effects associated secondary complications such as decreased organ function with use such and increased cancer incidence (2, 15, 17, 59). as increased The Dietary Supplement Health and Education Act (DSHEA), mortality (43-48). signed in 1994, classified dietary supplements as a subcategory of food. There is a history of controversy over this legislation, specifically, because it relies solely on the dietary supplement manufacturers to insure the quality and safety of their products. Vitamin supplements require no standardization or testing for safety to be submitted to the US FDA before they are made available on the market (57).
The leniency of the DSHEA also allows labels on vitamin bottles to include claims about structure and function of the vitamins as long as they do not claim to treat or cure certain diseases (57). This leads to confusion for consumers who are trying to decipher between
30 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 marketing claims and scientific descriptions on labels; knowledges, strategies”, Thousand Oaks: Sage (1996). many consumers have been misled to believe that dietary 8. Fries, C.J. “Governing the health of the hybrid self: integrative supplements including vitamins can be substituted for physician medicine, neoliberalism and the shifting biopolitics of subjectivity”, prescribed medications (60). Health Soc. Rev, 17, 353-67 (2008). 9. Geller, G., Bernhardt, B. A., Holtzmn, N. A. “The media and public reaction to genetic research”, JAMA, 287-773 (2002). Lastly, unlike in most European countries where the daily 10. Story, M., French, S. “Food advertising and marketing directed at dosage of a vitamin supplements cannot exceed 300% children and adolescents in the U.S”, Int J Behav Nutr Phys Act, 1, of the recommended daily allowance as regulated by 3-17 (2004). the government, marketers can dictate dosage and use, 11. Nagler, R. H. “Adverse outcomes associated with media exposure to bypassing any dosage regulations that are normally in place contradictory nutritional messages”, J. Health Commun, 19, 42-40 for determining the use of drugs in the United States (61). (2014). 12. Scheufele, D.A. “Framing as a theory of media effects”, J. Commun, There have been attempts to regulate the dietary supplement 49, 103-22 (1999). industry more closely, such as the proposition of The Dietary 13. Toufexis, A. “The New Scoop On Vitamins”, TIME, 54-59 (1992). 14. The National Academies Press Dietary Reference Intakes: Guiding Supplement Safety Act of 2010 by Senators John McCain and Principles for Nutrition Labeling and Fortification, Washington, DC, Byron Dorgan that would have required dietary supplement (2003). manufacturers to register all products with the US Food and 15. Fulgoni, V. L., Keast, D. R., Bailey, R. L., et al. “Foods, fortificants, and Drug Administration (FDA) and fully disclose all product supplements: Where do Americans get their nutrients?” J Nutr. (2011). ingredients upon registration (62). The Dietary Supplement 16. Honein, M. A., Paulozzi, L. J., Mathews, T. J., et al. “Impact of Safety Act never became law. Fortification of the US Food Supply on the Occurrence of Neural Tube Defects”, JAMA 285:2981-2986 (2001). 17. Datta, M., Vitolins, M. “ Food Fortification and Supplement Use- Are CONCLUSION There Health Implications?” Crit. Rev. in Food Science and Nutr. (2014). 18. Challem, J “The Past, Present and Future of Vitamins”: https://web. High dose vitamin supplements are challenging to study archive.org/web/20051130103653/http://www.thenutritionreporter. because of different study conditions, durations, doses and com/history_of_vitamins.html (Last viewed March 4th, 2015). agents used, diseases measured, and dietary histories of 19. Semba, R. “The Discovery of Vitamins”, Int J Vitam. Nutr. Res., 82(5), subjects involved in the research conducted thus far. One can 310-315 (2012). easily search the literature and locate studies showing both 20. Chick, H. “Studies of Rickets in Vienna 1919 – 22” (Report to the positive, negative and null findings. While those contradictions Accessory Food Factors Committee appointed jointly by the continue to exist (as common with many emerging research Medical Research Council and the Lister Institute). His Majesty’s topics), what many experts can agree on is the best way to Stationery get our vitamins is from the real foods that are naturally rich in 21. Lieberman, S., Bruning N. “The Real Vitamin & Mineral Book. NY: vitamins, minimally processed, and contain countless beneficial Avery Group, 3, ISBN 0-89529-769-8 22. Drincic, A., Armas, L., Van Diest, E., et al. “Volumetric dilution rather chemicals beyond vitamins. This method for primary vitamin than sequestration best explains the low vitamin D status of obesity.” intake insures a built-in system of “checks and balances” Obesity (Silver Spring), 20 (7), 1444-8 (2012). because while there is variability in nutrient intakes and needs 23. Earthman, C., Beckman, L., Masodkar, K., et al. “The link between across the population, it is difficult to reach vitamin toxicity obesity and low circulating 25-hydroxyvitan D concentrations: levels from naturally vitamin dense foods alone. In conclusion, Considerations and implications.” Int J Obes (Lond), 36 (3), 387-96 the hype for vitamins may still remain, but the hype should shift (2012). from reaching into bottles for capsules of vitamins to reaching 24. Paganini-Hill, A., Kawas, C. H., Corrada, M. M. “Antioxidant vitamin a lifestyle balance that incorporates healthy levels of vitamins intake and mortality: The leisure world cohort study” Am. J Epidem, into the diet, preferably in moderation from natural food 181(2), 120-126 (2014). sources. 25. Dachner, N., Mendelson, R., Sacco, J., et al. “An examination of the nutrient content and on-package marketing of novel beverages”, J. Appl Physiol Nutr. Metab, 40 (2), 191-8 (2015). 26. United States Department of Agriculture National Agriculture Library: REFERENCES http://www.nutrition.gov/smart-nutrition-101/dietary-reference- intakes-rdas (Last checked on March 4th 2015) 1. Bailey, R. L., Gahche, J. J., Lentino, C. V., et al. “Dietary Supplement 27. Clarke, J. D., Hsu, A., Riedl, K., et al. “Bioavailability and inter- Use in the United States 2003–2006”, J Nutr., 141, 261-266 (2011). conversion of sulforaphane and erucin in human subjects consuming 2. Bailey, R. L., Fulgoni III, V. L., Keast, D. R., et al. “Examination of Vitamin broccoli sprouts or croccoli supplement in a cross-over study Intakes among US Adults by Dietary Supplement Use”, J. Acad Nutr design.”, Pharmacol Res, 64(5), 456-463 (2011). Diet, 112, 657-663 (2012). 28. Fortmann SP, Burda BU, Senger CA, et al. “Vitamin and mineral 3. Radimer, K., Bindewald, B., Highes J., et al. “Dietary supplement use supplements in the primary prevention of disease by US adults: Data from the National Health and Nutrition cardiovascular and cancer: an updated systematic evidence review for the U.S. Examination Survey 1999-2000”, Am J. Epidemiol., 160 (4), 339-349 Preventive Services Tasl Force. Ann Intern Med 159:824-34 (2013). (2004). 29. Dias M, Frei B, Vita J, et al. “antioxidants and atherosclerotic heart 4. Denham, B. E. “Dietary Supplements—Regulatory Issues and disease. “ N Engl J Med. 337(6) 408-416 (1997). Implications for Public Health”, JAMA, 306, 428-429 (2011). 30. Steinberg D, Witztum J. “Is the oxidative modifican hypothesis 5. Mursu, J., Robien, K., Harnack, L. J., et al. “Dietary Supplements and relevant to human atherosclerosis? Do the antioxidant trials Mortality Rate in Older Women: The Iowa Women’s Health Study”, conducted to date refute the hypothesis?” Circulation. 105 (17) Arch Intern Med, 171, 1625-1633 (2011). 6. Nichter, M, Thompson, JJ. “For My Wellness, not just my illness: North 2107-2111 (2002). Americans’ use of dietary supplements”, Cult Med Psychiatry, 20, 175-222, (2006). Readers interested in a full list of references are invited to visit our 7. Petersen, A, Lupton D. “The new public health: discourses, website at www.teknoscienze.com.
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 31 PRODUCT FOCUS Agro FOOD Industry Hi Tech customer's publication
Importance of protein quality for the ageing population
Introduction amino acids absorbed from the gut in a certain chemical It is clear the world population gets older. We are constantly form. However, during the processing of food, some amino adding more years to our lives. This mainly is due to good acids such as lysine and alanine are susceptible to chemical medical care. But how do we keep the older generation modification. Lysine is especially important, because lysine is one healthy? The last decade the key word was prevention; exercise of nine essential amino acids that humans cannot synthesize and good nutrition. To be more specific; protein especially themselves. A fixed pattern of these essential amino acids is in combination with exercise was mentioned several times in required to maximize protein synthesis in tissues, such as muscle scientific research to stay healthy during ageing. and bones. In a grain-rich diet, lysine is often the first-limiting amino acid (3). New evidence shows that older adults need more dietary Lysine has a side chain amino group which can easily react protein than younger adults to support good health, promote with reducing sugars such as fructose, lactose and glucose. recovery from illness and maintain functionality (1). To help During processing (especially heat treatment) and during older people (>65 years) maintain and regain lean body mass storage, Maillard reactions occur (5). A Maillard reaction is a and function, it is recommended to ingest at least 1-1,2 gram chemical reaction between reducing sugars and amino acids. of protein per kg body weight per day. This is the opposite of The Maillard reaction can be divided into three different stages: what is happening in the diet of elderly; due to less appetite and initial, intermediate and late. A late or advanced Maillard low energy intake, protein ingestion decreases during aging reaction is recognized in browning when baking products. (2). Therefore it is of crucial importance that all amino acids in The early Maillard reaction results in the formation of Amadori a protein are available as a building block for the muscle and compounds in which lysine is no longer nutritionally available (6). other tissue. Unfortunately due to processing and differences in Loss of available lysine is the most significant consequence of quality, protein quality can be reduced. the Maillard reaction and it is of great importance in those foods where lysine is the limiting amino acid (7). Protein is not just protein. The quality of a protein source can be determined through three characteristics: the amount of protein How to preserve amino acids availability in food, the amount of indispensable amino acids in the protein An easy strategy to prevent early Maillard reactions is to choose and digestibility. a protein source with low or no reducing sugars. This means no Dietary protein quality can be assessed using protein quality sugar is naturally present (such as lactose in different protein parameters. The FAO/WHO recommends PDCAAS or Protein sources) or reducing sugars are added to the product. Also Digestibility Corrected Amino Acid Score as a suitable method check table 1 for the protein and lactose content of different for determining dietary protein quality (Figure 1). This score is protein sources. calculated from the first-limiting amino acid in digestible dietary protein relative to the amino acid requirement in humans (3).
Damage of protein after processing and storage Figure 1. Changes in total 1RM Figure 2. Changes in the during 12 weeks of resistance type quadriceps muscle CSA after 12 Figure 3. Amount of amino acids The available exercise in healthy young men weeks of resistance type exercise released in to the bloodstream with amino acids that who did or did not receive protein in young men who did or did not casein and whey protein. Casein protein has a prolonged release of can be used for supplementation. receive protein supplementation. **Different from PLA group P<0.05. **Different from PLA group P<0.05. amino acid. protein synthesis Adapted from (5). Adapted from (5). Adapted from (6). (2) are dietary
32 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 The body cannot use blocked lysine for protein synthesis It is assumed that blocked lysine is not biologically available because the covalent bond between lysine and sugar cannot be split by digestive enzymes (8). Although blocked lysine can be partly absorbed from the digestive tract, it has no nutritional value and will be excreted with urine (5). Hence, the vast majority of blocked lysine (> 70%) enters the colon where it is predominantly utilized by microorganisms and also affects microbiota composition. This means that blocked lysine will not be used for body protein synthesis in order to maintain or develop structural proteins in muscle and bone (8, 9).
Conclusion It is increasingly acknowledged that not only amino acid composition, but also the availability of essential amino acids, contributes to the quality of dietary proteins. Lysine damage impacts the availability of essential amino acids, especially lysine, for protein synthesis in tissues such as muscle.
The older population is decreasing dietary protein intake, so for this population it is of extreme importance that all the protein that are ingested is available for the body. Milk protein is known as a high quality protein source. Also proteins extracted from milk, such as caseinate are suitable as a protein enrichment in the diet of elderly. Caseinate has several benefits; it has a low lactose content, it has all amino acids available and the quality remains after processing.
FrieslandCampina DMV At Frieslandcampina we take great pride in 140 years of milk processing heritage. As a dairy co-operative, FrieslandCampina has unique control of the complete value chain ‘from grass- to-glass’. Together with our strong nutritional know-how we produce high quality nutritional proteins. For more information: www.dmv.nl or www.frieslandcampina.com
Literature (an extended list of references is available on request via [email protected]): 1. Bauer, 2013 2. Tieland, 2012 MIEKE ACDA 3. FAO protein quality [email protected] 4. Rutherfurd, 2012 FrieslandCampina DMV 5. Moughan, 2005 6. Erbersdobler, 2001 7. Rutherfurd, 2008 8. Rerat, 2008 9. Erbersdobler, 2001
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 33
NEWS NEWS NEWS NEWS NEWS from FIE
Meet the world’s leading ingredients suppliers in Paris at FIE
The Barry Callebaut studio: feeling and tasting the chocolate trends of tomorrow Booth 6K16 With sustainable cocoa and chocolate at the very heart of their business, Barry Callebaut, Global Chocolatier, presents deliciously stimulating chocolate experiences. Step into the Inspiration zone, where skillful and passionate Craftsmen and women inspire you with tailor-made solutions to suit today’s and tomorrow’s consumer demands. Their creations will highlight current trends including personalized, premium, wholesome & clean, clear and responsible chocolate dreams. www.solutions.barry-callebaut.com
Booth 6M5 Proven effectiveness in weight management Weight management ingredients are increasing in popularity. Recent consumer insights showed that the European consumers’ major health concern is weight management. At FiE 2015 BENEO will demonstrate how its ingredients can help manufacturers to develop tasty food and drink solutions for weight management that are validated and backed by sound science. www.beneo.com
Booth 6L73 CHS Inc. Soy & Canola Products Improving Food & Health CHS Inc. offers Soybean and Canola Oil, Defatted Soy Flour, Soy Protein Isolate, Textured Soy Flour and Analogs, and Soy Protein Isolate. We offer both GMO and Non-GMO options and have received Non-GMO Project Verification status as well as Cert ID EU Certification on many of our soy products. www.chsinc.com/foods
Booth 6D79 DKSH - Innovative food and beverage ingredients DKSH is a leading food and beverage ingredients distributor and global provider of Market Expansion Services for the beverage, dairy, confectionery and bakery, nutritional supplements, processed food and food service industries. www.dksh.com/fbi
Booth 6I5/6 DSM brings digestive health solutions to FIE 2015 At this year’s FiE, DSM brings its range of digestive solutions, highlighting nutritional and functional ingredients and enzymes backed by science to support digestive health. This allows products to be tailored to popular consumer health requirements. Visit DSM’s stand to find out its latest solutions for digestive health, sugar reduction and more. www.dsm.com/food
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 35 NEWS NEWS NEWS NEWS NEWS NEWS NEWS from FIE
Meet the world’s leading ingredients suppliers in Paris at FIE
Booth 6G110 Evonik - a premier supplier of advanced food ingredients, processing aids and functional coatings From the anti-caking agent in paprika to natural flavor extracts, amino acids, sodium reduction and functional coatings for food supplements — Evonik is widely represented in the food industry. Our unique ingredients bring value into nutritional products and in the process promote health and well-being. Potential applications range from infant formulas to sports nutrition and dietary supplements. www.evonik.com/food-ingredients
Booth 6K19 FrieslandCampina Domo introduces Vivinal GOS powder at Fi Europe Vivinal GOS Powder is a new product for infant nutrition applications. This galacto-oligosaccharide high purity powder contains high levels of galacto-oligosaccharides and low levels of mono-saccharides. This product gives more flexibility in recipe formulation, provides higher process flexibility and reduction in operational costs. www.vivinalgos.com
Booth 6K12 Team up with the expert Collagen Proteins are bridging the gap between different Food categories. GELITA, the expert in all areas of collagen protein applications can merge solutions and create synergies between www.gelita.com
Booth 6M74 Protein Fortified: Let us show you how We deliver the promise of dairy – whey protein, lactose and milk powder. From bars to beverages, our products provide flexible fortification and nutrition. Hilmar Ingredients is SQF certified and all facilities meet requirements of Global Food Safety (GFSI). We manage our business to balance economic, social and environmental sustainability. www.hilmaringredients.com
Booth 6F46 HOCHDORF – for healthy nutrition The HOCHDORF Group is a leading Swiss company in development and production of healthy ingredients manufactured from milk, whey, cereals and oil seeds. HOCHDORF are focused on customized casein and whey proteins, dairy fat specialties, base powders, wheat germ specialties (e.g. as a nut replacement) and organic vegetable oils. www.hochdorf.com
Indena at Food Ingredients Europe 2015 Booth 6F114 Indena is further expanding its presence in the food sector with a new and dedicated food division. Oplodex™, a new technology platform for food preservation, will be launched this year at Food Ingredients Europe. Based on innovative combinations of natural polyphenols endowed with strong antioxidant and chelating properties, Oplodex™ range of food ingredients aims to meet industry’s needs and consumers’ requests. www.indena.com
36 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 NEWS NEWS NEWS NEWS NEWS NEWS NEWS from FIE
Meet the world’s leading ingredients suppliers in Paris at FIE
Innophos-Ingredients for Life™ Booth 6K76 Innophos, Inc. is a global manufacturer of specialty phosphates with applications in food, beverage, and dietary supplement end markets. Innophos will be featuring solutions for sodium reduction. Innophos Nutrition offers bioactive mineral and premium botanical ingredients to the nutritional and natural products industries. www.innophos.com
Booth 6L45 Jungbunzlauer at Fi Europe 2015 Jungbunzlauer showcases innovative solutions to the global food trends sodium reduction and mineral fortification. A further highlight will be sugar reduction: The recent approval of the European Commission of erythritol for beverages opens new opportunities for Jungbunzlauer’s ERYLITE® in the field of energy reduced and “with no added sugars” flavoured drinks. www.jungbunzlauer.com
LECICO will present a range of sunflower lecithin at the FIE Europe Booth 7E18 LECICO is a highly specialized company with 30 years of experience and a 100% focus in the lecithin market. At the FIE Paris the lecithin expert focuses on the increasingly oo o important niche of sunflower lecithin – available in fluid, de-oiled or organic quality. L e c i t h i n + P h o s p h o l i p i d s Also special lecithin products from rapeseed, soy and milk will be showcased. www.lecico.de
Lesaffre Human Care to introduce their newly organic-certified Booth 6M49 nutritional yeast at FIE 2015 LHC will launch at the upcoming FIE, Lynside® NUTRI ORGANIC. In fact, the company recently obtained an organic certification from Ecocert for this traditional superfood. These nutritional yeasts are an excellent source of protein, B vitamins, fibers and minerals, making it a great nutritional partner for athletes, the elderly, expectant mothers as well as everyone who aspire to lead a healthy lifestyle. www.lesaffrehumancare.com
At FIE 2015 Nexira will focus on Fibregum™, an all-natural & Booth 6G19 GMO-free source of prebiotic soluble dietary fiber Fibregum™ offers a guaranteed minimum of 90% soluble fiber with proven prebiotic benefits, exceptional digestive tolerance and low caloric value. New results demonstrate the beneficial and innovative health benefits of Fibregum™ on the intestinal barrier. The Fibregum™ range is organic certified and verified by Non-GMO project. www.nexira.com
Booth 6Q55 Omya - Functional Minerals and beyond... With over 130 years of expertise, Omya provides a portfolio of pure natural minerals according to the most stringent regulatory and quality standards. These are required by the food, pharmaceuticals and cosmetics industries in particular. Omya‘s minerals portfolio covers a wide range of applications with established functions. www.omya.com/fpc
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 37 NEWS NEWS NEWS NEWS NEWS NEWS NEWS from FIE
Meet the world’s leading ingredients suppliers in Paris at FIE
Booth 6B36 Plantex, first time at FIE Plantex develops, manufactures and supplies natural ingredients for the Food industry. Specialist in liquid and dry botanical extracts in conventional and Organic Quality, Plantex is your tailor made solution provider for your food applications. Expert of vanilla Bourbon extracts Plantex will present for the first time at FIE 2015 its new FOOD SIGNATURE range of coffee and cocoa extracts. www.plantex.fr
Booth 6D25 Futurals, the next generation of coloring foodstuff ROHA, the leading manufacturer of colors, is showcasing at FIE Paris the Futurals brand of coloring foodstuffs with the attributes demanded by the market: natural origin and E-number free: • Futurals PAPRIKA: labeled as “Paprika Extract” • Futurals BLACK: black coloring • Futurals RED RANGE: bright red shades for coating www.roha.com
Booth 6K29 Rousselot presents Gelatin and Collagen Peptides Solutions at FIE 2015 Explore the unique and multifunctional properties of gelatin, uncover tailor-made protein solutions and a revolutionary nutrient / food supplement delivery concept with Rousselot. Stop by stand 6K29 to gain expert insights from the global leader in gelatin and collagen peptides. www.rousselot.com
Booth 6P24 Innovative concepts for appealing products According to its motto, “We bring life to products,” the Sensient team invites visitors to experience a whole new world of natural flavors and extracts, as well as a broad portfolio of sauces, inclusions, fillings and on-trend value added flavor systems. The company’s portfolio will cover versatile solutions for sweet and savory applications as well as beverages. www.sensientflavors.com
Booth 6L111 Groupe SOLACTIS solution: how to match with the hot trends Groupe SOLACTIS: a rapidly growing bioscience-based ingredients supplier, presents at FIE new breakthroughs in SOLACTIS® range: COMBO, a unique blend of milk derived oligosaccharides, new health benefits of its “pilar” GALACTOFRUCTOSE and ACTIVATOR for symbiotic compositions www.solactisgroup.com
Booth 7B2 Fat free ingredient helps to lower cholesterol Vitae Naturals is the main manufacturer of phytosterols esters and the first European producer of certified non-GMO IP naturally sourced Vitamin E. Vitasterol® S-80 WDP is mixed phytosterols microencapsulated especially developed for dairy products. This functional ingredient helps to lower blood cholesterol levels. It is a fat free ingredient. www.vitaenaturals.com
38 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 NEWS NEWS NEWS NEWS FROM SCIENCE
Collagen peptide supplementation in combination with resistance training improves body composition and increases muscle strength in elderly sarcopenic men: a randomised controlled trial
Protein supplementation in combination with resistance strength (IQS) of the right leg was determined and sensory training may increase muscle mass and muscle strength in motor control (SMC) was investigated by a standardised elderly subjects. The objective of this study was to assess one-leg stabilisation test. Following the training programme, the influence of post-exercise protein supplementation with all the subjects showed significantly higher (P<0·01) levels collagen peptides v. placebo on muscle mass and muscle for FFM, BM, IQS and SMC with significantly lower (P<0·01) function following resistance training in elderly subjects levels for FM. The effect was significantly more pronounced with sarcopenia. A total of fifty-three male subjects (72·2 in subjects receiving collagen peptides: FFM (TG +4·2 (sd (sd 4·68) years) with sarcopenia (class I or II) completed 2·31) kg/PG +2·9 (sd 1·84) kg; P<0·05); IQS (TG +16·5 (sd 12·9) this randomised double-blind placebo-controlled study. All Nm/PG +7·3 (sd 13·2) Nm; P<0·05); and FM (TG –5·4 (sd 3·17) the participants underwent a 12-week guided resistance kg/PG –3·5 (sd 2·16) kg; P<0·05). Our data demonstrate that training programme (three sessions per week) and were compared with placebo, collagen peptide supplementation supplemented with either collagen peptides (treatment in combination with resistance training further improved group (TG)) (15 g/d) or silica as placebo (placebo group body composition by increasing FFM, muscle strength and (PG)). Fat-free mass (FFM), fat mass (FM) and bone mass the loss in FM. (BM) were measured before and after the intervention using Daniel Königa et al., British Journal of Nutrition, DOI: dual-energy X-ray absorptiometry. Isokinetic quadriceps http://dx.doi.org/10.1017/S0007114515002810
Nano in food and agriculture: regulations require collaboration to ensure safety
An overview of regulatory solutions worldwide on the use of through microbial nanoparticles. nanotechnology in food and feed production shows a differing As with any other regulated product, applicants applying for approach: only the EU and Switzerland have nano-specific market approval have to demonstrate the safe use of such provisions incorporated in existing legislation, whereas other new products without posing undue safety risks to the consumer countries count on non-legally binding guidance and standards and the environment. Some countries have been more active for industry. Collaboration among countries across the globe is than others in examining the appropriateness of their regulatory required to share information and ensure protection for people frameworks for dealing with the safety of nanotechnologies. As and the environment, according to a JRC co-authored paper. a consequence, different approaches have been adopted in The paper Regulatory aspects of nanotechnology in the agri/ regulating nano-based products in the agri/feed/food sector. feed/food sector in EU and non-EU countries reviews how The analysis shows that the EU along with Switzerland potential risks or the safety of nanotechnology are managed are the only ones which have introduced binding in different countries around the world and recognises that this nanomaterial definitions and/or specific provisions for some may have implication on the international market of nano- nanotechnology applications. An example would be the enabled agricultural and food products. EU labelling requirements for food ingredients in the form Nanotechnology offers substantial prospects for the of 'engineered nanomaterials'. Other regions in the world development of innovative products and applications in many regulate nanomaterials more implicitly mainly by building on industrial sectors, including agricultural production, animal feed non-legally binding guidance and standards for industry. and treatment, food processing and food contact materials. The overview of existing legislation and guidances published While some applications are already marketed, many other as an open access article in the Journal Regulatory nano-enabled products are currently under research and Toxicology and Pharmacology is based on information development, and may enter the market in the near future. gathered by the JRC, RIKILT-Wageningen and the European Expected benefits of such products include increased efficacy Food Safety Agency (EFSA) through literature research and a of agrochemicals through nano-encapsulation, enhanced dedicated survey. bioavailability of nutrients or more secure packaging material European Commission Joint Research Centre
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 39 NEWS FROM SCIENCE NEWS NEWS NEWS
Scientists have developed rice with high folate stability
Researchers from Ghent University succeeded in stabilizing cell by specific enzymes (molecular machines) that add folates in biofortified rice, which can offer a solution to consecutive changes to a certain start product until a folate serious health problems caused by folate deficiency in molecule is formed. This process is comparable to a car developing countries. assembly line. By stimulating the production of two enzymes in the folate biosynthesis, researchers created the first Essential nutrient generation of rice lines containing high folate levels. The human body is unable to make vitamin B9, better known as folate. Adults need approximately 400 microgram Two strategies of folates per day to remain healthy, a number which is Now, they were able to stabilize this high folate content increased to 600 microgram for pregnant women. Folates in a new rice prototype. They applied two strategies. A are abundant in green leafy vegetables (folium is Latin for first strategy comprised the binding of folates with a folate leaf), such as spinach and legumes (e.g. beans). Most staple binding protein. This protein is unknown in plants, but well crops, such as rice and other cereals, contain very low studied in mammals. It occurs in e.g. milk and protects amounts of this vitamin. folate from degradation. This is also the way intact folates are passed on from the mother to her infant, to support its Inadequate folate intake can have severe effects on development. By expressing a synthetic gene, based on a human health. In addition to certain forms of anemia, folate binding protein from bovine milk, in the rice grain, the folate deficiency in pregnant women can result in an same principle is applied and folate content remains stable impaired development of the neural tube (the precursor upon long term storage. of the spinal cord) of the embryo. These developmental A second strategy consisted of the stimulation of the last problems often result in spina bifida: the so-called “cleft step in folate production. This step extends the tail of the spine”. Folate deficiency is also associated with Alzheimer folate molecule. This promotes cellular retention and binding disease, cardio-vascular diseases and the development to folate dependent proteins. Besides enhancing folate of a range of cancers. Due to the marginal levels of folate stability, the new gene combinations also resulted in folate in rice, consumed by about half the world population as levels that are up to 150 fold those found in normal rice. sole energy source, folate deficiency is highly prevalent in Since all genes which were used in this study, were placed developing countries. Several studies show that in certain next to each other on a single piece of DNA, this piece of regions of e.g. China and India the occurrence of neural genetic material can easily be transferred to edible rice tube defects is at least 10-fold higher than in Western varieties. Moreover, it is fairly easy to make combinations with countries. other interesting traits, such as the enhancement of other vitamins or certain minerals, such as iron. This technology Unstable molecules can also be used in other crops, both cereals (e.g. wheat, Vitamins are unstable molecules, that degrade easily upon sorghum) and non-cereals (e.g. potato, banana). contact with oxygen, light, humidity, increased temperatures and changes in acidity. For this reason, it is important to Collaboration consume food products, such as vegetables and fruit, as This investigation is the result of a close collaboration fresh as possible. A lot of vitamins get lost, not only during between the labs of prof. Dominique Van Der Straeten food processing and preparation, but also during storage. (development and characterization of the new rice Evidently, these problems occur in harvest products that are prototypes), prof. Willy Lambert and prof. Christophe Stove stored for a longer period, such as rice grains. These stability (development of analytical methods to measure folates) problems become more severe in developing countries and dr. Hans De Steur and prof. Xavier Gellynck (study of where the storage in high temperature and high humidity is the socio-economic impact of folate rice). These results inevitable. have been published in the renowned journal Nature In 2007, a research team from Ghent University (Belgium), Biotechnology (Blancquaert et al., 2015). coordinated by prof. Dominique Van Der Straeten, reported the development of a first generation of rice lines with Pioneers 100-fold higher folate levels as compared to normal rice. The stability issue is often underestimated or even neglected This result was achieved through metabolic engineering, in biofortification programs. It is obvious that not only high, the modulation of the biosynthesis pathway of a plant but also stable vitamin levels are important to tackle vitamin compound. Their new study shows that about half of the deficiencies. Not only does this study describe the effect folate content in these rice lines degrades after half a year. of long term storage on the folate levels in the first rice prototypes, it also provides an elegant solution to the stability Assemblage lines problem. This solution can be applied, in a customized To tackle this problem, researchers from this lab developed form, to other crops and vitamins and opens the door for a new rice prototype, in which the folate content awareness and consideration of vitamin stability in future remains stable upon long term storage. Again, metabolic biofortification studies. engineering was applied. Folate is produced in a plant Ghent University
40 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 NEWS NEWS NEWS NEWS FROM SCIENCE
Cocoa flavanols lower blood pressure and increase blood vessel function in healthy people
New studies by the EU-funded FLAVIOLA research of the study (by 33% in the younger age group and 32% in consortium show that cocoa flavanols could help maintain the older age group over the control intervention). In the cardiovascular health as we age older age group, a statistically and clinically significant Two recently published studies in the journals Age and the decrease in systolic blood pressure of 4 mmHg over control British Journal of Nutrition (BJN) demonstrate that consuming was also seen. cocoa flavanols improves cardiovascular function and lessens the burden on the heart that comes with the ageing Improving cardiovascular health and lowering the risk of and stiffening of arteries. The studies also provide novel data CVD to indicate that intake of cocoa flavanols reduces the risk of In the second study, published in BJN, the researchers developing cardiovascular disease (CVD). extended their investigations to a larger group (100) of As we age, our blood vessels become less flexible and less healthy middle-aged men and women (35-60 years) with able to expand to let blood flow and circulate normally, low risk of CVD. The participants were randomly and blindly and the risk of hypertension also increases. Arterial stiffness assigned into groups that consumed either a flavanol- and blood vessel dysfunction are linked with cardiovascular containing drink or a flavanol-free control drink, twice a day disease -- the number one cause of deaths worldwide. for four weeks. The researchers also measured cholesterol "With the world population getting older, the incidence of levels in the study groups, in addition to vasodilation, arterial cardiovascular disease, heart attacks and stroke will only stiffness and blood pressure. increase," says Professor Malte Kelm, Professor of Cardiology, "We found that intake of flavanols significantly improves Pulmonary Diseases and Vascular Medicine at University several of the hallmarks of cardiovascular health," says Hospital Düsseldorf and Scientific Director of FLAVIOLA. "It Professor Kelm. In particular, the researchers found that is therefore pivotal that we understand the positive impact consuming flavanols for four weeks significantly increased diet can have on cardiovascular disease risk. As part of flow-mediated vasodilation by 21%. Increased flow- this, we want to know what role flavanol-containing foods mediated vasodilation is a sign of improved endothelial could play in maintaining the health of the heart and function and has been shown by some studies to be blood vessels." associated with decreased risk of developing CVD. In Cocoa flavanols are plant-derived bioactives from the addition, taking flavanols decreased blood pressure (systolic cacao bean. Dietary intake of flavanols has been shown by 4.4 mmHg, diastolic by 3.9 mmHg), and improved the to have a beneficial effect on cardiovascular health but blood cholesterol profile by decreasing total cholesterol (by the compounds are often destroyed during normal food 0.2 mmol/L), decreasing LDL cholesterol (by 0.17 mmol/L), processing. Earlier studies have demonstrated that cocoa and increasing HDL cholesterol (by 0.1 mmol/L). flavanol intake improves the elasticity of blood vessels The researchers also calculated the Framingham Risk Score - and lowers blood pressure -- but, for the most part, these a widely used model to estimate the 10-year cardiovascular investigations have focused on high-risk individuals like risk of an individual - and found that flavanol intake reduced smokers and people that have already been diagnosed with the risk of CVD. "Our results indicate that dietary flavanol conditions like hypertension and coronary heart disease. intake reduces the 10-year risk of being diagnosed with CVD These two studies in Age and BJN are the first to look at the by 22% and the 10-year risk of suffering a heart attack by different effects dietary cocoa flavanols can have on the 31%," says Professor Kelm. blood vessels of healthy, low-risk individuals with no signs or The combined results of these studies demonstrate that symptoms of cardiovascular disease. flavanols are effective at mitigating age-related changes in blood vessels, and could thereby reduce the risk of Cocoa flavanols increase blood vessel flexibility and lower CVD in healthy individuals. The application of 10-year blood pressure Framingham Risk Scores should be interpreted with caution In the study published in Age, two groups of 22 young (<35 as the duration of the BJN study was weeks not years and years of age) and 20 older (50-80 years of age) healthy men the number of participants was around 100, not reaching consumed either a flavanol-containing drink, or a flavanol- the scale of the Framingham studies. That being said, free control drink, twice a day for two weeks. The researchers Professor Kelm comments that "the reduction seen in risk then measured the effect of flavanols on hallmarks of scores suggests that flavanols may have primary preventive cardiovascular aging, such as arterial stiffness (as measured potential for CVD." Other longer-term studies, such as the by pulse wave velocity), blood pressure and flow-mediated 5-year COcoa Supplement and Multivitamin Outcomes vasodilation (the extent to which blood vessels dilate in Study (COSMOS) of 18,000 men and women, are now response to nitric oxide). underway to investigate the health potential of flavanols on They found that vasodilation was significantly improved in a much larger scale. both age groups that consumed flavanols over the course University Hospital Düsseldorf
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 41 NUTRACEUTICALS
ELENA VEZZOLI1,2,3, VERONICA DI PIETRO4, ROSSELLA CALÒ3, DAVIDE MARCHESI1, ELENA DE VECCHI5, DOMENICO BIJNO4, LAURA MARABINI3, SIMONA RODIGHIERO1*, MAURA FRANCOLINI1,2* *Corresponding authors 1. Fondazione Filarete for Biosciences and Innovation, Viale Ortles 22/4, 20139 Milano, Italy 2. Università degli Studi di Milano – Dept. of Medical Biotechnology and Translational Medicine, Via Vanvitelli 32, 20129 Milano, Italy 3. Università degli Studi di Milano – Dept. of Pharmacological and Biomolecular Sciences, Via Vanvitelli 32, 20129 Milano, Italy 4. KolinPharma SpA, Viale Ortles 22/4, 20139 Milano, Italy 5. IRCCS Galeazzi Orthopaedic Institute - Laboratory of Clinical Chemistry and Microbiology, Via Galeazzi 4, 20161 Milano, Italy
Evaluation of nutraceuticals effect from in vitro assays
KEYWORDS: in vitro models, human cell lines, nutraceuticals.
The healthy effects of numerous bioactive natural compounds and the possibility to take advantage of Abstract their beneficial physiological functions are highly valued. Although the efficacy of a number of nutraceutical products has already been demonstrated in humans, the properties of many compounds are still unknown and, importantly, there is little information on the properties and potential of their combined use. For these reasons in vitro assays are necessary to identify the mechanisms underlying the protective role of a nutraceutical ingredient alone or in combination with other bioactive molecules. Here we describe examples of in vitro models and methodological approaches used to characterize and evaluate a number of effects of nutraceuticals in living cells.
INTRODUCTION that measures cell viability and cell proliferation rate (5). In our experiment, we performed a MTT test on human Studies aimed to identify the potential beneficial use of monocytes (THP-1 cells) treated with a nutraceutical (6) nutraceuticals have been growing in the last few years. at different concentrations (10-50-75-100-500 µM) for 16 Nutraceuticals are of considerable interest due to their hours. In Figure 1A, the results are shown. A strong reduction potential nutritional effects as dietary supplements (1-2) and in viability is seen in cells treated with the higher dose (500 their production should be accompanied by appropriate µM) of the compound, as indicated by the lower value quality assessment measures (3). Studies are thus needed to of the optical density (OD). In cells treated with the lower asses both their quality and their biological effects when used concentrations (from 10 to 100 µM) the OD is comparable to alone or in combination. For in vitro screening, a crucial step control and vehicle. is the choice of the appropriate model that best represents the cellular target of the relevant compound. Human cell lines, with organ-specific properties represent such important ANTI-INFLAMMATORY ACTIVITY experimental systems. Macrophages for acute/chronic inflammation and pain; chondrocyte for articular pain, Inflammatory response are characterized by the release uroepithelial cells for urinary tract infection, and many others. of pro-inflammatory cytokines and other mediators, as free In vitro models and assays are used to define concentration/ radicals, and prostaglandin E2 (PGE2), which are synthesized doses of compounds and to evaluate the cellular pathways by inducible nitric oxide synthase (iNOS) and cyclo-oxygenase involved in their action. (COX-2). Anti-inflammatory agents can act by modulating the production of cytokines and all the other mediators (7). The anti-inflammatory properties of several natural compounds CYTOTOXICAL SCREENING has long been known and, some of them as curcumin, flavonoids, saponins are generally recommended in the The first step to establish a cellular assay is the evaluation diet of patients suffering from inflammatory diseases such of the potential cytotoxic activity of compounds on the as fevers, pain and arthritis (8-9). Inflammatory conditions selected cell line. Different human cell lines were screened (i.e. arthritis and chronic pain) can be mimicked in in vitro for the potential cytotoxic effect of nutraceuticals through models by stimulating cells with bacterial lipopolysaccharide the measure of cell viability and proliferation. These tests are (LPS) or IL-1β-induced inflammation. Upon exposure to LPS or used to define the concentrations of the compound that do IL-1β, macrophages and chondrocytes, release a number not affect cell viability. These assays focus on the evaluation of immunoregulatory molecules including tumor-necrosis- of one of the following features: membrane integrity, cellular factor-α (TNF-α), IL-6, arachidonic acid metabolites and metabolite content, mitochondrial and lysosomal functions Nuclear-Factor-Kappa-B (NF-κB) is translocated into the and cell apoptosis (4). Here we described the MTT assay nucleus where it regulates the expression of inflammatory
42 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 genes. ELISA test and NF-κB Translocation-Assay are used we used, NHAC-kn cells with and without stimulation to measure these inflammatory responses in cultured cells. treated with vehicle. Cells were then fixed and labeled We next describe how we applied these two assays to with NF-κB-p65 antibodies and secondary antibodies study the anti-inflammatory properties of nutraceuticals in conjugated with AlexaFluor488, DAPI was used as a monocyte-like cells (THP-1) and Normal-Human-Articular- nuclear stain (Figure 2A). NF-κB translocation is calculated Chondrocytes (NHAC-kn) upon stimulation with LPS and IL-1β. by measuring the “ratio” of the average nuclear intensity to average cytoplasmic intensity (Nuc/Cyt Ratio). We demonstrated that cells treated with 500 μM Compound-B ELISA test have less nuclear NF-κB (reduction of inflammation) than The Enzyme-Linked-Immunosorbent-Assay (ELISA) has cells treated with vehicle and with an anti-inflammatory been designed for the in vitro quantification of a range drugs (Figure 2B). of cytokines. In the experiment illustrated in figure 1B, THP-1 cells, treated with the bioactive substance (Compound-A) for 16 hours, in basal condition or after ANTI-OXIDANT ACTIVITY stimulation with 1 mg/ml LPS for 2 hours, were centrifuged and the cell-free supernatants were collected. Sandwich Reactive oxygen species (ROS) include free radicals ELISA was performed on the cell supernatant to and other molecular species. They are generated during determine IL-1β concentration, in accordance with the incomplete metabolic reduction of oxygen to water. In manufacturer’s instructions (Life-Technologies). ELISA physiological conditions, Intracellular ROS are beneficial, as test in cells stimulated or not with LPS, were performed they protect against invading pathogens, and ROS levels to compare the effect of the compound with the basal are precisely controlled by various enzymatic activities. concentration of the cytokine. In figure 1B the results Under pathological conditions, however, intracellular ROS obtained in cells without LPS stimulation are shown (left); levels increase due to their increased production or impaired these cells present a low basal level of IL-1β (around elimination (oxidative stress). Oxidative stress causes cell 5-10 pg/ml). LPS stimulation induced an increase of the damage and eventually death. ROS play an important role concentrations of the IL-1β in all experimental conditions in the progression of several diseases including inflammation, (right). This increase is reduced upon treatment with the atherosclerosis, aging, and age-related degenerative bioactive Compound-A and, the effect of the nutraceutical on IL-1β production is more pronounced than the effect anti-inflammatory drugs (Nimesulide and Ibuprofen).
NF-kB translocation assay NF-κB is generally thought to be constitutively active and located in the cytoplasm in most cell types, until induced by a stimulus to migrate to the nucleus. Translocation of NF-κB is a critical step in the coupling of extracellular stimuli to the transcriptional activation of specific target genes. NF-κB is activated by pro- Figure 1. (A) MTT proliferation assay on THP1 treated with vehicle or increasing doses of Compound-A (10–50–75–100-500 μM) for 16 hours. Viability is expressed as OD value ± SEM. inflammatory cytokines (IL-1α, IL-1β, TNF-α); (B) ELISA of IL-1β in the supernatant of THP1 cultures unstimulated (left) or stimulated (right) bacterial toxins (LPS, exotoxin B); viral with LPS for 120 minutes. Experiments were done in quadruplicates. products (HIV-1, HTLV-1, EBV); and cell death stimuli (O2-free radicals, UV light, γ-radiation) (10-11). Upon cell stimulation, the nuclear localization signal on NF- κB becomes exposed and the protein translocate to the nucleus, where it turns on transcription factors and induces specific gene expression. In cells, the redistribution of NF-κB from the cytoplasm to the nucleus following cytokine stimulation can be easily monitored with fluorescence microscopy. In our experiment we used Normal- Human-Articular-Chondrocytes (NHAC- kn) stimulated with 10 ng/ml IL-1β for 16 Figure 2. (A) NHAC-kn cells unstimulated or stimulated with IL-1β. Unstimulated cells ((-IL-1β) hours and treated with increasing doses show a stained cytoplasm containing NF-κB-p65 with a dark nucleus. Following activation of of Compound-B (12) (125-250-500 μM) NF-κB (+IL-1β)), the nucleus is stained while the cytoplasm is no longer fluorescent indicating for 16 hours and anti-inflammatory drugs that the protein translocated from the cytoplasm to the nucleus. (B) NF-κB translocation expressed as Nuc/Cyt ratio in cells treated with compound-B at different concentrations (Celecoxib and Diclofenac) as positive (125–250–500 μM), Celecoxib and Diclofenac, and vehicles. (*p < 0.05, ***p < 0.001). controls for 16 hours. As negative controls
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 43 disorders (13). We used the fluorescent probe CellROX® Deep Red Reagent (ex. 640nm, em. 665nm) to quantify oxidative stress in cells as, upon interaction with oxygen species, the probe becomes brightly fluorescent. These cell-permeant dyes are weakly fluorescent while in a reduced state and exhibits photostable fluorescence upon oxidation by reactive oxygen species (ROS). We tested the antioxidant activity of three concentrations compound-A (50-75-100 μM) on THP-1 cells comparing its effect with two antioxidant molecules: Vitamin E and B6 (14), after 2 hours stimulation of cells with H2O2. Compound-A is able to decrease the levels of ROS in THP-1 cells after stimulation with H2O2, as shown in Figure 3A, D, and quantified in the histogram (Figure 3E).
ANTI-BACTERICAL ADHESIVE ACTIVITY
The antimicrobial activity of natural compounds is considered as one of their most valuable beneficial effects for the prevention and the treatment of human diseases (15-16). We investigated the antimicrobial activity of nutraceutical (Compound-C) (17) gains urinary pathogens in an in vitro model mimicking the first step in setting up of infection that is adhesion to Figure 3. Representative images of THP-1 cells, in fluorescence (A-C) and transmission host cells. After determination of the Minimal microscopy (B-D), treated with H2O2 for 2 hours. The fluorescence of CellROX indicates Inhibitory Concentration (MIC) (the lowest the presence and amount of ROS in untreated (A) and treated cells (C). (E) Mean fluorescence of CellROX in cells treated with compound-A at different concentrations concentration of the compound able to (50–75–100 μM), vitamins (E and B6), and vehicles. (*p < 0.05). (F) Quantification of the inhibit the growth of selected bacterial strains) number of adherent bacteria on uroepithelial cells after 12 hours of incubation with the by micro-dilution broth method according compound-C at two concentration ½MIC and ¼MIC. (G-I) Representative scanning electron microscopy images of the samples. to international guidelines. The in vitro ability of sub-inhibitory concentrations of the compounds under testing to inhibit adhesion of bacteria (10 Escherichia coli strains) to uroepithelial cells from healthy donors was evaluated. We did this by incubating bacteria previously grown with compound at concentrations: ½MIC and ¼MIC with uroepithelial cells, and counting adhered bacteria per cells at optical (Figure 3F) and electron microscope level (Figures 3G-I). As shown in figure the number of adhered bacteria, are reduced after strains were treated with sub-inhibitory concentrations of the tested compound. Figure 4. (A-E) Examples of DNA comet test, in (A) an undamaged cell with intact nucleoid; in (B-E) comets from damaged cells. (F) Antigenotoxic effect of Thymus vulgaris L. extract and Thymol in NCTC 2544 UVB-irradiated. Left panel shows clearly that 1h cell pretreatment with both substances increase with different potency, the amount of ‘A’ DNA DAMAGE – COMET TEST nucleoids in all UVB doses used, compared to the unpretreated cells. Right panel shows that the increase of undamaged cells is parallel to the decrease of B, C, D and E (ghost) nucleoids in all UVB doses, with different potency. Natural agents, besides their roles as antioxidant, anti-inflammatory, anti- mutagenic, anti-carcinogenic and green tea (19-20), the pretreatment of human reconstructed immunomodulators. Particularly, they have been also skin with pomegranate-derived products (21), or the exposure investigated for their activity in the prevention of UV-induced of keratinocytes cells to delphinidin and anthocyanidin, skin damage at the cellular and molecular levels (18). Their resulted in a marked reduction of UVB-induced DNA damages role in protection in different in vivo and in vitro models has (22). We analyzed the effect of two phenolic compounds been documented. It has been demonstrated that the oral Thymus vulgaris leaf extract and its major component feeding of hairless mice with pomegranate fruit extract or Thymol against UVB- induced DNA damage in the human-
44 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 keratinocytes-cell line NCTC 2544, using the ‘comet assay’ 5. Mosmann, T., “Rapid colorimetric assay for cellular growth and (Figure 4F). The substances tested are commonly used for survival: application to proliferation and cytotoxicity assays”, their expectorant, antiseptic, antispasmodic, antimicrobial, J. Immunol. Methods., 65 (1-2), 55-63 (1983). antifungal, antioxidative effects, but their genotoxic effect 6. Compound A: Xinepa 7. Leung, T. K., Liu, Y. C., Chen, H., et al “In Vitro Cell Study of Possible is still unclear. The comet test is a sensitive technique for the Anti-inflammatory and Pain Relief Mechanism of Far-infrared detection of DNA damage at the level of the single cells. Cells Ray-emitting Ceramic Material”, J. Med. Biol. Eng., 33 (2), 179-184 embedded in agarose on a microscope slide are lysed with (2012). solution containing detergent and high salt to form nucleoids 8. Yuan, G., Wahlqvist, M. L., He, G., Yang, M. and Li, D., “Natural of supercoiled loops of DNA. The DNA is allowed to unwind products and anti-inflammatory activity”,Asia Pac. J. Clin. Nutr., 15 under alkaline conditions in electrophoresis buffer. Following (2), 143-152 (2006). unwinding, the DNA undergoes electrophoresis at high pH 9. Pan, M. H., Lai, C. S., Ho, C. T., “Anti-inflammatory activity of natural (pH>13), allowing fragmented DNA to migrate away from the dietary flavonoids”, Food Funct., 1 (1), 15-31 (2010) nucleus. Once the slides are dried, they are stained with a 10. Trask, O. J., “Nuclear Factor Kappa B (NF-κB) Translocation Assay DNA-specific fluorescent dye such as propidium iodide and Development and Validation for High Content Screening”, Assay Guidance Manual Bethesda (MD): Eli Lilly & Company and the observed by fluorescence microscopy. Undamaged cells National Center for Advancing Translational Sciences, 2004- 2012 appears as intact nucleoids (Figure 4A), damaged cells as Oct 01. comets (Figure 4B-E). The extent of DNA migrated from the 11. Noursadeghi, M., Tsang, J., Haustein, T., Miller, R. F., Chain, B. M., head of the comet is directly proportional to the amount Katz, D. R., “Quantitative imaging assay for NF-kappaB nuclear of DNA damage. Brighter and longer is the tail, higher is translocation in primary human macrophages”, J. Immunol. the level of DNA damage. Results obtained in the present Methods, 329 (1-2), 194-200 (2008). study should be considered as predictive of future studies, 12. Compound B: Dolasix useful to identify new plant-derivate active molecules, with 13. Oyinloye, B. E., Adenowo, A. F., Kappo, A. P., “Reactive antioxidant properties and UV protective effects that could oxygen species, apoptosis, antimicrobial peptides and human be introduced in the organism by diet as food supplements or inflammatory diseases”, Pharmaceuticals (Basel), 8 (2), 151-75 (2015). used as additional components in sunscreens. 14. Kim, H. H., Kim, J. H., Gao, X., et al. “Analgesic effect of vitamin E is mediated by reducing central sensitization in neuropathic pain”, Pain, 122, 53-62 (2006). CONCLUSION 15. Moghadamtousi, S. Z., Kadir, H. A., Hassandarvish, P., et al. “A Review on Antibacterial, Antiviral, and Antifungal Activity of In this paper, we described some examples of specificin Curcumin”, Biomed. Res. Int., Epub 2014 Apr 29. vitro assays that can be used to test the biological effects of 16. Drago, L., De Vecchi, E., Nicola, L., et al. “In vitro antimicrobial nutraceutical compounds. These assays represent only a fraction activity of a novel propolis formulation (Actichelated propolis)”, J. of all possible in vitro studies to evaluate the different properties Appl. Microbiol., 103 (5), 1914-21 (2007). of nutraceuticals; indeed, as nutraceutical development and 17. Compound C: Ivuxur 18. Afaq, F., “Natural Agents: Cellular and Molecular Mechanisms of related techniques of investigation are rapidly expanding new in Photoprotection”, Arch. Biochem. Biophys., 508 (2), 144-51 (2001). vitro analytical tools are constantly required. 19. Afaq, F., Khan, N., Syed, D. N., et al.“Oral feeding of pomegranate fruit extract inhibits early biomarkers of UVB radiation-induced carcinogenesis in SKH-1 hairless mouse epidermis” Photochem. REFERENCES AND NOTES Photobiol., 86 (6), 1318-26 (2010). 20. Lu, Y. P., Lou, Y. R., Li, X. H., et al. “Stimulatory effect of oral 1. Nasri, H., Baradaran, A., Shirzad, H., et al. “New concepts in administration of green tea or caffeine on ultraviolet light-induced nutraceuticals as alternative for pharmaceuticals”, Int. J. Prev. increases in epidermal wild-type p53, p21(WAF1/CIP1), and apoptotic Med., 5 (12), 1487-1499 (2014). sunburn cells in SKH-1 mice”, Cancer Res., 60 (17), 4785-91 (2000). 2. Das, L., Bhaumik, E., Raychaudhuri, U., et al. “Role of nutraceuticals 21. Afaq, F., Zaid, M. A., Khan, N., et al. “Protective effect of in human health”, J. Food Sci. Technol., 49 (2), 173-83 (2012). pomegranate-derived products on UVB-mediated damage in 3. Pferschy-Wenzig, E. M., Bauer, R., “The relevance of human reconstituted skin” Exp. Dermatol., 18 (6), 553-61 (2009). pharmacognosy in pharmacological research on herbal medicinal 22. Afaq, F., Syed, D. N., Malik, A.,et al. “Delphinidin, an anthocyanidin products”, Epilepsy Behav, Epub 2015 Jul 10. in pigmented fruits and vegetables, protects human HaCaT 4. Albert, P. L., “Preclinical in vitro screening assays for drug-like keratinocytes and mouse skin against UVB-mediated oxidative properties”, Drug Discov. Today Technol., 2 (2), 179-85 (2005). stress and apoptosis”, J. Invest. Dermatol., 127 (1), 222-32 (2007).
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 45 SENIOR NUTRITION
CHRIS STOTT, CHRIS STIRLING KPMG LLP, 15 Canada Square, Canary Wharf, London, United Kingdom
Chris Stott Chris Stirling Ageing population trends and the flourishing nutraceuticals market
KEYWORDS: Health, nutrition, ageing, nutraceuticals, food, beverage, pharmaceuticals.
KPMG has recently written two reports which discuss the ageing consumer and the Nutraceuticals Abstract market. Partners from KPMG discuss how Nutraceuticals companies stand to make huge profits from their older consumers if they position themselves correctly in this fast evolving and high growth market. Nutraceuticals is a market sitting between Food & Beverage and Pharmaceuticals and thus poses interesting challenges for this demographic.
INTRODUCTION The Nestlé R&D team is already testing their products to identify barriers like these, including difficult packaging, which As much of the world faces rising rates of chronic lifestyle are preventing older consumers from using their products. The illness, in the UK alone, the potential cost of this to the NHS is company has invested in a machine hand which mimics the over £10bn annually. The incentive for governments to focus effects of arthritis and, while it is not marketing this directly to on preventative care and for consumers to become more consumers, the research is being used to make alterations to health-conscious is immense. Couple this with the fact the packaging which will benefit all shoppers. ageing population (65 and over) is set to double globally by 2035 to 1.1bn and then again by 2050 to 2bn, there is a fast growing market of functional foods, nutraceuticals and dietary NUTRACEUTICALS supplements aimed towards this specific group of consumers. In two recent reports, KPMG explores these trends and how When looking beyond the packaging to what’s inside food and pharmaceutical companies are perfectly placed to the wrapper, there is growing demand from consumers capitalise. for nutraceuticals products. The term nutraceuticals was coined in the 1980s to describe food products that have a medicinal benefit, and the sector has exploded in recent AGEING POPULATION TRENDS years to include functional foods such as vitamin-enriched The most recent report from KPMG (1), which focuses on products, nutritional supplements, sports drinks and medically the UK market, predicted that by 2020 the 65-and-over age formulated foods (3). group will outnumber under-fives for the first time. Added KPMG’s report; Nutraceuticals: The future of intelligent food (4) to this, in the last decade, spending among over 50s has highlights that future superfoods could potentially tackle the grown on average 4.4% a year – faster than any other age underlying causes of conditions such as diabetes, obesity and group – and they also earn more than any other segment cardiovascular disease, by linking diet to the human genome. of the population (2). This therefore means, a greater It is therefore unsurprising that the global nutraceuticals concentration of both money and consumers at the older market is predicted to be worth $250 billion by 2018 as a result end of the spectrum. of increased uptake by health savvy consumers. Brands looking to capitalise on these trends need to also bear Food companies have so far taken the lead in the in mind that the older generations place far more value on nutraceuticals space, thanks to a greater understanding quality and experience meaning they are far less discount- of nutrition, food formulation and decades of consumer driven than younger shoppers. This group is also relatively research to support growth in this area. However, food brand loyal. companies lack the scientific resources to achieve game- Nestlé is a good example of a company adapting to the changing breakthroughs with innovation tending to involve ‘silver’ market. Of the 1,261 consumers surveyed by KPMG modest improvements, such as reducing sugar content. and YouGov, over 70% of those aged 70+ were likely to have trouble opening packaging and have difficulty reading On the other side of the coin, pharmaceutical companies are labels. That said, nearly 50% of those aged 20 to 60 agreed. also making attempts to gain ground in the nutraceuticals
46 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 space. For example, there have been a number of deals in the pharmaceutical and consumer healthcare sectors in recent years. In 2010 Nestlé claimed to have pioneered a new industry between food and pharma with the creation WHEY PROTEIN of Nestlé Health Science and Nestlé Institute of Health Fortified Sciences. More recently, in March 2015, Novartis and GSK for an agility advantage agreed to create a consumer healthcare business through a joint venture between Novartis OTC and GSK Consumer Healthcare. While these moves have not yet led to a nutraceuticals leader being established, the direction of travel is pretty clear.
CHARACTERISTICS OF SUCCESS: DARING TO WIN
Currently, it seems neither pharmaceuticals nor food companies have all the ingredients to take the lead in the nutraceuticals market. Yet with the sector set for huge growth over the coming years it seems inevitable that, at some point, a nutraceuticals powerhouse will emerge - but what will it take to build one?
Technology Technology is going to be crucial. The nutraceuticals market is likely to benefit from the trend of personalised medicine, and developing technology which aids this movement is key, including taking advantage of the huge amount of data that will be created by an increasingly connected healthcare system. Consumer companies have the intelligence available to personalise dietary Maintain muscle to stay active advice, and with prudent partnerships, pharmaceutical companies may be able to personalise over-the-counter Older adults, as they age, benefit from more nutraceuticals products to supplement consumer lifestyles. protein in their diets to help counter the natural loss of muscle. Beverages fortified with Compliant marketing high-quality whey protein provide an easy way The market for nutraceuticals is not globally uniform: countries vary in the way their regulatory systems recognise to consume some of the protein required to nutraceuticals – as pharmaceuticals versus food, while meet the recommended dietary allowance. consumers themselves cannot agree which category these products fit into best. Successful companies will have to Whey protein delivers: develop a highly nuanced global marketing campaign that can exploit economies of scale while adapting to a myriad of • Building blocks for localised preferences, regulations and buying behaviours. lean muscle • Complete protein with Regulation high biological value Regulations around nutraceuticals vary from one country to another and are changing all the time. In the EU, products • Hunger satisfaction that claim to be a nutraceutical have to be certified by the • Easy digestibility European Food Standards Authority, and a similar system operates in Canada. However, this contrasts with the US and Japan, two markets that currently account for over half the Visit us at world’s demand for nutraceuticals, where products do not have to pass stringent government tests as long as they do not claim to treat or prevent a specific disease. Manufacturers FiE will have to work closely with governments to anticipate market opportunities that can arise when rules are altered. Booth 6M74 Supply chain Learn more: With increasing consumer concerns about food safety, hilmaringredients.com the quality of raw materials used to create nutraceutical products have come under intense scrutiny, so companies or give us a call: +001-209-667-6076 must consider carefully how to access and assure supplies of essential ingredients or flavorings. A number of recent © 2015 HILMAR INGREDIENTS acquisitions of food and beverage companies are aimed
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 47 at securing stable, high quality supplies of raw materials. For example, many in the older generation who are Vertical integration of supply chains, for example, gives house-bound are commonly found to be deficient in greater certainty of supply, and lays a foundation for vitamin D due to the lack of sunlight exposure. In fact, developing new uses of existing materials, or to produce a 2014 study found that vitamin D deficiency was highly new, related ingredients. prevalent and associated with increased mortality among the elderly in Swedish nursing homes. (5) Product strategy Blockbusters in this industry may be few and far between. It is clear that nutritional dietary supplementation is Recently some companies have been re-balancing their extremely important for ageing consumers and makes product portfolios and actually abandoning product them a key target market for nutraceuticals companies. lines that do not fit their strategy. GSK are an example Added to this, the silver generation no longer want to be of this, having sold Ribena and Lucozade in 2013 to seen as “senior” and “ageing” and prefer to be seen as Suntory. Competitive advantage will be gained by those full of vitality and enjoying the finest years of their lives. companies with a superior product strategy: juggling ingredients, technology and labelling to optimise the Looking ahead, there is a huge market of health conscious product portfolio in each country. ageing consumers who want to do all they can to prolong their lives but not only that, they want to live and enjoy Deal making these extra years in as healthily a position as possible. Use of Companies can make up for their weaknesses in certain nutraceuticals is one option this group have to take some of areas by being quick and decisive corporate deal-makers. the control back into their own hands in terms of their health It will only take one sizable acquisition to change the and wellbeing in their later years. dynamics of the industry, and other big companies in the field will be forced to respond. Big pharma and big With a high level of disposable income and the willingness consumer goods companies are not necessarily known to spend this on products they trust and believe in, for being nimbly entrepreneurial, so they will have to do correctly targeted nutraceuticals brands are set for huge a number of things that may go against their corporate success in the silver market if they get the ingredients and cultures: be bold, supple and patient, all at the same time. recipe right for this slice of the demographic cake.
THE RELEVANCE OF NUTRACEUTICALS FOR THE AGEING REFERENCES AND NOTES POPULATION 1. KPMG/YouGov Consumer Insights Panel: http:// There is a plethora of products already on the market consumerinsightspanel.org/assets/docs/Turning_Silver_Into_ targeted towards the older consumer and an increasing Gold_Report_CIP.pdf (last checked on Sep. 16th 2015) number of these focus on health and wellness. 2. KPMG/YouGov Consumer Insights Panel: http://www. kpmg.com/uk/en/issuesandinsights/articlespublications/ As people get older, their bodies deteriorate slowly as part newsreleases/pages/over-60s-will-be-key-driver-of-consumer- of the aging process but their requirement for nutrients goods-sales-over-the-next-decade-predicts-kpmg-you-gov- th does not decrease to match. Ageing people also often consumer-insights-panel.aspx . (last checked on Sep. 16 2015) eat less and do not digest or absorb the nutrients from 3. Wikipedia: https://en.wikipedia.org/wiki/Nutraceutical (last their food as efficiently as they used to in their younger checked on Sep. 16th 2015) years. 4. KPMG Nutraceuticals: The future of intelligent food: https:// author.ema.kworld.kpmg.com/UK/en/IssuesAndInsights/ Joints, muscles, bones and the nervous system are a ArticlesPublications/Pages/nutraceuticals-the-future-of- few examples of parts/areas of the body which tend to intelligent-food.aspx (last checked on Sep. 16th 2015) deteriorate noticeably with ageing but are also areas that 5. Pub Med: http://www.ncbi.nlm.nih.gov/pubmed/24520134 can be supported by the correct type of supplementation. (last checked on Sep. 16th 2015)
48 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 WOMEN'S HEALTH
TOMMASO CAI Department of Urology, Santa Chiara Regional Hospital, Largo Medaglie d'Oro 9, Trento, Italy
Tommaso Cai Eve and the apple: the origin of original sin Apple comsumption and female sexual quality of life: an intriguing relationship
KEYWORDS: Apple, polyphenols, mediterranean diet, quality of life, female sexual function, FSFI questionnaire.
The relationship between food and women’s sexual health is very intriguing. Several authors stated that Abstract the daily use of some foods or beverages, such as chocolate or red wine, are associated with a better female sexual quality of life in terms of sexual desire or lubrication. Recently, a national multicenter cross-sectional study suggest a potential relationship between regular daily apple consumption and better sexuality in our young women population, highlighting the role of some phytoestrogen such as phloridzin, a dihydrochalcone glycoside, that is contained under the apple pee. Here, we evaluate the role of daily apple consumption in the female sexual quality of life, by using a narrative review of the current literature.
INTRODUCTION Moreover, the vaginal capillaries of microcirculation are filled with blood and the increased hydrostatic pressure “[...] and when the woman saw that inside them forces out a plasma transudate (ultrafiltrate) in the tree was good for food, and a the interstitial space around the blood vessels (1). Continued delight to the eyes, and to be desired formation of this transudate fills up the interstitial space to make one wise, she took of its fruit, and then passes through and between the cells of vaginal and gave it to her husband. And epithelium to leak onto the surface wall of the vagina as the their eyes were open and they were vaginal lubrication (1). Another important factor determining conscious that they had no clothing normal vaginal lubrication and, generally, female sexuality, and they made themselves coats of is estradiol. It is well known that post-menopausal reduction leaves stitched together” [from the in estradiol is commonly associated with vaginal dryness, Book of Genesis, 3:6-8] (Figure 1). which improves with its replacement (5). Recently, some authors have highlighted the fact that apples represent Female sexuality is regulated by several anatomical, an important source of polyphenols and antioxidant in neurobiological, and psychological mechanisms and their the Western diet. It has been suggested that their regular interaction characterizes the state of women’s sexual intake may result in human health benefits (6). Moreover, it response (1). Several studies have suggested that some appears that apples contain phloridzin, a dihydrochalcone foods could also have an intriguing impact on female glycoside, a common phytoestrogen (7). It is well known that sexual quality of life (2-3). Mondaini et al. found that regular phytoestrogens, such as phloridzin, can produce estrogen- moderate intake of red wine is associated with higher FSFI like effects because of their structural similarity to estradiol, scores for both sexual desire, lubrication and overall sexual and can combine with estrogen receptors in mammalians or function as compared to teetotaller status (3). Recently, humans, resulting in antiestrogenic or estrogen-like activity Salonia et al. showed that those women who reported eating (8). In this sense, daily apple intake should improve female ≥1 chocolate cubes daily have higher FSFI scores for both sexual quality of life. Cai et al., recently, by using a cross- sexual desire and overall sexual function than women who sectional study in a large cohort of young sexually active did not report eating chocolate (2). The authors hypothesized women, find that daily apple use is associated with higher that reason for the impact of these foods on female sexuality Female Sexual Function Index (FSFI) scores in sexually active may be the fact that containing polyphenols and antioxidant female patients thus increasing their lubrication and overall can stimulate peripheral vasodilation via activation of the sexual function (9). The correlation between daily apple nitric oxide system (2-3). During sexual arousal, in fact, the intake and higher FSFI scores for lubrication and overall central reduction of sympathetic tone and the release of two sexual function as compared to those who did not report vasodilator neurotransmitters (vasoactive intestinal peptide – eating apples is intriguing (9). The aim of the present study VIP; nitric oxide – NO), create an increase in the blood flow to is to evaluate the role of apple consumption in the female the external genitalia and vagina and promote relaxation of sexual quality of life, by using a narrative review of the the smooth muscle of the cavernous sinuses in the clitoris (4). current literature.
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 49 MATERIALS AND METHODS while 388 were included in Group B. Group A women reported a daily median apple intake of 1 (ranging from 1 to 2). Group We conducted a search of the English-language literature A had a significantly higher total (p=0.001; Cohen’s d=3.39) and from 1960 through December 2014 with use of the Medline lubrication domain (p=0.03; Cohen’s d=3.02) FSFI scores than computerized database of the US National Library of Medicine participants in Group B. Multivariate analysis demonstrated (http://www.ncbi.nlm.nih.gov/pubmed). The Medline search has that daily apple intake must be considered as an independent been carried-out by using the following Medical Subject Headings parameter (p=0.002) in predicting a better score at questionnaire and free text terms: apple, polyphenols were combined with the examination, in their female population (9). In this sense, this study terms women health, female sexual function, FSFI questionnaire is the first to find a relationship between apple consumption and and then limited to humans and female. Moreover, we searched female sexuality. In this study, moreover, the authors find a good reference lists of articles to identify potential additional references. correlation between apple intake and physical activity, even if it All original paper and review studies on female sexual function is not statistically significant (9). It is probably due to the fact that have been considered for this review. From an initial literature women who constantly practice physical activity are those who search with 20 unique citations, a total of 6 articles were selected eat more vegetables and fruit. It is important to highlight the fact for the present review. Finally, a matched research between that daily physical exercise associated with fruit and vegetable apple consumption and female sexual function (exploded) has consumption are essential components of the Mediterranean found 2 articles. life-style (20). Recently, a Mediterranean-style diet carried out for 2 years was claimed to be effective in ameliorating sexual function in women with metabolic syndrome but the exact mechanism RESULTS AND DISCUSSION remains unclear (21). The authors claim that food rich in fibres and antioxidants is the key point of female sexual function changes in Apple and health: compound and pharmacological proprieties multivariate analysis (21). Apples have commonly been described as a healthy food. Moreover, apples are a fruit of specific interest, since they are Apple and sex: future directions cheap and easy to store and transport, and thus are the most Up to the moment, only one prospective study has been frequently consumed fruit. It is well known that increasing the conducted in the analysis of the impact of apple consumption on consumption of fruit and vegetables is a key component of women sexual quality of life (9). Data available from the current obesity prevention, as their consumption reduces the risks of literature shows several limitations that can be taken into account. several diseases, such as type 2-diabetes, cardiovascular diseases First of all, we cannot exclude that unexpected selection bias or colorectal cancer (10-11). Apples, for instance, contain a lot due to cultural and socioeconomic status may have affected of pharmacologically active substances such as phytoestrogens, participation in the study. Furthermore, we cannot exclude that poliphenols and antioxidants (6, 12-13). In fact, apples are large respondents who agree to complete the questionnaires might contributors of phenolic compounds in European and North have been more sexually active per se, that is, have the highest American diets (6). In particular, apples, when compared with sexual desire profile, and also hold different food-related attitudes other commonly consumed fruits, had the second highest level of than those who did not agree to complete the questionnaires. antioxidant activity (after cranberries) and phenolic compounds, Moreover, as suggested by Salonia, because both health and and the highest level of free phenolic compounds (12, 14). For taste factors are important in the food choice process, dedicated these proprieties, apple intake has been studied and negatively tools for studying these aspects of food choices are required (1). associated with lung cancer incidence (15), coronary and However, further studies will be necessary to clarify all molecular total mortality (16), symptoms of chronic obstructive pulmonary mechanisms involving the relationship between apple intake disease (17), and risk of thrombotic stroke (18). Moreover, it is also and female sexuality. Further epidemiological investigations well known that the concentration of total phenolic compounds is prospectively evaluating larger cohort populations of women much greater in the peel of apples than in the flesh (19). are needed to confirm the precise role of apple intake in the context of female sexual response. However, the present data Apple and sex: the phloridzin can allow the development of future research for identifying new Apples contain phloridzin, a dihydrochalcone glycoside, a compounds and food supplements to use in the female sexuality common phytoestrogen (7). It is well known that phytoestrogens, recovery. such as phloridzin, can produce estrogen-like effects because of their structural similarity to estradiol, and can combine with estrogen receptors in mammalians or humans, resulting in CONCLUSIONS antiestrogenic or estrogen-like activity (8). In this sense, daily apple intake should improve female sexual quality of life. Cai In conclusions apple consumption is related with better sexual et al. found that a regular daily apple consumption is related quality of life in sexually active female patients. Although these to a better sexuality in young sexually active women (9). They findings need to be interpreted with some caution due to the enrolled in a cross-sectional study 731 women (mean age 31.9, limited number of published studies, daily apple intake could range 18-43), which completed anonymously the Female Sexual improve sexual quality of life in young sexually active women, thus Function Index [FSFI questionnaire] and were asked to report on improving sexual satisfaction and the couple’s quality of life. their amount of daily apple consumption. On the basis of apple consumption all women were split into two groups: Group A – regular daily apple consumption, Group B – no regular apple REFERENCES consumption (less than one apple/day). The main outcome 1. Salonia, A., Giraldi, A., Chivers, M.L., Georgiadis, J.R., Levin, R., measure was the Female Sexual Function Index questionnaire Maravilla, K.R., McCarthy, M.M.. “Physiology of Women’s Sexual result (9). They found the followings results: 343 women reported Function: Basic Knowledge and New Findings”. J Sex Med, 7:2637– a regular daily apple intake and were classified in Group A, 2660 (2010).
50 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 2. Salonia, A., Fabbri, F., Zanni, G., Scavini, M., Fantini G.V., Briganti, A., Naspro, R., Parazzini, F., Gori, E., Rigatti, P., Montorsi, F. “Chocolate and Women’s Sexual Health: An Intriguing Correlation”. J Sex Med, 3:476–482 (2006). 3. Mondaini, N., Cai, T., Gontero, P., Gavazzi, A., Lombardi, G., Boddi, V., Bartoletti, R. “Regular moderate intake of red wine is linked to a better women’s sexual health”. J Sex Med, 6(10):2772-7 (2009). 4. Maravilla, K.R., Heiman, J.R., Garland, P.A., Cao, Y., Carter, W.O., Peterson, B.T., Weisskoff, R.M. “Dynamic MR imaging of the sexual arousal response in women”. J Sex Marital Ther, 29(1 suppl):71–6 (2003). 5. Rymer, J., Morris, E.P. “Extracts from ““Clinical evidence”: Menopausal symptoms”. BMJ, 321(7275):1516-9 (2003). 6. Wolfe, K., Wu, X., Liu, R.H. “Antioxidant activity of apple peels”. J Agric Food Chem, 51(3):609-14 (2003). 7. Lauren, D.R., Smith, W.A., Adaim, A., Cooney, J.M., Wibisono, R., Jensen, D.J., Zhang, J., Skinner, M.A. “Chemical composition and in vitro anti-inflammatory activity of apple phenolic extracts and of their sub-fractions”. Int J Food Sci Nutr, 60 Suppl 7:188-205 (2009). 8. Wang, J., Chung, M.H., Xue, B., Ma, H., Ma, C., Hattori, M. “Estrogenic and antiestrogenic activities of phloridzin”. Biol Pharm Bull 33(4):592-7 (2010). 9. Cai, T., Gacci, M., Mattivi, F., Mondaini, N., Migno, S., Boddi, V., Gacci, P., Detti, B., Gontero, P., Chiodini, S., Mereu, L., Tateo, S., Mazzoli, S., Malossini, G., Bartoletti, R. “Apple consumption is related to better sexual quality of life in young women”. Arch Gynecol Obstet, 290(1):93-8 (2014). 10. Forwood, S.E., Walker, A.D., Hollands, G.J., Marteau, T.M. “Choosing between an apple and a chocolate bar: the impact of health and taste labels”. PLoS One 8(10):e77500 (2013). 11. Cooper, A.J., Sharp, S.J., Lentjes, M.A.H., Luben, R.N., Khaw, K-T. Wareham, N.J., Forouhi, N.G. “A prospective study of the association between quantity and variety of fruit and vegetable intake and incident type 2 diabetes”. Diabetes Care, 35:1293-1300 (2012). 12. Boyer, J., Liu, R.H. “Apple phytochemicals and their health benefits”.Nutr J 3:5 (2004). 13. Martin, J.H., Crotty, S., Warren, P., Nelson, P.N. “Does an apple a day keep the doctor away because a phytoestrogen a day keeps the virus at bay? A review of the anti-viral properties of phytoestrogens”. Phytochemistry 68(3):266-74 (2007). 14. Gallus, S., Talamini, R., Giacosa, A., Montella, M., Ramazzotti, V., Franceschi, S., Negri, E., La Vecchia, C. “Does an apple a day keep the oncologist away?” Ann Oncol, 16(11):1841-4 (2005). 15. Knekt, P., Järvinen, R., Seppänen, R., Hellövaara, M., Teppo, L., Pukkala, E., Aromaa, A. “Dietary flavonoids and the risk of lung cancer and other malignant neoplasms”. Am J Epidemiol, 146(3):223-30 (1997). 16. Knekt, P., Jarvinen, R., Reunanen, A., Maatela, J. “Flavonoid intake and coronary mortality in Finland: a cohort study”. BMJ, 312(7029):478-81 (1996). 17. Tabak, C., Arts, I.C., Smit, H.A., Heederik, D., Kromhout, D. “Chronic obstructive pulmonary disease and intake of catechins, flavonols, and flavones: the MORGEN Study”.Am J Respir Crit Care Med, 164(1):61-4 (2001). 18. Knekt, P., Isotupa, S., Rissanen, H., Heliövaara, M., Järvinen, R., Häkkinen, S., Aromaa, A., Reunanen, A. ”Quercetin intake and the incidence of cerebrovascular disease”. Eur J Clin Nutr, 54(5):415-7 (2000). 19. Escarpa, A., González, M.C. “High-performance liquid chromatography with diode-array detection for the determination of phenolic compounds in peel and pulp from different apple varieties”. J Chromatogr A, 823(1-2):331-7 (1998). 20. Trichopoulou, A., Costacou, T., Bamia, C., Trichopoulos, D. “Adherence to a Mediterranean diet and survival in a Greek population”. N Engl J Med, 348(26):2599-608 (2003). 21. Esposito, K., Ciotola, M., Giugliano, F., Schisano, B., Autorino, R., Iuliano, S., Vietri, M.T., Cioffi, M., De Sio, M., Giugliano, D. “Mediterranean diet improves sexual function in women with the metabolic syndrome”. Int J Impot Res 19(5):486-91 (2007).
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 FUNCTIONAL FOOD
SARA MONTOYA1*, A. C. SALGADO1, M. L. LORENZO2, E. ORTEGA1 *Corresponding author 1. Department of Edaphology and Agricultural Chemistry, Pharmacy College, University of Granada, Campus Cartuja, s/n. 18071 Granada, Spain 2. Department of Nutrition and Bromatology, Pharmacy College, University of Granada, Campus Cartuja, s/n. 18071 Granada, Spain
Montoya Sara Sodium / potassium relationship in biofortified cucumber
KEYWORDS: Biofortification, potassium, sodium, cucumber, seed, seedlings.
Fortification policies are increasing to achieve cost savings as a way of enriching plant products For Abstract this reason in our study we have increased potassium content in cucumber and we studied the relationship of this element with sodium. Cucumber was chosen for its easy use, including in many culinary preparations, in addition to its low calorie input. Twenty-eight samples biofortificated with different potassium sulfate concentrations (0.014 mg / L , 1 g / L, 2 g / L and 4 g / L) were collected in three different stages from two different greenhouses, one cultivated from seed and the other one from seedlings. We conclude potassium content of biofortified cucumbers has increased over time. After the comparison of the potassium average content in the fruits obtained in the different collections, we see that in the case of those from seedlings it is higher in the first two collection moments equaling in the last one.
INTRODUCTION the intake of sodium in the general population is above the daily recommendations, whereas the intake of potassium is Nowadays, consumers do not only demand a high insufficient. production of food but they also require nutritive products American countries recommend a higher intake of that contain the macro- and micronutrients needed by the potassium, fiber, vitamin D and calcium, and a lower intake body to improve/restore the body’s physiological functions of calories from saturated fats and simple sugars (13). Since and reduce the risk of disease (1). Policies are being the cost of potassium is fairly expensive, it is estimated that developed to produce cost-saving enriched vegetables (2) increasing the intake of potassium-rich products to meet and improve the efficiency of infertile soils (3). Biofortification daily recommendations would increase consumer costs an is considered a new tool for reducing deficiencies of average of €279/year (14) in their study on the relationship micronutrients (4). between the intake of sodium and potassium and food There are recent studies about biofortifiction with different expenditure concludes that diets with beneficial effects on elements, such as (5), which assessed the effect of enriching cardiovascular health (those high in potassium and low in seeds of different plant foods (cucumber, among others) sodium) tend to be more expensive. Therefore, the intake of with ZnO on germination. The results of the study revealed potassium-enriched products would be more viable from an that the uptake of ZnO increased germination by 10% in economic point of view. cucumber, whereas germination decreased in other plants. We chose cucumber for its easy handling and because it is Another example is (6). This study revealed that biofortification used in many dishes worldwide; also, it is a low calorie food of broccoli and carrot crops with selenium –an element with –12 kcal / 100g according to (15) – which is important for important health benefits– increased Se concentrations in overweight and obese people, who require a high input of these food crops, which is especially relevant for Se-deficient potassium. regions. The aim of this study was to assess whether potassium A higher intake of potassium has beneficial effects on concentrations in a greenhouse-grown cucumber crop human health. According to (7) hypokalemia is a risk factor could be increased through biofortification by adding for heart, arterial, cerebrovascular and renal disease. an extra amount of potassium sulfate to regular fertilizers. Increasing the intake of potassium reduces mortality from A secondary aim was to assess the effect of potassium cardiovascular disease since it reduces blood pressure biofortification on the sodium concentration of cucumber and has a direct effect on the cardiovascular system (8). and determine whether increasing the potassium In accordance with (9), excessive sweating during exercise concentration of cucumber results in a reduction in the can cause an electrolyte imbalance that causes dizziness, sodium concentration due to the competition between vomiting, bloating, muscle weakness, cramps ... Recent these two cations (16). Most of the studies reviewed studies conducted in different countries (10-12) confirm that analyzed potassium and sodium concentrations either in the
52 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 ground or in the plant, but not in the fruit (17, 18). S1 and P1 abbreviations correspond to 0.014mg/L Other studies have demonstrated that some elements limit potassium sulfate, S2 and P2 to 1g/L, S3 and P4 are the absorption of others, for example, green tea catechins 2g/L; finally, S4 and P4 represent a concentration of reduce cholesterol due to an interaction mecanism (19) thus 4g/L. The acronyms S and P indicate that the fruits were resulting in a heart protective effect. grown either from seeds (S) or from seedlings grown in seedbeds (P). Solutions were added with PVC bottles adapted as MATERIAL AND METHODS irrigation dispensers that were placed next to the plant root system (Figure 3) and released the solution according The cucumber samples (Cucumis sativus L) used were Almeria to plant needs and soil humidity. type of the Valle seed variety (RZ code 24-165) developed by Rijk Zwaan and other seeds of the same variety collected from seedlings grown in seedbed and transplanted to the greenhouse in soil blocks. The greenhouses were located in the Punta Entinas-Sabinar Natural Park (Almeria). Twenty-eight samples were collected in three different stages. The first crop was collected on December 12, 2012 (Crop 1), the second on February 25, 2013 (Crop 2) and the last on April 3, 2013 (Crop 3). Of the 28 samples from each batch, fourteen had been grown from seeds in a greenhouse and Figure 3. Bottles with the rest from seedlings grown in seedbed. We collected dispenser location. samples from three different plants fertilized with the same potassium sulphate concentration added to the usual fertilizer, plus two controls. Between each treated sample was left at least a white side to prevent possible contamination of the treatments, as shown in Figures 1 and 2. Samples from each treated plant were collected randomly.
Potassium and sodium determination by flame photometry Sodium and potassium concentrations were determined by flame photometry following the “Official Methods of Analysis of Food” (20). Sample preparation. Getting ashes: 5g cucumber (in the case of samples without skin they were previously peeled with a disinfected knife) were put in a tarred quartz capsule and heated slowly in a sand bath until most of the organic substance was carbonized. Then the capsule was inserted into the muffle at 525°C for eight hours, cooled in a desiccator for 30 minutes and weighed. Potassium: Appropriate sample dilution was performed with distilled water. Readings of the 766-770 nm wavelength flame photometer were performed against Figure 1. Greenhouse seeds. reference solutions. Measurements were taken three times to obtain a real value by calculating the average of the three values obtained. Potassium concentrations were calculated from the average value obtained by deducting the average value from the equation of the calibration curve taking into account the dilution made. The results reported are for the 5g samples analyzed. Conversions were performed to express results We used a standard solution of potassium* concentration of 1g/L for the calibration curve. From this solution, we prepared solutions of 0, 1, 2, 3, 5 and 7mg/L. The 0mg/L concentration was prepared only with miliQ water. Sodium: Sodium concentrations were measured using the same method as that used for measuring potassium concentrations, although measurements were made at a 590 nm wavelength. For the calibration curve a standard solution of sodium** 1g/L was used. Solutions of 0, 2, 5, 10, 15 and 20mg/L were prepared from the previous solution. The white solution Figure 2. Greenhouse seedlings. was done with miliQ water.
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 53 RESULTS AND DISCUSSION concentration of potassium was the same for both groups in Crop 3 (Figure 4). Student’s t test was performed for independent samples in order to determine whether there were significant differences regarding the potassium contents between the cucumbers grown from seeds shed in a greenhouse and the cucumbers grown from seeds shed in seedbed and transplanted with the root ball, the results are expressed in mg K+/100 g cucumber. We found statistically significant differences at a 95% confidence level in the average potassium concentration, which was higher in the samples from the seedlings (Table 1).
Figure 4. Evolution of potassium concentration of samples from seeds and seedlings over time.
Next, we analyzed whether there were significant differences Table 1. Average of potassium concentrations (mg K+/100g) in potassium concentrations among samples with and without skin from the three crops. The two types of culture were analyzed separately. In Crop 1 we observed differences in potassium We assessed whether there were significant differences concentrations among the samples from the seeds and the among the samples from the three crops. Friedman’s test seedlings; thus, the average concentration of potassium was was performed for comparison of related samples n>2 and higher in the samples with skin for both groups (Table 4). revealed significant differences between the two groups over time at a 99% confidence level. Table 2 shows the results for the samples from seeds, and Table 3 displays the results for the samples from the seedlings.
Table 4. Average potassium concentration for samples with and without skin in Crop 1 (mg K+/100g).
Table 2. Average, maximum and minimum potassium concentrations in the seed samples over time (mg K+/100g). In Crop 2, no significant differences were found concerning potassium concentrations among the cucumbers from seeds with or without skin at a 95% confidence level. On the other hand, differences were found between the seedling samples with and without skin, at the same confidence level. Thus, the average concentration of potassium was higher in the seedling samples with skin in Crop 2 (Table 5).
Table 3. Average, maximum and minimum potassium concentrations in the seedling samples over time (mg K+/100g). Table 5. Average concentration of potassium for Table 2 shows that average values were higher for Crop 1, samples with which means that potassium concentrations progressively and without skin increased over time reaching an increase of 24% in Crop 2 in Crop 2. (mg K+/100g). from the first crop. As to minimum values, they also increased gradually over time. Finally, there was also an increment in maximum values for Crops 2 and 3, although the maximum In Crop 3, the initial differences between samples with and value for Crop 2 was higher as compared to Crop 3. without skin had disappeared in both groups. In crop 3, the In table 3 we can see that average values were higher for average concentration was slightly lower in the seedling samples, Crops 2 and 3 although it was higher for Crop 2 as compared as opposed to what happened in Crops 1 and 2 (Table 6). to Crop 3, which showed an increase of only 1.9%. However, the maximum potassium concentration was reached in Crop 3; in fact, standard deviation was quite significant in Crop 3; Table 6. Average this would explain that the average potassium concentration potassium concentrations was not higher in the samples from the seedlings in Crop 1 as in Crop 3 (mg K+/100g). it occurred in greenhouse seeds. In Crop 3 maximum values increased by 40% with respect to Crop 1. Comparing the average potassium concentration in fruits in To sum up, potassium concentrations were higher in the the different crops, we see that potassium concentrations samples with skin of both groups in Crops 1 and 2. However, were higher in the fruits from the seedlings as compared such differences between the samples with and without skin to those from seeds in Crops 1 and 2. However, the disappeared in Crop 3.
54 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 As to sodium concentrations, after verification by Student’s t 2. Lenoir-Wijnkoop I, Jones PJ, Uauy R, Segal L and Milner J. (2013). test, no significant differences were observed between the Nutrition economics – food as an ally of public health. British seed and the seedling samples. Friedman’s test revealed Journal of Nutrition, 109: 777–784. significant differences in the amount of sodium absorbed 3. Graham RD, Welch RM, Saunders DA, Ortiz-Monasterio I, Bouis HE, Bonierbale M, de Haan S, Burgos G, Thiele G, Liria R. (2007). over time at a 99% confidence level. Thus, in Table 7 we Nutritious subsistence food systems. Advances in Agronomy, 92: can see that average sodium concentrations were greater 1–74. in Crops 2 and 3, which means that -as it occurred with 4. Bouis, H. E., Hotz, C., McClafferty, B., Meenakshi, J. V., & Pfeiffer, potassium concentrations- sodium concentrations increased W. H. (2011). Biofortification: a new tool to reduce micronutrient over time. We can also observe that maximum and minimum malnutrition. Food & Nutrition Bulletin, 32(Supplement 1), 31S- concentrations increased. 40S. 5. De la Rosa G, López-Moreno ML, De Haro D, Botez CE, Peralta- Videa JR, and Gardea-Torresdey J. (2013). Effects of ZnO nanoparticles in alfalfa, tomato and cucumber at the germination stage: Root development and X-ray absorption spectroscopy studies. Pure Appl. Chem., 85 (12): 2161–2174. 6. Bañuelos, G. S., Arroyo, I., Pickering, I. J., Yang, S. I., & Freeman, J. L. (2015). Selenium biofortification of broccoli and carrots Table 7. Average, maximum and minimum sodium concentrations grown in soil amended with Se-enriched hyperaccumulator for the three crops (mg K+/100g). Stanleya pinnata. Food chemistry, 166, 603-608. 7. Macías Garrido E, García Frade F. (2008). Hipopotasemia en pacientes hospitalizados. Med Int Mex, 24: 3-7. Initially, we expected that sodium concentrations would 8. He FJ, MacGregor GA. (2008). Beneficial effects of potassium on drop with increased potassium concentrations, as these human health. Physiol Plant, 133(4):725-735. two cations would compete for absorption . However, 9. Marins JC, Dantas EH, Zamora S. (2000). Variaciones del sodio y statistical analysis revealed that sodium concentrations potasio plasmáticos durante el ejercicio físico: factores did not drop with increased potassium concentrations. asociados. Universidad Brasil/Murcia. Educación física y Deportes, 62: 48-55. Nevertheless, we think that increasing potassium 10. Rodrigues S. L, Baldo M.P, Machado R.C, Forechi L et al. (2014). concentrations in cucumber may be beneficial at High potassium intake blunts the effect of elevated sodium metabolical level as it would mitigate the negative effects intake on blood pressure levels. Journal of the American Society of sodium intake, as it has been shown in many studies of Hypertension, 8(4): 232–238. performed in the past (21) and at present (10). The latter, 11. Yang Q, Liu T, Kuklina EV, Flander D, Hong Y, Gillespie C et al. which was performed on a large population sample (1,285 (2011). Sodium and potassium intake and mortality among US healthy subjects ranging from 25-64 years), reported that a adults: prospective data from the Third National Health and high sodium intake was not associated with higher blood Nutrition Examination Survey. Arch Intern Med, 171: 1183–1191. pressure in the subjects who had increased potassium intake 12. Donfrancesco C, Ippolito R, Lo Noce C, Palmieri L, Lacone R, through diet. Thus, this study concluded that a high intake of Russo O et al. (2013). Excess dietary sodium and inadequate fresh vegetables, fruits and whole grains rich in potassium can potassium intake in Italy: results of the MINISAL study. Nutr Met Cardiovasc Dis, 23: 850–856. help reduce the harmful effect of a sodium-rich diet. 13. Monsivais P, Aggarwal A, Drewnowski A. (2011). Following federal guidelines to increase nutrient consumption may lead to higher food costs for consumers. Health Aff (Millwood), 30: 1471– CONCLUSIONS 1477. 14. Drewnowski A, Rehm CD, Maillot M and Monsivais P. (2014). The In the light of the results obtained, we recommend relation of potassium and sodium intakes to diet cost among US greenhouse-grown cucumber biofortification with potassium adults. Journal of Human Hypertension, 1–8. sulfate without removing the skin. Since no significant 15. Mataix Verdú J. (2003). Tabla de composición de alimentos. 4ª differences were observed among samples in potassium Edición. Editorial Universidad de Granada. 555 p. concentrations, we recommend using the minimum 16. García Morales S. (2007). Respuestas agronómicas y fisiológicas del pepino a distintas concentraciones de cloruro sódico. Tesis concentration required for obtaining the same result Doctoral. México. 148 p. (0.014g/l) in order to save costs. Moreover, we recommend 17. Kaya C, Kirnak H, Higgs D. (2001). Effects of supplementary the biofortification of cucumber from seedlings to get a higher potassium and phosphorus on physiological development and concentration of potassium during the entire culture period. mineral nutrition of cucumber and pepper cultivars grown at The reason is that the samples from the seeds needed 90 days high salinity (NaCl). Journal of plant nutrition, 24(9): 1457-1471. to reach a potassium concentration similar to that found in 18. Ersin I, Simsek M. (2004). Ameliorative effects of potassium the samples from the seedlings, which only needed 60 days. and calcium on the salinity stress in embryo culture of Unexpectedly, sodium concentrations were not reduced with cucumber (Cucumis sativus). Journal of Biological Sciences, increased potassium concentrations. 4 (3): 361-365. 19. Makoto K, Masato N, Nao I, Takahiro H, Masahito Y, Yuichi U, Yuko MS, Takayuki D, Tsutomu N,⊥ Shigenori K and Ikuo I. 2014. Epigallocatechin Gallate Decreases the Micellar Solubility of REFERENCES Cholesterol via Specific Interaction with Phosphatidylcholine J. * Potassium Panreac Standard solution. K = 1.000 ± 0.002 g / L ICP Agric. Food Chem, 62: 2881-2890. ** Panreac Standard Sodium solution. Na = 1.000 ± 0.002 g / L AA 20. MAPA. (1994). Métodos Oficiales de Análisis de Alimentos. 1. Cortés M, Martelo Y.J, Rodríguez E. (2011). Valoración de Madrid. Mundiprensa Libros S.A. 342-343. atributos de calidad en pepino (cucumis sativus l.) fortificado 21. Meneely GR and Ball CO. (1958). Experimental epidemiology of con vitamina E. Biotecnología en el Sector Agropecuario y chronic sodium chloride toxicity and the protective effect of Agroindustrial, 9(1): 24 – 34. potassium chloride. Am J Med, 25(5): 713-725.
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 55 PLANT INGREDIENTS
NESLI SOZER, KAISA POUTANEN VTT Technical Research Centre of Finland, Tietotie 2, 02044 VTT, Finland
Nesli Sozer Kaisa Poutanen Plant protein ingredients for future foods
KEYWORDS: Plant proteins, food ingredients, cereals, pulses, agro-side streams, protein functionality.
The demand for animal protein is expected to increase drastically in the future because of the global Abstract growth of population and income. The increasing need for good quality protein cannot be met only with animal based proteins without drastic adverse impacts for environment and humanity. Finding new sources of food proteins and especially plant proteins is a prerequisite to obtain sustainable and affordable foods for the growing global population. However, there are technological, nutritional and sensory challenges associated with the use of plant proteins. This article reviews the applicability of plant proteins as a food ingredient for the development of future foods and suggests strategies for improving their use in various food applications.
INTRODUCTION Furthermore majority of the soy protein is imported and thus interfering with the protein independency for Europe. In line The global population is forecasted to increase to 7.6-10.6 with its new agricultural policy, EU is stimulating production billion people by 2050. This rapid growth in combination and use of domestic plant proteins. Wheat proteins cannot be with expected increasing welfare will lead to an increased consumed by coeliac patients, and also currently suffer of the demand for animal protein which is forecasted to growing consumer trend of excluding gluten from the diet. increase by 52% between 2007 and 2030. Thus, additional alternative and sustainable plant protein sources with high The interest in plant proteins is globally increasing rapidly, technological, sensory and nutritional functionality are and an increasing number of commercial plant protein needed. Plant protein production requires less land and ingredients are already available (Table 1), with many more thus less resource intensive than that of animal protein, in pipeline. but plant proteins also pose challenges especially when compared to the properties of the current animal proteins. CEREAL PROTEINS: WHEAT, RICE AND OATS Protein is an essential part of human nutrition. Animal proteins are “complete proteins” since they provide Most commonly used cereal protein is gluten which plays a sufficient amounts of essential amino acids. Plant proteins key role in determining the unique quality of many cereal are generally “incomplete proteins” as they lack one or more of the essential amino acids. Besides nutritional value, proteins contribute to the technological functionality of in food matrices, such as foam-formation, gelation, rheological and texturizing characteristics. These properties, including sensory quality, are essential in applications of plant proteins as building blocks of food structure and in design of new high- protein foods. However, most of the plant based proteins don’t provide optimal techno-functional properties. The existing scientific knowledge is generally built around meat, dairy or egg proteins, and of the plant proteins, only soy and wheat protein are rather well studied. The use of soybean involves issues such Table 1. Major commercially available plant protein ingredients. as GMO which is a consumer concern.
56 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 based products. It provides cohesivity, viscosity and elasticity PULSES AS SOURCES OF PROTEIN to dough and has good water absorption capacity. Gluten proteins can be divided into two main fractions according to Pulses are an excellent, low cost source of protein, as well as their solubility in aqueous alcohols: the soluble gliadins and dietary fibre, minerals and vitamins (Table 2). the insoluble glutenins. Gliadins confer viscosity to dough whereas polymeric gluten protein, glutenin is responsible for dough elasticity. Many gluten-based products with tailored properties are currently available (Table 1).
In current grain and seed processing, side streams are often generated which are under-exploited or even wasted. Many of these are used as feed especially because of their high protein content. Valorisation of side streams in the form of upgraded protein ingredients will benefit food industry by development of sustainable protein ingredients alternative to animal derived ones. Processing of cereal grains for starch or bioethanol production generates substantial volumes of co-streams rich in protein. Wheat bran, 167 million tons of which is produced annually, is currently used as cattle feed, biofuel production or even discarded totally. In wheat the bran fraction is around 14–19% of the whole kernel, and contains 8.3–19.3% protein (1). The aleurone layer which makes half of the wheat bran is rich in protein and has superior amino acid composition compared to wheat endosperm (low in methionine, sulphur-rich proteins and cysteine; high in essential amino acids valine, threonine and especially lysine (2). Rice bran is undervalued side stream of rice starch or milling industries. Rice bran is generally not consumed as food because of its high fibre content, possible hull contamination and rancidity issues due to lipase activity (3). Table 2. Protein content of major pulse crops and cereals. Thus, it is mainly used for feed or fuel. Ten percent of the rice kernel is rice bran which contains from 12% (native bran) to They are a recognised protein source in human nutrition 20% (defatted bran) protein (4). In rice bran, water soluble already now especially in Asian countries, and can offer albumins comprise the major protein fraction (37% of bran an interesting alternative and addition to animal protein proteins) together with globulins (36% of bran proteins) which when used in new applications. Legumes, particularly beans may render rice bran proteins with good functional properties have been called as the “poor man’s meat” due to their compared to other cereal proteins as well as rice endosperm high protein content, ranging from 19 to 36%, and excellent proteins in water-rich food matrices. nutritional profile and yet low cost. Faba beans f.ex. have good amino acid composition, and are rich in essential Oats are an important minor crop world-wide with the amino acids isoleucine, leucine, lysine, phenylalanine, global production of 21 million tons per year, 62% of which threonine and valine (7). When combined with cereals (rich in EU. Use of oats in food is still limited, and finding new uses in sulphur-containing amino acids) they offer a balanced would diversify the consumption of grains in Europe, now plant protein source with nutritionally adequate amino acid dominated by wheat. Concentration of oat beta-glucan in composition. Gluten-free fermented faba bean flour could a dry fractionation process makes also available a stream be applied to obtain high protein bread recipes together rich in oat protein (5). Oats is exceptional within cereal crops with corn starch (Figure 2). with respect to nutritional value because of higher lysine and Among the pulses, soybeans have been in the centre basic amino acid content (6). Oat protein has also potential during the past 15-20 years not only due to their high protein because it can be in many countries used in gluten-free diet content but also as a source of a group of phytochemicals (Figure 1). called isoflavones. About 85 percent of the world’s soybeans are processed, or “crushed,” to yield oil and meal, 98 percent of the latter going to animal feed.
Figure 2. Gluten free faba bean bread made with a) 50 % fermented faba flour and 50% Figure 1. High protein oat bread. corn starch b) 70% fermented faba flour and 30% corn starch.
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 57 The rest is consumed in various forms of protein ingredients. PROTEINS FROM OIL CROPS Soy concentrates (70% protein) and isolates (90% protein) are obtained from defatted soybean meal through a series The annual worldwide growth of canola production is predicted of acidic or ethanol extractions, rendering them variable to exceed 15 million tonnes by 2015. Australia and Canada are functional properties as the conditions might cause protein the biggest exporters of canola seed. The growing demand for denaturation. Soy concentrates are used for bakery products canola oil worldwide offers a good source of canola proteins, whereas soy isolates are used in meat and dairy foods where the major constituents of oil press cake. Canola proteins have emulsifying, thickening and gelling properties are crucial (8). comparable or even better emulsifying properties compared to soy proteins which are dependent on extraction methods. Pea protein, extracted from yellow pea, is becoming popular Acid precipitated protein isolates have good foaming and after soybean it is the second most used pulse protein. It properties (14). They are balanced in all essential amino acids, is one of the few plant protein ingredients without bitter taste. having a better amino acid profile than soybean protein However, the expensive solvent extraction method used for isolates. The major disadvantages: antinutritional factors, colour pea protein is complex. Pea protein concentrate (51-55% and taste could be resolved by targeted extraction procedures. protein) and isolate (85% protein) applications include meat, fish, processed foods, soups, sauces, baked goods cereals, Dehulled sunflower meal has protein content similar to soy snacks, nutraceuticals, sports, and clinical nutrition. meal but higher levels of crude fibre and ash. Sunflower protein concentrate and isolate contain 69 and 88% protein, Dry beans (eg. white, black, navy, kidney, pinto, faba and respectively. Sunflower seed proteins are mainly globulins field bean) are high in fibre, calcium, and iron, but also a great (70-80%) exhibiting low protein solubility below pH 8 but high source of protein (15-25% wb) and soluble fibre. Dry bean dispersability in dilute salt solutions (15). Lysine is the major proteins remain largely underexploited. They include the water- limiting amino acid in sunflower protein, which does not bring soluble (albumins) and dilute salt-soluble (globulin) storage along any anti-nutritive and toxical compounds. In processing proteins and some of the metabolic proteins such as the of sunflower proteins colour problems occur resulting from finely enzyme inhibitors and lectins. Most bean proteins have acidic ground particles of dark hulls and polyphenolic acids which isoelectric points because of large amounts of glutamic and are easily oxidized and converted into brown polymerics. aspartic acids, have water holding capacity varying 5-6 times Sunflower proteins could be applied in infant formulas, milk of their own weight and oil holding capacity < 5 g oil/g (9). For extenders and milk substitute, bakery products, spreads and gel formation from bean proteins higher protein concentration salad sauces. (10 to 20% w/v) and temperature (> 70°C for several plant proteins compared with 4° C for gelatin) is required compared to animal proteins. Several dry bean storage proteins have FUNCTIONALITY OF PLANT PROTEIN INGREDIENTS excellent emulsion properties, both emulsion capacity and stability, and therefore have been used successfully in many Compared with animal proteins, notably dairy and meat countries (eg. India, Egypt, Turkey, China) to prepare local proteins, research on plant proteins, with the exception of foods. soy proteins, has so far been limited. Solubility or dispersability in the form of stable colloidal aggregates is one of the main Lupine, is an ancient leguminous seed originated more requirements for functionality of proteins as emulsifying, than 3500-4000 years ago back, is being increasingly used foaming or gel forming agents. Plant proteins generally in food formulations due to its nutritional and functional show low solubility (or dispersability) at pH values relevant to properties (10). The protein and dietary fibre content in lupine most foods. Furthermore, processes such as oil extraction or accounted for 29-34 and 50-40 g/100 g d.m depending on high temperature treatments for enzyme inactivation may the variety. Lupine protein can replace egg proteins and their induce denaturation which further decreases solubility. These potential applications are in the category of cereals, snacks, physico‐chemical properties restrict full exploitation of plant baby formula, soups and salads. However, the most critical proteins in different foods. Despite the low solubility of plant problem with lupine is that many people with peanut allergies, proteins, the so called soluble aggregates (colloidal particles) one of the most common allergies, can cross-react to lupine. at certain environmental conditions (pH, ionic environment) have shown good surface active properties. For example, Anti-nutritional factors (ANFs) cause the main limitation for the soluble aggregates of oat globulins were reported to lower food and feed use of legumes. Some factors, such as tannins surface tension at air-water interface which was comparable and proteinase inhibitors, can reduce the rate of protein and to milk proteins (16). It was recently shown that colloidal zein starch digestibility (the latter is a benefit, though), and some, (corn protein) particles (∼ 70 nm) had good wetting properties such as phytic acid, can decrease the bioavailability of at the oil-water interface at various environmental conditions minerals (e.g. zinc, calsium and iron) (11). Thus the ANFs often rendering them good inherent surface activity (17). In fact, lower the nutritional value of many legumes. In some cases the the remarkable capability of particles to stabilize foams and ANFs can also have more severe and toxic effects. For example emulsions (pickering emulsions) is well documented (18-19). the main anti-nutritional compounds of faba bean, vicine and convicine, are known to cause haemolytic anaemia disease For solid food applications such as bread, the addition of called favism for people who suffer from glucose-6-phosphate isolated or concentrated plant proteins in the formulation dehydrogenase deficiency (12), and lectins, found in most impacts on the rheological properties of the dough as well legumes, can cause gastrointestinal tract distress (13). Cereal as the characteristics of the final products. In bread making, and pulse proteins can be utilized as hybrid proteins to reach to the enrichment with isolated proteins (e.g. lupine, sesame, complete aminoacid (aa) profile as due to lack of essential aa, barley) increases water absorption of the dough and makes methionine in legumes and lysine in cereals. it less stable and less resistant to prolonged kneading (20-22).
58 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 As these proteins are not as elastic as gluten, they do not form 3. Fabian, C., Ju, Y. H. “A review on rice bran protein: its properties and a network and thus do not allow cell expansion, so the crumb extraction methods”, Critical reviews in food science and nutrition, appears more compact. Harder crumb is probably a result of 51(9), 816-827 (2011). the thickening of the crumb walls surrounding the air cells and 4. Ali, R., Shih, F.F, Riaz, MN “Processing and functionality of rice bran proteins and peptides”, Chapter 16 In Bioactive Proteins and Peptides strengthening of the crumb structure by the non-gluten-forming as Functional Foods and Nutraceuticals Ed by Mine, Y., Li-Chan, E. protein particles (22). However, different types of protein Jiang, B. (2010). isolates, depending on their origin, extraction methods and 5. Sibakov, J., Myllymäki, O., Holopainen, U., et al. ”Lipid removal pre-treatments, have different effects on the quality of baked enhances separation of oat grain cell wall material from starch and products (23). protein” Journal of Cereal Science, 54(1), 104-109 (2011). 6. Lasztity, R. The chemistry of cereal proteins. Boca Raton CRC Press, Boca Raton, FL, (1996). STRATEGIES FOR MODIFICATION OF FUNCTIONALITY 7. Perez-Maldonado R. A., Mannion P. F., Farrell D. J. “Optimum inclusion of field pies, faba beans, chick peas and sweet lupins in poultry diets. Degree of purity is important for the functionality of a protein I. Chemical composition and layer experiments. British Poultry Science ingredient. Protein-carbohydrate interactions at some 40(5):667-673 (1999). 8. Kinsella, John E. “Functional properties of soy proteins.” Journal of the conditions might even have a positive impact on improving the American Oil Chemists’ Society 56.3: 242-258 (1979). techno-functional properties. The separation of protein by dry 9. Sathe, S. K. “Dry bean protein functionality.” Critical Reviews in fractionation techniques depends largely on the interactions Biotechnology 22.2 : 175-223 (2002). of cell walls, protein and starch components in the plant cell 10. Duranti, M. , Consonni, A., Magni, C., et al. “ The major proteins of lupin matrix. Milling is usually needed to enhance the dissociation seed: Characterisation and molecular properties for use as functional between starch, protein and fibre rich particles, and it is and nutraceutical ingredients. Trends in Food Science and Technology important to selectively break up plant cell components without 19(12):624-633 (2008). causing additional damage to other compounds (24). The 11. Carbonaro, M. Role of pulses in nutraceuticals. In: Tiwari BK, Gowen yield and purity of protein-rich fractions during the separation A, McKenna B. Pulse Foods - Processing, Quality and Nutraceutical of protein from the other cell components depends strongly on Applications. London: Elsevier Inc. p 385-418. (2011) 12. Belitz, H. D., Grosch, W., Schieberle, P. Food Chemistry. 3 ed. Berlin: the milling operation and the milling quality. Springer. p. 1070 (2004) 13. Miyake, K., Tanaka, T., McNeil, P.L. Lectin-based food poisoning: The applicability of plant proteins in foods can also be improved A new mechanism of protein toxicity. Published online: Plos One. by modification of the protein itself. Potential food-compatible Available at: http://www.plosone.org/article/fetchObject.action?uri= technologies for improving the technological functionality of info%3Adoi%2F10.1371%2Fjournal.pone.0000687&representation=PDF proteins include complete or limited hydrolysis, protein cross- (2007) linking and protein functionalization by enzymatic means, 14. Tan, S. H., Mailer, R. J., Blanchard, C. L., et al. Canola proteins for human shear-induced processing, microfluidization, complexation consumption: extraction, profile, and functional properties Journal of with polysaccharides or with other protein (16, 25). Enzymatic Food Science, 76: R16–R28 (2011). hydrolysis improved the water and fat absorption ability, 15. Sosulski, F. Food uses of sunflower proteins JAOCS, 56 (3):438-442, (1979). solubility, emulsifying and foaming properties but not foam 16. Ercili-Cura, D., Miyamoto, A., Paananen, et al. Adsorption of oat proteins to air-water interface in relation to their colloidal state. Food stability of rice bran proteins (26). Hydrocolloids, 44:183-190 (2014). 17. De Folter, J. W., Van Ruijven, M. W., Velikov, K. P. Oil-in-water Pickering emulsions stabilized by colloidal particles from the water-insoluble FUTURE PROSPECTS protein zein. Soft Matter, 8(25), 6807-6815 (2012). 18. Dickinson, E. Food emulsions and foams: Stabilization by particles. As animal protein sources become limited, the use of more Current Opinion in Colloid & Interface Science, 15(1), 40-49 (2010). plant protein is inevitable. The offering of plant protein 19. Vignes-Adler, M., Weaire, D. New foams: Fresh challenges and ingredients will increase rapidly. Building on the existing protein opportunities. Current Opinion in Colloid & Interface Science, 13(3), 141- ingredients, there is vast activity to find new sources of plant 149 (2008) proteins by valorising side streams of current grain and seed 20. Alu’datt, M. H., Rababah, T., Ereifej, K., et al. Effects of barley flour and barley protein isolate on chemical, functional, nutritional and biological processes, but also through introduction of new protein crops properties of Pita bread. Food Hydrocolloids, 26(1), 135-143 (2012). with good nutritional and technological properties. Plant 21. El-Adawy, T. A. Effect of sesame seed protein supplementation on the proteins have several challenges, related to their limited nutritional, physical, chemical and sensory properties of wheat flour solubility and functionality, and delivery of essential amino bread. Food Chemistry, 59(1), 7-14 (1997). acids. The former can be overcome by modifications at 22. Paraskevopoulou, A., Provatidou, E., Tsotsiou, D., et al. Dough rheology different structural level, and much research is warranted here. and baking performance of wheat flour–lupin protein isolate blends. The latter can be tackled by establishing a variety of proteins Food Research International, 43(4), 1009-1016 (2010). sources to be combined in the diet. Little is as yet known about 23. Ziobro, R., Witczak, T., Juszczak, L., et al. Supplementation of gluten-free the digestibility of plant proteins, and how processing changes bread with non-gluten proteins. Effect on dough rheological properties it. Taste aspects are the major consumer concern, and flavour and bread characteristic. Food Hydrocolloids, 32(2), 213-220 (2013). and sensory design will be an important part of future work. 24. Tyler, Robert T. “Impact milling quality of grain legumes.” Journal of Food Science 49.3: 925-930 (1984) 25. Nordlund E., Katina K., Aura A., et al. Changes in bran structure by bioprocessing with enzymes and yeast modifies the in vitro digestibility REFERENCES and fermentability of bran protein and dietary fibre complex. Journal of 1. Safdar, M. N. “Composition of wheat grain”, Wheat milling news. (2005). Cereal Science, 58(1), 200-208 (2013). 2. Buri, R. C, Von Reding, W., Gavin, M. H. “Description and 26. Zhang, H. J., Zhang, H., Wang, L., et al. Preparation and functional characterization of wheat aleurone”, Cereal Foods World, 49 (5), properties of rice bran proteins from heat-stabilized defatted rice bran. 274–282 (2004). Food Research International, 47(2), 359-363 (2012).
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 59 FOOD ANALYSIS
BOŽANA ODŽAKOVIĆ1*, NATALIJA DŽINIĆ2, SLAVICA GRUJIĆ1, SNEŽANA KRAVIĆ2, MARIJA JOKANOVIĆ2 *Corresponding author 1. Faculty of Technology, University of Banja Luka, Stepe Stepanovića 73, 78000 Banja Luka, Bosnia and Herzegovina 2. Faculty of Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia
Božana Odžaković Sensory and physico-chemical evaluation of commercial coffees consumed in Banja Luka (Bosnia and Herzegovina) – Part 2*
*Part 1 - Agro FOOD Industry Hi Tech, 26(4), 52-55 (2015).
KEYWORDS: traditional black coffee quality, descriptive sensory analysis.
The objective of this study was to evaluate the quality and establish the sensory profile of five traditional Abstract coffee beverages prepared with commercial roasted ground coffee blends of known trademarks available on the market in Banja Luka (Bosnia and Herzegovina) in 2014. Descriptive sensory analyse was used to identify and evaluate properties that influence coffee beverage preference. Caffeine, fat and fatty acids content and CIEL*a*b* colour parameters of roasted ground coffee samples were analysed. Differences between analysed coffee beverages were determined in sensory properties colour, bitterness, acidity and sweetness and aroma attributes. Consumers preferred dark brown with orange- reddish hue coffee beverages, with moderately expressed coffee-like and less pronounced roasted, caramel-like and nutty aromas, and moderately pronounced bitterness, less pronounced acidity, and mild sweetness. Cereal and malty aromas, as less desirable were moderately expressed. Burnt, dusty and earthy aromas were undesirable and less pronounced in coffee beverage.
INTRODUCTION and to avoid the aging, caused by hydrolysis and oxidation of triacylglycerols (9). Among the most important fatty acids Roasted ground coffee has a long tradition of preparing for coffee freshness are oleic (C18:1n-9), linoleic (C18:2n-6) and consuming worldwide, as a warm beverage, due to its and linolenic (C18:3n-3) acids (10). Quality of roasted coffee characteristic sensory properties. Freshly brewed coffee has cannot be evaluated only by chemical analysis results (11), been associated with pleasure and enjoyment by stimulating because of the high importance of sensory properties of our senses, from the alluring smell of aroma volatiles to the the coffee beverage prepared of it. Aroma and taste of first sip inducing a well-balanced taste impression, centring coffee are determined by a complex mix of components, on bitterness and acidity (1-2). It is usually prepared using especially those formed during the beans roasting, and they varieties of Arabica and Robusta species, or their blends in have always been recognized as important attributes for their known trademarks commercially available on the market (3- sensory quality (12). The combined sensation of aroma, taste 5). The economic success of the food processing enterprises and mouth feel interact in flavour forming (13). The first sip in a competitive environment depends on their capability to of warm coffee should leave an impression of a well- offer food products that are tuned to the wishes of consumers, balanced taste centred on pleasant bitterness and to satisfy their expectations and so increase productivity and acidity that characterize this product (1). competitiveness (6). The quality and acceptability of coffee is The objective of this study was to evaluate the quality correlated with its sensory properties. The descriptive sensory and establish sensory profile of five traditional coffee analysis may be used for quality control and comparative beverages prepared from commercial roasted assessment of samples, to determine sensory properties, ground coffee blends of known trademarks, which impact on acceptance or rejection of the coffee available on the market in Banja Luka beverage. The quality of coffee beverage depends on (Bosnia and Herzegovina) in 2014. Descriptive the green and roasted coffee beans composition, which sensory analysis was used to evaluate are affected by conditions of drying, storage, roasting and selected sensory properties that influence grinding and method of the coffee beverage preparation coffee beverage preference. Caffeine and (7-8). Chemical composition, as caffeine, fat and fatty acids fat content, fatty acids composition and content in the coffee bean depends on the coffee variety CIEL*a*b* colour parameters of roasted ground (5). The integrity of the beans is important to keep coffee fresh coffee samples were analysed.
60 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 MATERIAL AND METHODS Each assessor is presented a specific subset of the 3 of 5 analysed samples in a randomized order, according to the Coffee samples balanced – incomplete block design (15, 20). Five commercial samples of roasted ground coffee from different producers randomly selected, purchased from local Chemical and physical attributes market in Banja Luka (BA) in 2014, were used for experiment. Caffeine was extracted from roasted ground coffee with Each group of samples (5x200g coffee for each sample) was dichloromethane. The absorbance of the extract was from the same production batch. The compared samples determined by UV/Vis spectrophotometer (PerkinElmer had approximately uniform shelf life, and were properly Lambda 25) at the 274.7 nm wavelength (22). Fat content packed and labelled. All coffee samples were blends of was determined by the Soxhlet method (23). To determine Arabica and Robusta. the fatty acids content in coffee fat, samples were derivatized according to the methodology of Kravić et al. (24). The Descriptive sensory analysis analyses of fatty acid methyl esters were performed on a Descriptive sensory analysis of coffee beverage samples Hewlett-Packard (HP) 5890 gas chromatograph coupled were conducted in the Laboratory for sensory analysis of with a HP 5971A mass spectrometer detector. The results foods, designed according to the ISO 8589:2007 (14). The of all analyses were expressed as the mean value of three coffee samples were ranked in previous research (2), measurements. based on overall quality preference from 1st place the Colour parameters (CIEL*a*b*), as indicators of lightness, sample with most, to 5th place, the sample with the least redness and yellowness were determined using a tristimulus pleasant quality (15). The panel for the coffee beverages colorimeter (Minolta CP410) with standard illumination D65, descriptive analysis was formed by 32 selected and trained and colorimetric normal observer angle of 2°. The results assessors, and they worked individually in the booths (16-19). were expressed as the mean value of five measurements. Each assessor participated in two sessions, and 3 samples per session were presented, according to the balanced Statistical analysis – incomplete – 3 of 5 block design (15, 20). Each sample The data obtained by descriptive sensory analysis were was analysed 21 times. Bottled drinking water and neutral statistically analysed using one-way analysis of variance crackers were used for the panellist’s receptors regeneration. (ANOVA) and t-test was performed to determine if List of descriptors (attribute with verbal description) for statistically significant difference between the mean values each selected sensory property and scales were previously of analysed parameters at the p<0.05 confidence level prepared for the study and used for establishing coffee exist. Descriptive Statistical Analysis of all data for caffeine, beverages sensory profile (17-19). Colour was descriptively fat, fatty acids content and colour parameters of coffee described. Aroma, bitterness, acidity and sweetness were samples were realised by Microsoft Office Excel 2007 and analysed by sipping the coffee from the cup and evaluating presented as mean values ± standard deviations. descriptors intensity on the 5 point interval scale. Values for expressed intensity were defined on scale from 5 to 1 (5=intensively; 4=markedly; 3=moderately; 2=less; 1=mildly) RESULTS AND DISCUSSION with the possibility of using semi-points. Colour of coffee depends on the degree of roasting. Preparation and presentation of coffee samples Comparing visually assessed brown colour and hue of the Coffee beverage samples were prepared according to the evaluated coffee beverage samples, it could be concluded ISO 6668:2008 (21). The water was heated to boiling point that the basic colour was dark brown. Orange shade was (90°C), poured into the special coffee pot containing coffee identified in all coffee samples with variation in intensity and heated to boiling, making coffee beverage with rich and proportion of yellowish and reddish hue. The colour of foam. The beverage was removed from the hotplate, left the samples G and D was dark brown with orange and discreet coffee to precipitate partially in coffee pot (3-5 minutes) and yellow hue. The samples B and P were dark brown with an poured into a thermos bottle (pre-heated with hot water). orange hue and with slight reddish tones. Sample F was dark Each sample (~50 ml/cup) was served warm brown with a striking orange shade. (~55°C) in white arkopal coffee cups (130 ml volume) pre-heated to 50°C. Samples The sample B was the best evaluated in preference test were prepared shortly (2). Results of descriptive analysis showed that sample B before the sensory had the highest intensity of the coffee-like aroma (Table analysis and 1), as one of the most appreciated in coffee beverages labelled with a (25). Statistically significant differences (p<0.05) were 3-digit code. found between sample B and other coffee beverages. Caramel-like aroma was less pronounced and identified only in sample B, and it had less pronounced nutty aroma, significantly different (p<0.05) comparing with other samples. Roasted aroma was less pronounced in sample B similar as in samples F, P and D. Cereal and malty aromas were moderately expressed, and less burnt, dusty and earthy. Sample F was on the second and sample G was on the third place in preference test. Pleasant aroma of roasted ground coffee and cereal and malty aromas were characteristic attributes of sample F. Less desirable burnt and dusty aromas
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 61 were moderately expressed and coffee-like and nutty less. Grassy aroma was identified only in sample F. Cereal and malty aromas were the most expressed in sample G, but burnt aroma was moderately expressed and significantly different (p<0.05) of other samples. Dusty aroma, associated with aroma of toasted coffee was moderately expressed. Less pronounced were earthy and pleasant roasted and nutty aromas. Samples D and P had the least preferred aroma. The dusty aroma was expressed in all samples, but the highest was in sample D. Pleasant nutty aroma was moderately expressed and dominated in the sample P comparing to the other samples. Undesirable drugs-like aroma were identified only in samples P and D (Table 1). Earthy and drugs-like aromas had the highest intensity in sample P coffee beverage, impacting on its lowest preference (2).
The sensory analysis of bitterness, acidity and sweetness in traditional coffee beverages Table 1. Results of descriptive sensory analysis of coffee beverage prepared from five showed different intensity. Bitterness of coffee commercial samples of roasted ground coffees from local market in Banja Luka (BA) in 2014. beverage is highly related to the roasting degree and arises from many compounds, partly from caffeine (8). Bitterness intensity was moderately expressed in samples B, P and F, and they were significantly different (p<0.05) of sample G. Acidity of the coffee samples was characterized as moderately expressed in samples G, D and P, and with slightly lower intensity in samples F and B. The relatively high acidity was in samples G, P and D. A mild note of sweetness was identified in all samples (Table 1). Descriptive analysis crossed with preference test showed that coffee beverages samples B and F, with the moderately expressed bitterness, less pronounced acidity and mild sweetness were the favourite. Sample G was on the third place, with moderately expressed acidity, less bitter and with the Table 2. Caffeine, fat, fatty acids content and colour parameters (CIEL*a*b*) for five highest sweetness. Sample P had more commercial samples of roasted ground coffees from local market in Banja Luka (BA) in 2014. expressed bitterness than sample D, and both had moderately expressed acidity, mild sweetness (Table 1) and the lowest level of overall quality are able to solve that problem, and descriptive sensory analysis impression (2). could evaluate the product properties and identify attributes related to the preferences. Producers should incorporate the Caffeine, fats and fatty acids content in roasted ground coffee data in the specification as important quality parameters for depends on the used varieties and their relation in coffee blends roasted coffee production and quality control, and offer in the (5). The main fatty acids of six analysed were linoleic and palmitic market roasted ground coffe which has quality harmonized with acids (Table 2). Similar results of analyses for linoleic and palmitic the expectations of consumers on the target market. acids reported Vila et al. (26). Other fatty acids content, identified in coffee samples, had lower average values. Similar results were presented in literature (4, 26-27) for content of fatty acids in CONCLUSION roasted Arabica and Robusta coffee. CIEL*a*b* colour parameters for roasted ground coffee samples were measured to Established sensory profile for analysed coffee beverage characterize it (Table 2). Measured values for L* (lightness) and b* identify expected and preferred quality characteristic on (yellowness) were the highest on sample P, the highest values for target market. Consumers prefer dark brown, with orange- a* (redness) had, as follows: samples D, F, B, G and F. Identified reddish hue coffee beverages with moderately expressed difference in a* values could be related to the raw coffee colour, coffee-like and less pronounced roasted, caramel-like and which depends on its variety and processing (28-29). nutty aromas. Producers should identify which coffee products consumers like They like moderately expressed bitterness, less pronounced and dislike, and why. Quality and preference analyses techniques acidity and mild sweetness in coffee beverages. Cereal and
62 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 malty aromas, as less desirable and grassy, earthy, dusty, determination by UV/HPLC. Journal of Food Composition and burnt and drugs-like aromas were undesirable. The coffee Analysis, 20, 440–448 (2007). companies could use results of the study for standardisation 13. Burseg M. M. K, De Hoog E., Huppertz T. Flavour optimization of of ground roasted coffee quality and fulfill consumers’ beverages. AgroFOOD Industry Hi Tech., 23(4), 5-7 (2012). 14. ISO 8589:2007 (E). Sensory analysis – General guidance for the design expectations on target market. of test rooms. 15. ISO 8587:2006 (E). Sensory analysis – Methodology – Ranking. 16. ISO 8586-1:1993 (E). Sensory analysis – general guidance for the REFERENCES selection, training and monitoring of assessors – part 1: selected assessors 1. Kreppenhofer S., Frank O., Hofmann T. Identification of (furan-2-yl) 17. ISO 13299:2003 (E). Sensory analysis - Methodology - General methylated benzene diols and triols as a novel class of bitter guidance for establishing a sensory profile. compounds in roasted coffee. Food Chemistry, 126, 441-449 (2010). 18. ISO 11035:1994 (E). Sensory analysis - Identification and selection of ž ć ž ć ć ć ć 2. Od akovi B., D ini N., Gruji S., Kravi S., Jokanovi M. Sensory and descriptors for establishing a sensory profile by a multidimensional physico-chemical evaluation of commercial coffees consumed in approach Banja Luka (Bosnia and Herzegovina) – Part1. Agro FOOD Industry Hi 19. ISO 11037:2011 (E) Sensory analysis - General guidance and test Tech, 26(4), 52-55 (2015). method for assessment of the colour of foods 3. Fujioka K. and Shibamoto T. Chlorogenic acid and caffeine contents 20. Stone H. and Sidel J. Sensory Evaluation Practices. 3rd Edition, Elsevier, in various commercial brewed coffees. Food Chemistry, 106, 217–221 Printed in Italy (2004). (2008). 21. ISO 6668:2008 (E). Green coffee – preparation of samples for use in 4. de Souza R. M. N. and Benassi M. T. Discrimination of Commercial sensory analysis. Roasted and Ground Coffees According to Chemical Composition. J. 22. Belay A., Ture K., Redi M., et al.: Measurement of caffeine in coffee Braz. Chem. Soc., 23, 1347-1354 (2012). beans with UV/vis spectrometer. Food Chemistry, 108, 310–315 (2008). 5. Martin M. J., Pablos F., A. Gonzalez G., et al. Fatty acid profiles as 23. Ismail I., Anuar M. S., Shamsudin R. Effect on the physico-chemical discriminant parameters for coffee varieties differentiation. Talanta, 54, properties of liberica green coffee beans under ambient storage. 291–297 (2001). International Food Research Journal, 20(1), 255-264 (2013). ć ć 6. Gruji S. and Gruji R. Food product development as opportunity for 24. Kravić S., Marjanović N., Suturović Z., et al. Determination of trans fatty success or survival in the market. 6th Central European Congress on acid content of Serbian shortening by gas chromatography-mass Food. CEFood. Novi Sad, Serbia, 1202-1206 (2012). spectrometry. Acta Alimentaria, 39, 413-423 (2010). 7. Oliveira S. D., Franca A. S., Gloria M. et al. The effect of roasting on the 25. Perez-Martines M., Sopelana P., Paz de Pena M., et al. Application of presence of bioactive amines in coffees of different qualities. Food multivariate analysis to the effects of additives on chemical and Chemistry, 90, 287–291 (2005). sensory quality of stored coffee brew. J. Agric. Food Chem. 56, 11845- 8. Ribeiro J.S., Ferreira M.M.C., Salva T.J.G. Chemometric models for the 11853 (2008). quantitative descriptive sensory analysis of Arabica coffee beverages 26. Vila M. A., Andueza S., Paz de Pena M., et al. Fatty acid evolution using near infrared spectroscopy. Talanta, 83, 1352-1358 (2010). during the storage of ground, roasted coffees. JAOCS, 82(9), 639-646 9. Toci A. T., Neto V. J. F. M., Torres A. G., et al. Triacylglicerols changes (2005). during the storage of roasted coffee. Proc. 22nd Int. Conf. Coffee Sci. 27. Hurtado A.B. and Dorado D. A. Study of the yield and fatty acid profile ASIC. Campinas, SP,Brazil, 504–507 (2008). of coffee (Coffea arabica) oil from roasted beans obtained with 10. Toci A. T., Neto V.J.M.F., Torres A. G., et al. Changes in triacylglycerols supercritical carbon dioxide. III Iberoamerican Conference on and free fatty acids composition during storage of roasted coffee. Supercritical Fluids Cartagena de Indias (Colombia), 1-10 (2013). LWT - Food Science and Technology, 50, 581-590 (2013). 28. Mendes Luciane C., Hilary C. de Menezes, et al: Optimization of the 11. Alvarado R. A. and Linnemann A. R. The predictive value of a small roasting of robusta coffee (C. canephora conillon) using acceptability consumer panel for coffee-cupper judgment. British Food Journal, 112 tests and RSM. Food Quality and Preference 12 (2001) 153-162. (9), 1023-1032 (2010). 29. Souto U. T. C. P., Pontes M. J.C., Silva E. C., et al.: UV–Vis spectrometric 12. Rodrigues C. I., Marta L., Maia R., et al. Application of solid-phase classification of coffees by SPA–LDA. Food Chemistry 119 (2010) 368– extraction to brewed coffee caffeine and organic acid 371.
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 63 FOOD ANALYSIS
ANA ALIMPIĆ1*, DEJAN PLJEVLJAKUŠIĆ2, KATARINA ŠAVIKIN2, VLADO MATEVSKI3,4, PETAR D. MARIN1, SONJA DULETIĆ-LAUŠEVIĆ1 *Corresponding author 1. Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology, University of Belgrade, Takovska 43, Belgrade, Serbia, 2. Institute for Medicinal Plants Research “Dr. Josif Pancić”, Tadeuša Košćuška 1, 11000 Belgrade, Serbia, 3. Institute of Biology, Faculty of Natural sciences and mathematics, University "Ss. Cyril and Methodius", 1000 Skopje, Macedonia 4. Macedonian Academy of Sciences and Arts, 1000 Skopje, Macedonia Ana Alimpić Salvia jurisicii Košanin and Salvia amplexicaulis Lam. Chemical composition of the essential oils
KEYWORDS: Salvia, essential oil, GC-FID, GC-MS, sesquiterpenes, spathulenol.
Numerous Salvia species were proven to be biologically active due to their chemical composition, and Abstract also promising in searching for new medicinal, cosmetics, or food ingredients of natural origin. The chemical composition of the essential oils of endemic species Salvia jurisicii as well as of S. amplexicaulis growing wild in Macedonia was analyzed using GC-FID and GC-MS. The main class of compounds in S. jurisicii oil were sesquiterpenes, consisting of spathulenol (12.3%), ß-bourbonene (7.0%), n-nonanal (5.1%), γ-muurolene (4.8%) and thujopsan-2-α-ol (4.5%) as dominant components. Sesquiterpenes were also the most abundant class in S. amplexicaulis oil, with caryophyllene oxide (11.3%), germacrene D (7.8%), thujopsan-2-α-ol (7.0%), germacra-4(15),5,10(14)-trien-1-α-ol (5.4%), α-cadinol (5.3%) and spathulenol (5.1%) as the most abundant components.
INTRODUCTION Gram-positive bacteria was noticed comparing to Gram- negative bacteria (5). Essential oils have been widely used since the middle ages for The aim of this study was to characterize chemical bactericidal, antiviral, fungicidal, antiparasitic, insecticidal, composition of the essential oils of Salvia jurisicii, endemic medicinal and cosmetic applications, especially nowadays in species of great horticultural interest, which to the best of sanitary, pharmaceutical, cosmetic, food and agricultural our knowledge has not yet been investigated previously, industries (1,2). It contains a variety of volatile molecules such and Salvia amplexicaulis, which has not been investigated as terpenes and terpenoids, phenol-derived aromatic and from its natural localities in Macedonia. This study has not aliphatic components (2). Pharmacological investigations been performed with the ambition to characterize showed its antioxidant, antibacterial, antifungal, antiviral, population chemical properties, but to anchor early stage cytotoxic, antiinflammatory and tumorigenesis-preventing chemical data on two scarcely observed Salvia species. and other activities (2-5) as well as ecological significance such as pest-toxic and repellent activity (3). Salvia jurisicii Košanin (Balkan sage, Jurisic’s cutleaf sage) is herbaceous perennial endemic to the southern regions of MATERIAL AND METHODS the former Yugoslavia (Macedonia) (6). Our previous study of S. jurisicii herb comprised antioxidant activity, total Plant material phenolic and flavonoid content of different extracts (7). To Aerial parts of Salvia jurisicii Košanin were collected in natural the best of our knowledge, data on chemical composition habitat, near city of Štip, while S. amplexicaulis plant material or biological effects of its essential oil, have not been was collected in locality Pletvar (Macedonia). Both species reported previously. were collected during period of full flowering stage, in July Salvia amplexicaulis Lam. (stem clasping violet sage) is 2012. Due to the lack of plant material, and protection legacies herbaceous perennial that is native to southeastern Europe on these species, plant material was collected in bulk from (6). Our previous investigations showed strong antioxidant about 20-50 plants (approximately 500 g, depending of activity of S. amplexicaulis extracts correlated to high species) per site, thus creating a unique sample per species for amount of phenolics and flavonoids (8). It has been reported further analysis. Plant material was air-dried and kept in shadow on high amounts of sesquiterpenes in essential oil of S. at room temperature for further processing. Voucher amplexicaulis, collected from mountainous region of south- specimens were deposited in the Herbarium of the Institute of east Serbia (5,9). Also, antimicrobial activity of the essential Botany and Botanical Garden “Jevremovac”, Faculty of oil of S. amplexicaulis growing in Serbia was reported, and Biology, University of Belgrade (BEOU; voucher No. 16674 and better inhibition of the growth of Candida albicans and BEOU; voucher No. 16673, respectively).
64 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 Essential oil isolation Air-dried aerial parts (100g) of Salvia jurisicii and S. amplexicaulis, randomly taken from whole collected sample of each species, were grounded. Volatile oils were obtained by hydrodistillation using a Clevenger type apparatus during 3h (10).
Essential oil analysis Qualitative and quantitative analysis was carried out using GC–FID and GC–MS. In the first instance model HP-5890 Series II gas chromatograph equipped with a split-splitless injector, HP-5 capillary column (25 m × 0.32 mm, film thickness 0.52 μm) and a flame ionization detector (FID), was employed. Hydrogen was used as carrier gas (1 ml min-1). The injector was heated at 250 °C, the detector at 300 °C, while the column temperature was linearly programmed from 40 to 260°C (4° C min-1). GC–MS analyses was carried out under almost the same analytical conditions, using HP G 1800C Series II GCD analytical system, equipped with HP-5MS column (30 m × 0.25 mm × 0.25 μm). Helium was used as carrier gas. The transfer line (MSD) was heated at 260 °C. The EI mass spectra (70 eV) were acquired in the scan mode in the m/z range 40–400. In each case 1 μl of sample solution in ethanol (10 μl ml-1) was injected in split mode (1:30). The identification of constituents was performed by matching their mass spectra and retention indices with those obtained from authentic samples and/or NIST/Wiley spectra libraries, using different types of search (PBM/NIST/AMDIS) and available literature data (11,12). The percentage compositions were obtained from electronic integration measurements using flame ionization detection (FID; 250 °C).
RESULTS AND DISCUSSION
The aerial parts of S. jurisicii yielded less than 0.01% of the essential oil (0.01 g EO/g of dry plant material), which chemical composition is presented in Table 1. Seventy-three compounds were identified representing 95.5% of the oil. Oxygenated sesquiterpenes (51.2%) and sesquiterpene hydrocarbons (23.6%) were dominant classes of the terpenes. Monoterpene hydrocarbons were present in lesser amount (4.0%) as well as oxygenated monoterpenes (3.6%), while oxygenated diterpenes were found in small percent (1.9%). Among hydrocarbons, aliphatic fraction was more abundant (6.5%) comparing to aromatic (4.6%). Spathulenol (12.3%), ß-bourbonene (7.0%), n-nonanal (5.1%), γ-muurolene (4.8%) and thujopsan-2-α-ol (4.5%) were characterized as main components.
The aerial parts of S. amplexicaulis also yielded less than 0.01% of the essential oil (0.01 g EO/g of dry plant material). Chemical composition of the essential oil is presented in Table 2. Fifty-two compounds, representing 97.5% of the total oil, were identified. Oxygenated sesquiterpenes and
a) Retention index relative to n-alkanes on HP-5 capillary column. * tentative identification n.i., not identified
Table 1. Chemical composition of Salvia jurisicii essential oil.
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 65 sesquiterpene hydrocarbons were CONCLUSIONS the most abundant classes of the terpenes respesented with 56.5% and The study showed that oxygenated 34.6%, respectively. sesquiterpenes were the most Monoterpene hydrocarbons were abundant components in the present only in oxygenated form essential oils of endemic S. jurisicii and (5.6%). The major constituents of this oil S. amplexicaulis aerial parts collected were caryophyllene oxide (11.3%), from natural habitats in Macedonia. germacrene D (7.8%), thujopsan-2-α-ol The chemical compositions of the (7.0%), germacra-4(15),5,10(14)-trien- investigated oils were qualitatively 1-α-ol (5.4%), α-cadinol (5.3%) and different regardless that samples were spathulenol (5.1%). taken in close collection sites and during the same season. Therefore, it Obtained results showed that oils of could be concluded that the both studied species were rich in differences are attributed to the oxygenated sesquiterpenes (more chemotaxonomic identity of species than 50% of total oil). Sesquiterpenes rather than to the ecological were also previously recognized as conditions. predominant class in Salvia oils with different main compounds, such as viridiflorol in S. albicaulis (4), ACKNOWLEDGEMENTS β-caryophyllene, germacrene B, caryophyllene oxide, cis-β-farnesene, Authors are grateful to the Ministry of germacrene D in S. nemorosa and Education, Science and (E)-β-ocimene, α-gurjunene, Technological Development of Serbia germacrene D in S. reuterana (13), for financial support (Projects No. viridoflorol, manool and α-humulene 173029 and 46013). in S. argentea (14), spathulenol in S. reflexa and caryophyllene oxide in S. nemorosa and S. glutinosa (15), germacrene, ß-caryophyllene, spathulenol and α-humulene in S. chionantha (16), viridoflorol, manool, α-thujone and α-humulene in S. argentea (17). Our findings are in accordance with previous studies of S. amplexicaulis collected from two different locations in Serbia, where essential oil was characterized by high quantity of sequiterpenes, with germacrene D, viridiflorol, caryophyllene oxide and a) Retention index relative to n-alkanes on HP-5 REFERENCES ß-caryophyllene as dominant capillary column. n.i., not identified components (5,9). Sesquiterpene 1. Burt, S. “Essential oils: their alcohol spathulenol was dominant Table 2. Chemical composition of Salvia amplexicaulis antibacterial properties and potential component of S. jurisicii oil (12.3%) essential oil. applications in foods - a review”, Int. J. and one of dominant components of Food Microbiol., 94, 223-253 (2004). S. amplexicaulis oil (5.1%). Several studies reported that 2. Bakkali, F., Averbeck, S., Averbeck, D., et al. ”Biological effects of essential oils - a review”, Food Chem. Toxicol., 46 (2), 446-475 spathulenol was very potent spasmolyitic agent (18), (2008). powerful against multidrug resistance cancer cells (19) and 3. Barićević, D., Bartol, T. The biological/pharmacological activity of very strong immunomodulatory compound through inhibition the Salvia genus, in Sage - the genus Salvia, Edited by Kintzios, of lymphocytes proliferation and inducing their apoptosis S.E., Ed. Harwood Academic Publishers, Amsterdam, Netherlands (20). The most abundant component of S. amplexicaulis oil (2000). (11.34%), caryphyllene oxide, was not present in S. jurisicii oil. 4. Kamatou, G.P.P., Viljoen, A.M., Figueiredo, A.C., et al. “Trichomes, This compound was recently reported as strong analgesic essential oil composition and biological activities of Salvia and anti-inflammatory as well as cytotoxic agent (21,22). albicaulis Benth. and S. dolomitica Codd, two species from the Germacrene D, identified only in S. amplexicaulis oil (7.79%), Cape region of South Africa”, S. Afr. J. Bot., 73, 102-108 (2007). was previously described (23) to be very important odor 5. Petrović, S., Pavlović, M., Tzakou, O., et al. “Composition and antimicrobial activity of Salvia amplexicaulis Lam. essential oil”, J. agent in plant-insect interactions, while β-bourbonene Essent. Oil Res., 21, 33-36 (2009). showed antiectoparasitic activity (24). Moreover, in the oil 6. Hedge, I.C. Salvia L., in Flora Europaea Vol. 3, Edited by Tutin, T.G., samples investigated in this study, compounds such as Heywood, V.H., Burges, N.A., Valentine, D.H., Walters, S.M., Webb, thujone and camphor were not present although such D.A., Eds. Cambridge University Press, Cambridge, (1972). compounds that could provoke convulsions in central 7. Alimpić, A., Duletić-Laušević, S., Matevski, V., et al. “Antioxidant nervous system are reported for some Salvia species (25). activity of Salvia jurisicii Košanin ethanol extracts”, Botanica
66 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 Serbica, 38 (2) (2014) (in press) Toxicol., 48, 3189-3193 (2010). 8. Alimpić, A., Oaldje, M., Matevski, V., et al. “Antioxidant activity 17. Ben Farhat, M., Landoulsi, A., Chaouch-Hamada, R., et al. and total phenolic and flavonoid contents of Salvia amplexicaulis ”Profiling of essential oils and polyphenolics of Salvia argentea Lam. extracts”, Arch. Biol. Sci., 66 (1), 307-316 (2014). and evaluation of its by-products antioxidant activity”, Ind. Crop. 9. Velićković, D., Ristić, M., Milosavljević, M., et al. “Chemical Prod., 47, 106-112 (2013). composition of the essential oils of Salvia austriaca Jacq. and 18. Perez-Hernandez, N., Ponce-Monter, H., Medina, J.A., et al. Salvia amplexicaulis Lam. from Serbia”, Agro Food Ind. Hi Tec., 23, ”Spasmolytic effect of constituents from Lepechinia caulescens 8-10 (2012). on rat uterus”, J. Ethnopharmacol., 115, 30-35 (2008). 10. Yugoslavian Pharmacopoeia, Pharmacopoea Jugoslavica editio 19. Martins, A., Hajdú, Z., Vasas, A., et al. “Spathulenol inhibit the quarta. Ph. Jug. IV. National institute for Health Protection, human ABCB1 efflux pump”, Planta Med., 76, 608 (2010). Belgrade, Serbia (1984). 20. Ziaei, A., Schneider, B., Ramezani, M., et al. “Identification of 11. Hochmuth, D.H. MassFinder 3: Software for GC/MS interpretation spathulenol in Salvia mirzayanii and the immunomodulatory and presentation, mass spectral library administration, Hamburg, effects”, Phytother. Res., 25, 557-562 (2011). Germany, (2006). 21. Chavan, M.J., Wakte, P.S., Shinde, D.B. “Analgesic and anti- 12. Adams, R.P. Identification of essential oil components by gas inflammatory activity of caryophyllene oxide from Annona chromatography/ quadrupole mass spectroscopy. Allured squamosa L. bark”, Phytomedicine, 17, 149-151 (2010).. publishing Co., Carol Stream, Illinois, USA (2001). 22. Jun, N.J., Mosaddik, A., Moon, J.Y., et al. “Cytotoxic activity of 13. Mirza, M., Sefidkon, F. “Essential oil composition of two Salvia β-caryophyllene oxide isolated from Jeju Guava (Psidium species from Iran, Salvia nemorosa L. and Salvia reuterana Boiss”, cattleianum Sabine) leaf”, Rec. Nat. Prod., 5, 242-246 (2011). Flavour Fragr. J., 14, 230-232 (1999). 23. Rostelien, T., Borg-Karlson, A.-K. Fäldt, J., et al. ”The plant 14. Couladis, M., Tzakou, O., Stojanović, D., et al., “The essential oil sesquiterpene germacrene D specifically activates a major type composition of Salvia argentea L.”, Flavour Fragr. J., 16, 227-229 of antennal receptor neuron of the tobacco budworm moth (2001). Heliothis virescens”, Chem. Senses., 25, 141-148 (2000). 15. Malenćić, Đ., Couladis, M., Mimica-Dukić, N., et al. “Essential oils of 24. Birkett, M.A., Abassi, S.A., Kröber, T., et al. ”Antiectoparasitic three Salvia species from the Pannonian part of Serbia”, Flavour activity of the gum resin, gum haggar from the East African plant, Fragr. J., 19, 225-228 (2004). Commiphora holtziana”, Phytochemistry, 69, 1710-1715 (2008). 16. Tel, G., Öztürk, M., Emin Duru, M., et al. ”Chemical composition of 25. Millet, Y., Jouglard, J., Steinmetz, M.D., et al. “Toxicity of some the essential oil and hexane extract of Salvia chionantha and essential plant oils. Clinical and experimental study”, Clin. Toxicol., their antioxidant and anticholinesterase activities”, Food Chem. 18 (12), 1485-1498 (1981). ANTIOXIDANTS
NILGÜN HAVVA BUDAK Department of Food Processing, Egirdir Vocational School, Süleyman Demirel University, Isparta, Turkey
Nilgün Havva Budak Antioxidant activity and phenolic contents Pomegranate vinegar
KEYWORDS: Pomegranate vinegar, ORAC, TEAC, antioxidant, phenolic substances, ellagic acid.
Pomegranate is well known for its attributed health beneficial properties. The aim of this study was to Abstract determine the effects of ethanol and acetic acid fermentations on total antioxidant activities and phenolic substances of pomegranate juice. Juices of two types of pomegranates and their 1:1 mixture with pomegranate pomace (25%) were used to make pomegranate wine and vinegar. Total solids, pH, total soluble solids, total sugar, total phenolic substances, ORAC and TEAC assays, and phenolic substances analyses were carried out. Total phenolic content of Hicaznar and Beynar pomegranate juices were 2471.77 mg/L GAE and 1765.11 mg/L GAE, respectively. ORAC values of pomegranate vinegar samples were between 15.93-28.58 µmol/mL. Concentration of ellagic acid was the highest in pomegranate juice in all samples.
INTRODUCTION manufacturing vinegar can be divided into two kinds; slow or traditional methods in which the culture of acetic acid Plant-based foods have a wide range of bioactive bacteria is placed on the surface of a wood barrel, and components that can provide significant beneficial health quick or industrial processes involving submerged culture effects when consumed by humans. Fruits are rich in bioactive where the oxygenation should be increased so that the phenolic compounds such as flavonoids, phenolic acids, process is achieved at faster rates of acetification (13). stilbenes, coumarins, and tannins (1). It was reported that a Previous studies have determined that wines and wine diet rich in flavonoids is associated with a reduction in the risk vinegars have significant antioxidant activity (8, 14-15). It was of hypertension, coronary heart disease and certain types of reported that pomegranate juice and pomegranate wine cancer (2). Pomegranate (Punica granatum) has high had significant protective effects toward low-density antioxidant activity due mainly to its inherent anthocyanins such lipoprotein oxidation (16). as delphinidin, cyanidine and pelargonidine, and ellagitannins such as ellagic acid, punicalagin and punicaline (3,4). In the Punica granatum L.cv. Hicaznar is a variety of pomegranate last decade, health benefits of pomegranate such as with a reddish peel and reddish arids and is the major reducing the risk of cardiovascular diseases and certain pomegranate cultivar grown in Turkey; Punica granatum L.cv. types of cancer, improving the insulin sensitivity and Beynar variety has a pale red-whitish color arils and is another preventing arthritis were reported (4,5). Lansky and Newman economically important cultivar in Turkey. In terms of flavor, (4) stated that there are over 1000 cultivars of Punica Punica granatum L.cv. Hicaznar is more sour than Punica granatum, mainly grown in the Mediterranean region, China, granatum L.cv. Beynar. India and California (USA). The aim of this study was to determine the changes in total antioxidant properties and phenolic substances as Fruit juices, wines and vinegars are good dietary sources of pomegranate juice is converted into pomegranate wine and antioxidants (6,7). Vinegar has positive effects on health (8). pomegranate vinegar. Vinegar has effect which reduction of blood pressure (9), blood glucose response, and serum insulin (10). In addition, a potential antitumor effect (11), affirmative effects on blood MATERIAL AND METHODS lipid levels, liver functions and steatosis, and body weight increase (8) of apple cider vinegars has been expressed. Ethanol and acetic acid fermentations of pomegranate juice Vinegar is obtained from agricultural raw materials Punica granatum L.cv. Hicaznar and Punica granatum L.cv. containing starch or sugars by a double-fermentation Beynar were harvested from Korkuteli, Antalya, Turkey. process; the first one is ethanol fermentation and the second Pomegranates were immediately transported to the is acetic acid fermentation (12). In general, methods of Department of Food Engineering, Suleyman Demirel
68 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 University, Isparta, Turkey. Sample codes and sample descriptions were given in Table 1. The flow scheme of the process used to collect pomegranate juice is Data expressed as presented in Figure 1. mean ± standard error
Proximate analysis a,b Values with the Collected pomegranate same lower-case juice was examined as letters in the same column do not differ follows; pH was measured significantly using a pH meter (WTW, (P>0.05) Inolab, USA), total solids (%) was determined gravimetrically, and total Table 1. Sample soluble solid (°Brix) content of descriptions and the samples was measured chemical analysis of using Abbe refractometer pomegranate juice, wine and vinegar (Bellingham Stanley Limit samples. 60/70 Refractometer, England) (17). Total sugar was determined according to the Luff-Schrool method in the pomegranate juice samples (17).
Total phenolic content Total phenolic contents of the pomegranate juice, wine and vinegar samples were determined spectrophotometrically according to the Folin- Ciocelteau method (18). The absorbance was determined at 760 nm using a spectrophotometer (Shimadzu Scientific Instruments, Inc., Tokyo, Japan).
Oxygen Radical Absorbance Capacity (ORAC) assay ORAC assay was carried out according to the hydrophilic ORAC-Florescein method (6). The method kinetically measures the total antioxidant capacity of the samples using 2,2’-Azobis (2-amidinopropane) dihydrochloride (AAPH) damage to fluorescent compound. The Trolox® was used as a standard and ORAC values were kinetically calculated according to Cao and Prior (19 ) using KC4™ Data Reduction Software (BioTek Instruments, Winooski, VT).
2,2’-azinobis (3-ethlybenzthiazoline)-6-sulfonic acid (ABTS) assay The 2,2’-azinobis (3-ethlybenzthiazolin-6-sulfonic acid) diammonium salt radical (ABTS) inhibition against Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was spectrophotometrically measured to determine Trolox Equivalent Antioxidant Capacity (20).
Quantification of phenolics Gallic acid, catechin, epicatechin, chlorogenic acid, syringic acid and elagic acid in pomegranate juice, pomegranate wine and pomegranate vinegar samples were quantified by High Performance Liquid Chromatography (Shimadzu, Kyoto, Japan) equipped with an autosampler (SIL–10AD vp), a reversed-phase column (Agilent Eclipse XDB-C18, 250x4,60 mm i.d., 5μm) Figure 1. Flow scheme of ethanol and acetic acid fermentations of and a diode array detector (lmax=278) (HPLC-DAD). pomegranate juice. The data were integrated and analyzed using the
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 69 Shimadzu Class-VP Chromatography Laboratory Automated Software system. Standard and sample chromatograms are presented in Figure 2 and 3.
Statistical analysis Differences among experimental data were analysed by ANOVA statistical program. Differences among experimental data were analysed by using SPSS for Windows (version 17.0, SPSS Inc.). Vinegar production was repeated two times. The significance was established at P < 0.05.
RESULTS AND DISCUSSION
The pH values, total solids (TS) and total soluble solids (TSS) of pomegranate juice, wine and vinegar samples, and total sugar content of Figure 2. A chromatogram of standard 1: Gallic acid 2: catechin 3: chlorogenic pomegranate samples are presented in Table 1. acid 4: caffeic acid 5: epicatechin 6: syringic 7: p-coumaric acid 8: ferulic acid The pH values of pomegranate juice obtained from 9: ellagic acid 10: hesperidin 11: apigenin-7-glucoside 12: rosmarinic acid 13: Hicaznar and Beynar varieties of fruit were 3.14 and eriodictyol 14: quercetin 15: naringenin 16: luteolin 17: apigenin 18: acacetin. 3.27, respectively (P>0.05). The pH values of wine samples made from Hicaznar, Beynar, and their mixture with pulp were similar (P>0.05). Vinegar samples had the lowest pH values ranging from 2.87 to 2.96 due to high content of acetic acid; the effect of acetic acid fermentation on pH was significant (*P<0.05). The content of total soluble solids (TSS) is a significant parameter for wine production; contents of TSS for Hicaznar and Beynar juices were 17.75 Brix and 14.85 Brix, respectively (Table 1). Kumoro et al. (21) stated that yeast and initial sugar concentrations affected the production of wine. Higher yeast and initial sugar concentrations were found to inhibit the growth of the yeast cell.
Total sugar contents of Hicaznar and Beynar pomegranate juices were 169.64 g/L and 179.04 g/L, respectively. Contents of TSS and total sugar in both pomegranate juices were suitable for wine production. Ordoudi (22) stated that the content in total sugars of commercial pomegranate varieties Figure 3. A chromatogram of WPJ sample 1: Gallic acid 2: catechin 3: chlorogenic (171 g/L) was higher than typical ones declared for acid 4: caffeic acid 5: epicatechin 6: syringic acid 7: p-coumaric acid 8: ferulic fresh apple (100 g/L), mango ( 36 g/L) that are also acid 9: ellagic acid used for vinegar production.Regardless of the pomegranate cultivar, wine and vinegar samples had a tendency to have lower TSS and TS than that of Data expressed as pomegranate juice samples (*P<0.05) mean ± standard due to the ethanol formation from error sugar during alcoholic fermentation a,b,c,d,e Values with and removal of sedimental ingredients the same lower- during wine making. case letters in the same column do not differ Total phenolic contents and TEAC significantly (ABTS-) /ORAC values of (P>0.05) pomegranate juices, wine and vinegar are presented in Figure 4 and Figure 5, respectively.TEAC and Table 2. Phenolic ORAC values of pomegranate juices substances of in Hicaznar and Beynar pomegranate samples. juices had 2471.77 mg GAE/L and 1765.11 mg GAE/L total phenolic
70 Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 were 28.58 µmol/mL and 15.93 µmol/mL (*P<0.05). The TEAC and ORAC values of MIXPV were significantly higher than that of WPV (P>0.05); the application of maceration resulted in increase of total antioxidant activity maybe due to the extraction of color substances from the reddish arids of Hicaznar pomegranate. All pomegranate vinegars had significant contents of total phenolic substances and total antioxidant activities in this study (*P<0.05); actually these results were higher than the values reported in the literature for vinegars made from different raw materials. The grape wine vinegar exhibited ORAC values between 4.5 µmol TE/mL and 11.5 μmol of TE/mL (6). Budak and Seydim (15) reported that total antioxidant activity values of traditional and industrial wine vinegars were 13.50 mmol/L and 10.37 mmol/L TEAC, Figure 4. Total phenolic substances of pomegranate juices, wine and vinegar. respectively. The beneficial health effects of fruit vinegars may in part be related to the process-induced changes in their phenolics and generation of new antioxidative phenolics during fermentation (7). Ellagic acid, gallic acid, catechin, epicatechin, chlorogenic acid, syringic acid, and p-coumaric acid were quantified in Hicaz and Beynar pomegranate juice, wine and vinegar samples (Table 2). Caffeic acid and ferulic acid were not dedected in pomegranate juice, wine and vinegar samples. Ellagic acid is known as a significant bioactive component (26). Pomegranate juices (WPJ, RPJ, MIXJ) had the highest content of ellagic acid in all samples, between 20.45 mg/L and 42.65 mg/L, respectively. Ellagic acid has also been identified and quantified in many fruits and fruit juices (27,28). For example, Amakura et al. (27) reported ellagic acid contents of blackberry, strawberry, raspberry, bayberry, pomegranate, pineapple, apple, grape, prune juices. In our study, both ethanol and acetic acid Figure 5. Antioxidant activity of pomegranate juices, wines, vinegars. fermentations of pomeganate juice resulted in significant destruction of ellagic acid (*P<0.05). contents (*P<0.05), and 48 µmol/mL and 19 µmol/mL ORAC Amakura et al. (27) stated that the content of ellagic acid values (*P<0.05), respectively. Pomegranate wine samples was 14.3 mg/g while gallic acid was not found in had the highest total phenolic contents among all the pomegranate juice. Gallic acid was 0.00 mg/L in RPJ; 53.2 samples including pomegranate juices and vinegars mg/L in WPJ; 58.35 mg/L in MIXJ. The significant differences (*P<0.05). Total phenolic contents of RPW, WPW, MIXPW observed between the contents of gallic acid in RPJ and WPJ were 4448.0 mg/L, 2958.0 mg/L, 4815.5 mg/L, respectively. (*P<0.05) may be due to cultivar variability, environmental The two pomegranate varieties displayed significant characteristics, period of harvesting or fruit maturity (26). differences in total antioxidant activities. Hicaznar has more Phenolics range from simple, low molecular weight, single reddish pigments while Beynar has pale red-whitish color. aromatic-ring compounds to the large and complex tannins The presence of more color pigments in Hicaznar might be and derived polyphenols. They are usually conjugated to the reason for the high total antioxidant activities in sugars and organic acids (1). Gallic acid is the most common Hicaznar and mixture samples. In a study conducted by phenolic acid and occurs widely as complex sugar esters in Mousavinejad et al. (23) total phenolic substances and gallotannins such as 2-O-digalloyltetra-O-galloyl-glucose (28). TEAC results of six pomegranate juice samples were However, ethanol fermentation of RPJ increased the amount reported and were in accordance with our results. Mixture of gallic acid in RPW. Gallic acid has been liberated following of both variety juices had similar results with Hicaznar juice to ethanol fermentation with sugar consumption by S. (P>0.05). However, the ORAC values of RPJ and MIXJ were cerevisiae (15). Pomegranate vinegar samples also contained significantly higher than the RPW and MIXPW, respectively; significant amounts of gallic acid. it should be noted that contents of ellagic acid were also significantly higher in RPJ and MIXJ samples than the Contents of epicatechin were similar in two varieties of contents of ellagic acid in pomegranate wine and vinegar pomegranate juices (P>0.05). It gradually decreased in WPW, samples (Table 2). Jin-wei (24) stated that there may not be WPV and MIXPW, MIXPV. Some phenolic substances may have a correlation between total phenolic contents and total been lost during resettling and racking during vinegar antioxidant activities since the potential antioxidant activity production. was not solely from the phenolic contents for different fruits. Lee and Renaker (25) stated that there was a significant Variety and also acetic acid fermentation become important variation in ORAC values of wines; for example, ORAC in the contents of catechin and chlorogenic acids since they values of Cabernet Sauvignon wine varied considerably were only identified in RPV and MIXPV (29). In particular, the from 6.0 μmol TE/mL to 87.0 μmol of TE/mL. o-quinones of (+)-catechin and (−)-epicatechin were seen to ORAC values RPV and WPV of pomegranate vinegar samples be much less stable than those of chlorogenic acid (30).
Agro FOOD Industry Hi Tech - vol. 26(5) - September/October 2015 71 CONCLUSION 15. Budak N.H., Guzel-Seydim Z. Antioxidant Activity and Phenolic Content of Wine Vinegars Produced by Two Different Techniques. Different varieties of pomegranate juices had different J Sci Food Agr, 90: 2021-2026 (2010). antioxidant properties and different profiles of phenolic 16. Aviram M., Dornfeld L., Kaplan M., et al. Pomegranate wine has greater protection capacity than red wine on low-density substances. The content of bioactive ellagic acid is very high lipoprotein oxidation. J Med Food, 10: 371-374 (2007). in both varieties of pomegranate juices. Ethanol and acetic 17. AOAC, Association of Official Analytical Chemists, Official acid fermentations resulted in changes in profiles of phenolic Methods of Analysis, 15th edition. AOAC, Washington DC. (1992). substances such as the destruction of ellagic acid. However, 18. Singleton V.L., Orthofer R., Lamuela-Raventos R.M. Analysis of total total antioxidant activities tended to increase in phenols and other oxidation substrates and antioxidants by pomegranate wine samples due to liberation of other means of Folin–Ciocalteu reagent. Meth Enzymol, 99: 152–178 phenolics. Pomegranate wine and vinegar could not only be (1999). unique products due to their sensory properties but also 19. Cao G., Prior R. Measurement of Oxygen Radical Absorbance provide significant contribution to health. Capacity in Biological samples, Oxidants and Antioxidants. Meth Enzymol, 299: 50-62 (1999). 20. Seeram N.P., Adams L., Henning S. In vitro antiproliferative, apoptotic and antioxidant activities of punicalagins, ellagic acid ACKNOWLEDGEMENT and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate This project was partially funded by TUBITAK (Project # 2209). juice. J Nutr Biochem, 16: 360–367 (2005). 21. Kumoro A.C., Sari D.R., Pinandita A.P.P., et al. Preparation of Wine from Jackfruit (Artocarpus heterophyllus lam) Juice Using Baker REFERENCES yeast: Effect of Yeast and Initial Sugar Concentrations. World Appl Sci J, 16: 1262-1268, (2012). 1. Crozier A., Jaganath I.B., Clifford, M.N. Dietary phenolics: 22. Ordoudi A.S., Mantzouridou F., Daftsiou, M., et. al. Pomegranate chemistry, bioavailability and effects on health. Nat Prod Rep, 26: juice functional constituents after alcoholic and acetic acid 1001–1043 (2009). fermentation. J Funct Foods, 8: 161–168 (2014).23 Mousavinejad 2. Ghosh D., Scheepens A.Vascular action of polyphenols. Mol Nutr 23. Emam-Djomeh Z., Rezaei K., et al. Identification and Food Res, 53: 322-331(2009). quantification of phenolic compounds and their effects on 3. Seeram N.P., Zhang Y., Reed J.D., et al. Pomegranate antioxidant activity in pomegranate juices of eight Iranian phytochemicals. In: Pomegranates: Ancient roots to modern cultivars. Food Chem, 115: 1274–1278 (2009). medicine, Edited by Seeram, N.P., Schulman R.N., Heber, D. CRC 24. Jin-wei L., Shao-dong D., Xiao-lin D. Comparison of antioxidant Press Taylor and Francis Group. Boca Raton, Fl. 3-26 (2006). capacities of extracts from five cultivars of Chinese jujube. Process 4. Lansky E.P., Newman R.A. Punica granatum (pomegranate) and Biochem, 40: 3607–3613 (2005). its potential for prevention and treatment of inflammation and 25. Lee J., Rennaker C. Antioxidant capacity and stilbene contents of cancer. J Ethnopharmacol, 109:177–206 (2007). wines produced in the Snake River Valley of Idaho. Food Chem, 5. Afaq F., Saleem M., Krueger C.G. Anthocyanin- and hydrolysable 105: 195–203 (2007). tannin-rich pomegranate fruit extract modulates MAPK and 26. Ancos, B., Gonzalez, E.M., Cano, M.P. Ellagic Acid, Vitamin C, and NF-kappaB pathways and inhibits skin tumorigenesis in CD-1 mice. Total Phenolic Contents and Radical Scavenging Capacity Int J Cancer, 113: 423–433 (2004). Affected by Freezing and Frozen Storage in Raspberry Fruit. J Agr 6. Dávalos A., Bartolomé B., Gómez-Cordovés C. Antioxidant Food Chem, 48: 4565-4570 (2000). properties of commercial grape juices and vinegars. Food Chem, 27. Amakura Y., Okada M., Tsuji S., et al. Determination of phenolic 93: 325–330 (2005). acids in fruit juices by isocratic column liquid chromatography. J 7. Shahidi F., McDonald J., Chandrasekara A., et al. Antioxidant Chrom B, 891: 183–188 (2000). properties of commercial grape juices and vinegars. Asia Pac J 28. Tanaka T., Matsuo Y., Kouno I. A novel black tea pigment and two Clin Nutr, 17: 380-382 (2008). new oxidation products of epigallocatechin-3-O-gallate. J Agr 8. Budak N.H., Doguc K.D., Savaş Ç.M., et al. Effects of Apple Cider Food Chem, 53: 7571–7578 (2005). Vinegars Produced with Different Techniques on Blood Lipids in 29. Tanaka T., Matsuo Y., Kouno I. Chemistry of Secondary High-Cholesterol-Fed Rats. J Agr Food Chem, 59: 6638-6644 (2011). Polyphenols Produced during Processing of Tea and Selected 9. Kondo S., Tayama K., Tsukamoto Y., et al. Antihypertensive effects Foods. Int J Mol Sci, 11: 14-40 (2010). of acetic acid and vinegar on spontaneously hypertensive rats. 30. Richard-Forget F.C., Rouet-Mayer M.A., Goupy P.M., et al. Biosci Biotechnol Biochem, 65: 2690-2694 (2001). Oxidation of chlorogenic acid, catechins, and 4-methylcatechol 10. Ebihara K., Nakajima A. Effect of acetic acid and vinegar on in model solutions by apple polyphenol oxidase. J Agr Food blood glocuse and insulin responses to orally administered sucrose Chem, 40: 2114–2122 (1992). and starch. Agr Biol Chem, 52: 1311-1312 (1988). 11. Abe K., Kushıbıkı T., Matsue H. Generation of Antitumor Active Neutral Medium-Sized α-Glycan in Apple Vinegar Fermentation. Biosci Biotechnol Biochem, 71: 2124-2129 (2007). 12. FAO/ WHO, Codex Alimentarus Comission. Draft European regional Standard for vinegar, (1982). 13. Garcia-Parilla M.C., Gonzalez G.A., Heredia F.J., et al. Differentiation of wine vinegars based on phenolic composition. J Agr Food Chem, 45: 3487-3492 (1997). 14. Burin V.M., Cost L.L.F., Rosier J.P., et al. Cabernet Sauvignon wines from two different clones, characterization and evolution during bottle ageing. Food Sci Technol, 44: 1931-1938 (2011).
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