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International Journal of Food Sciences and Nutrition ISSN: 0963-7486 (Print) 1465-3478 (Online) Journal homepage: http://www.tandfonline.com/loi/iijf20 Mineral essential elements for nutrition in different chocolate products Luciano Cinquanta, Cinzia Di Cesare, Remo Manoni, Angela Piano, Piero Roberti & Giancarlo Salvatori To cite this article: Luciano Cinquanta, Cinzia Di Cesare, Remo Manoni, Angela Piano, Piero Roberti & Giancarlo Salvatori (2016): Mineral essential elements for nutrition in different chocolate products, International Journal of Food Sciences and Nutrition, DOI: 10.1080/09637486.2016.1199664 To link to this article: http://dx.doi.org/10.1080/09637486.2016.1199664 Published online: 26 Jun 2016. Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iijf20 Download by: [95.245.225.155] Date: 27 June 2016, At: 07:01 INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION, 2016 http://dx.doi.org/10.1080/09637486.2016.1199664 RESEARCH ARTICLE Mineral essential elements for nutrition in different chocolate products Luciano Cinquantaa, Cinzia Di Cesareb, Remo Manonic, Angela Pianod, Piero Robertid and Giancarlo Salvatorib Downloaded by [${individualUser.displayName}] at 07:01 27 June 2016 a Department of Agricultural, Environmental and Food Science, Molise University, Campobasso, Italy; bDepartment of Medicine and Sciences for Health ‘‘V. Tiberio’’, Molise University, Campobasso, Italy; cARPAm Molise, Campobasso, Italy; dCOSIB, Termoli, Italy ABSTRACT ARTICLE HISTORY In this work, the essential mineral nutritional elements in cocoa beans, in chocolates at different cocoa percentage (60,70,80 and 90%) and in milk chocolate are evaluated. Dark chocolates are confirmed as an excellent source of magnesium (252.2 mg/100 g) and iron (10.9 mg/100 g): in chocolate containing 90% cocoa, their content corresponds to, respectively, 67.0% and 80.3 of Nutrient Reference Values (NRV) in the European Union. The chocolate containing 90% cocoa is also a good source of zinc (3.5 mg/100 g), which is important for the immune system, and selenium (0.1 mg/100 g). Three main components suitable to explain the mineral concentrations are analyzed by factor analysis. The component 1 can be interpreted as the contribution from the cocoa beans, owing to the mineral characteristics of the soil in which they have grown; the component 2 is mainly due to the manipulation and transformation of the cocoa in chocolate, while the component 3 represents the milk powder. Received 7 December 2015 Revised 4 June 2016 Accepted 6 June 2016 Published online 27 June 2016 Introduction Chocolate, the most popular among cocoa products, is reckoned as a luxury good, its composition varies, since many extra ingredients influence the final composition of the product (Steinberg et al. 2003). Data on consumption of chocolate vary among countries: in the United States mean chocolate intake has been calculated as 12 g per day, in Italy 9 g per day and in Brazil 4 g per day; in India 5.5 g per day for adult and 20 g per day for children (Sepe et al. 2001; Dahiya et al. 2005; Confectionery news 2014). The eight largest cocoa-producing countries are C^ote d’Ivoire, Ghana, Indonesia, Nigeria, Cameroon, Brazil, Ecuador and Malaysia. These countries represent 90% of the world cocoa production. After harvest, cocoa seeds (35–50 per baccate) and their surrounding sweet fruit pulp are placed in heaps or boxes and fermented under the influence of naturally occurring microbes that multiply using the sugar from the pulp as an energy source (Afoakwa et al. 2011). The seeds are then dried and the thin coats are removed from the embryonic tissue, which is then roasted, and milled into what is referred to as chocolate liquor. Chocolate has several beneficial health effects because of its high content of antioxidants: mainly a subclass of flavonoids, called flavanols, including especially the monomer epicatechin and CONTACT Luciano Cinquanta 86100 Campobasso, Italy [email protected] ß 2016 Informa UK Limited, trading as Taylor & Francis Group KEYWORDS Chocolate; cocoa; iron; magnesium; mineral; nutritional values catechins, and possibly procyanidins and metabolites (Fisher & Hollenberg 2005). Thus, it is worth recalling the potential cardiovascular beneficial effect of the flavan-3-ols content in chocolate, related to its percentage of cocoa. In fact, it is found consistent acute and chronic benefits of chocolate or cocoa on flow-mediated dilatation and promising effects on insulin and insulin resistance (Hooper et al. 2012). Cocoa is also an extremely rich source of many essential minerals and chocolate has the potential to provide significant amount of minerals in the human diet (Paoletti et al. 2012). As in any other plant food, the mineral content of cocoa reflects the soil in which it is grown, another possible source of variability may be contamination with copper as fungicides. Some heavy metal are essential nutrients (Cu, Ni, Zn) needed by our body, but all of them can be harmful if ingested in heavily metalcontaminated foods. Reports of heavy metals in cocoa beans and chocolates are of concern to cocoa producers (Dos Santos et al. 2005). Consequently, international legislative bodies, as well as chocolate manufacturing countries, have introduced new regulations for the protection of the health of their consumers (Dickson-Spillmann & Siegrist 2011). In the present study, the levels of Ca, P, Mg, K, Fe, Zn, Cu, Se and other microelements were evaluated in cocoa beans, different dark chocolates (60, 70, 80 and 90% cocoa) Department of Agricultural, Environmental and Food Science. Molise University, Via F. De Sanctis 2 L. CINQUANTA ET AL. and milk chocolate. The samples were also examined with respect to the reference levels of nutrients. Material and methods Downloaded by [${individualUser.displayName}] at 07:01 27 June 2016 Samples and extraction procedure Cocoa beans (Theobroma cocoa, var. hybrid) were picked from Seed Production Division of Cocobod at Goaso (Ghana); dark chocolates (at 60, 70, 80 and 90% cocoa) and milk chocolates were collected from the Dolceamaro srl company (Monteroduni, Italy). The samples (three of 100 g of each type), have been grinded and 1 g has been digested with 10 ml of nitric acid with mineralizer (SCP Science DIGIprep, Quebec H9X 4B6, Canada), with the following instrumentals condition: start at 40 C for 15 min; heating at 60 C in 15 min; stay at 60 C for 15 min; heating to 90 C in 20 min. The digested samples have been cooled and brought to a volume of 50 ml with bidistilled water and analyzed at the optical ICP (Inductively Coupled Plasma). Chemicals and reagents Ultrapure nitric acid for trace analysis, sulfuric acid (96%) and standard mono element in nitric acid 2% were purchased from Sigma Aldrich (20151-Milan, Italy). Analysis of minerals The determination of metals (Al, Ba, K, Ca, Cr, Co, Cu, Fe, Mg, Mn, Mo, Na, Ni, P, Se, Zn) in cocoa and chocolate samples was carried out with the technique of nitric mineralization and the analysis by spectrophotometry plasma emission (Varian ICP 710, OES, Inductively Coupled Plasma Optical Emission Spectrometers, Palo Alto, CA 94304-1038). Statistical analysis Statistical analysis was performed using the software SPSS for Windows 21.0 software (SPSS Inc., Chicago, IL). Statistical evaluation was carried out by means of exploratory factor analysis, because our hypotheses regarding the model are not very specific. In particular, we do not have specific predictions about the size of the relation of each observed variable to each latent variable. The cutoff loading value to determine which elements will be used at the clustering stage was set at the level >0.70. Results In our samples the amount of calcium is significantly greater in the milk chocolate (180.4 mg/100 g), compared to all the other types of chocolate investigated (Table 1). Hundred grams of this product fulfill the 22.5% of the daily intake for adults according to the Nutrient Reference Values (Table 2). The content of calcium increases from 64.4 mg/100 g to 90.8 mg/100 g, as the cocoa content increases from 60 to 90%. The Magnesium content in samples vary from a minimum of 52.3 mg/100 g in milk chocolate to a maximum of 252.2 mg/100 g in chocolate containing 90% cocoa (Table 1), corresponding to 67.0% NRV (Table 2). Similarly, the highest content in Potassium is observed in chocolate containing 90% cocoa (720.1 mg/100 g), corresponding to about 36.0% NRV (Table 2). The amount of some micronutrients (aluminum, barium, boron, cobalt, molybdenum) in different samples is reported in Table 1 (Sager 2012). The highest sodium values are observed in milk chocolate: 72.9 mg/100 g, and the lowest in chocolate containing 80% cocoa: 3.3 mg/100 g (Table 1). In the samples analyzed significantly higher Copper content is observed in chocolate containing 90% cocoa: 2.0 mg/100 g (Table 1), Table 1. Mean (±SD) of some elements (mg/100 g) in different chocolate and cocoa beans. Nutrient Ca Mg P K Na Cu Fe Se Zn Cr Mn Al Ba Ni Mo (mg/kg) Co (mg/kg) a,b,c,d,e Milk chocolate 180.43a ± 3.00 52.28a ± 2.03 198.91a ± 2.72 379.05a ± 10.94 72.91a ± 1.48 0.31a ± 0.00 1.19a ± 0.03 0.06a ± 0.01 0.94a ± 0.04 0.01a ± 0.00 0.31a ± 0.00 0.05a ± 0.01 0.10a ± 0.00 0.07a ± 0.00 0.06a ± 0.02 0.10a ± 0.02 Dark chocolate 60% 64.33b ± 1.01 158.78b ± 7.33 221.80a ± 7.48 465.55b ± 11.22 5.20 ± 0.11 1.43b ± 0.04 9.73b ± 0.02 0.08a ± 0.00 2.24b ± 0.08 0.04ac ± 0.00 1.65b ± 0.06 0.56b ± 0.02 0.54b ± 0.00 0.36b ± 0.01 0.09ab ± 0.02 0.33b ± 0.03 Dark chocolate 70% 79.51c ± 8.14 192.23b ± 16.87 279.90b ± 14.14 540.43c ± 30.11 4.20bc ± 0.45 1.83c ± 0.08 9.84b ± 0.29 0.09b ± 0.02 3.19c ± 1.01 0.07bc ± 0.02 1.98b ± 0.11 0.77b ± 0.29 0.64c ± 0.03 0.49bc ± 0.05 0.12bce ± 0.06 0.43c ± 0.06 Dark chocolate 80% 72.41bc ± 3.10 198.76b ± 10.88 297.63b ± 9.37 565.88c ± 20.17 3.30c ± 1.27 1.78c ± 0.10 11.24c ± 0.25 0.10b ± 0.01 2.85bc ± 0.14 0.07bc ± 0.02 1.85b ± 0.07 1.38c ± 0.04 0.68c ± 0.05 0.49bd ± 0.02 0.19c ± 0.02 0.46c ± 0.04 Scheffè test, different letters within same row indicate significant difference (p < 0.01). Dark chocolate 90% 90.83d ± 4.92 252.21c ± 21.58 396.51c ± 24.76 720.11d ± 49.91 3.50bc ± 0.53 2.02c ± 0.12 10.89c ± 0.13 0.10b ± 0.07 3.52c ± 0.16 0.08b ± 0.03 2.05b ± 0.15 1.32c ± 0.02 0.80d ± 0.04 0.61cd ± 0.03 0.07d ± 0.03 0.58d ± 0.04 Cocoa Beans 70.14bc ± 2.97 290.64c ± 55.15 515.84d ± 37.66 720.22d ± 37.35 3.40bc ± 0.36 3.12d ± 0.26 3.20d ± 0.09 0.07a ± 0.05 4.88d ± 0.33 0.01a ± 0.00 3.01c ± 0.55 0.69b ± 0.36 0.33e ± 0.04 0.60cd ± 0.16 0.07ae ± 0.05 0.26b ± 0.06 INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION 3 Table 2. Percentage of elements dietary intake from 100 g of chocolate compared with NRV. Nutrient Milk chocolate % NRV Dark chocolate 60% % NRV Dark chocolate 70% % NRV Dark chocolate 80% % NRV Dark chocolate 90% % NRV Recommen-dations NRV Adults (mg/day) 22.5 13.9 28.4 18.9 31.8 8.5 103.2 9.4 30.7 15.8 8.0 42.3 31.7 23.3 143.8 69.5 159.6 22.5 106.7 82.8 9.9 51.2 39.9 27.0 183.3 70.3 163.4 31.9 174.7 99.2 9.0 53.1 42.5 28.3 178.5 80.3 182.0 28.6 198.0 92.6 11.3 67.2 56.6 36.0 202.1 77.8 192.4 35.2 206.2 102.7 800 375 700 2000 1 14 0.055 10 0.04 102 Downloaded by [${individualUser.displayName}] at 07:01 27 June 2016 Ca Mg P K Cu Fe Se Zn Cr Mn corresponding to about 202.1% NRV (Table 2). Content of Fe in chocolate containing 80% cocoa is about 11.2 mg/100 g, corresponding to about 80.3% NRV (Table 2). In our research, chocolates containing 80% and 90% cocoa are also a good source of selenium, equal to about 0.10 mg/100 g, value far away by UL (upper limit ¼ 0.3 mg/day) (EFSA 2016). In our samples, the highest values of Zinc are observed in chocolate containing 90% cocoa and the lowest concentration in milk chocolate, with values respectively of 3.52 mg/100 and 0.94 mg/100 g. The Zn content observed in chocolate 90% cocoa corresponds to about 35.2% NRV (Table 2). In the samples studied, the amount of Chromium3þ varies from 0.01 mg/100 mg in milk chocolate to 0.08 mg/100 g in chocolate containing 90% cocoa (Table 1). The content observed in chocolate containing 90% cocoa, corresponds to about 206.2% NRV (Table 2). Finally, the lowest concentration in Manganese is observed in milk chocolate and the highest in 90% cocoa-containing chocolates (Table 1), with values, respectively, equal to 0.31 mg/100 g and 2.05 mg/ 100, corresponding to about 102.7% NRV (Table 2). Discussion The chocolate manufacturers often receive very heterogeneous batches of cocoa liquors, for these reasons, they have only rough expectations of the qualitative parameters by country of origin. In recent years, various handicraft companies are trying to buy the cocoa beans directly from the producers. In this context, our goal is to contribute to characterize the different raw materials (here, the minerals present followed by phenols present) and study the evolution of such compounds in chocolate-based products. The human body utilizes minerals for the proper composition of the bone and blood and maintenance of the normal cell function. The Regulation (EU) No 1169/2011 of the European Parliament and of the Council of 25/X/2011 on the provision of food information to consumers gives the daily reference intakes for vitamins and minerals (adults) and their NRVs. Calcium, the most abundant mineral in the body, is required for vascular contraction and vasodilatation, muscle function, nerve transmission, intracellular signaling and hormonal secretion. Milk, yogurt and cheese are rich natural sources of calcium, broccoli and spinach provides calcium but its bioavailability is poor (Borchers et al. 2000). The susceptibility of calcium to form complexes with oxalates and phytates interferes with its absorption. Our results show that milk chocolate could be an excellent source of calcium (Chekri et al. 2012). Magnesium is involved in catalyzing multitude biological reactions, including protein synthesis, transmission of nerve impulse, muscle relaxation, energy production, and bone and teeth adsorption. Chocolate is a strong source for Mg (up to 252.2 mg/ 100 g), with values similar to whole-meal bread and potatoes. Cocoa contains 4-5 times more Mg than peas, white wheat, corn and rice (Spiegel & Sager 2008). This component can be interpreted as the contribution from the cocoa beans indeed. Typically, foods rich in protein are a good source of phosphorus with about 15 mg P/g protein (Anderson et al. 2006). The chocolate is a good source of phosphorus, providing on an average about 200 mg/100 g of protein (Carnovale & Marletta 2001), and its concentration increases with cocoa percentage, as well as Potassium content. Potassium is essential for maintaining cellular osmolality and membrane potentials, thus playing a role in vascular tone and other biochemical pathways related to cardiovascular health. A diet high in processed foods and low in fresh fruits and vegetables is often high in sodium that is a huge problem throughout the world, as it contributes to blood pressure elevation, along with increased risk of cardiovascular diseases (Strazzullo et al. 2009). The minimum intake level necessary for proper bodily function is estimated to be as little as 200–500 mg/day (Shin et al. 2011; Taylor et al. 2011). Data from around the world suggest that the population average sodium consumption is well above the value recommended by the 2002 Joint (WHO/FAO) Expert Consultation (WHO Library 2012): 2 g sodium/day (equivalent to 5 g salt/day). 4 L. CINQUANTA ET AL. Table 3. Rotated component matrix after factor analysis of mineral concentrations found in cocoa beans and different chocolate samples. Downloaded by [${individualUser.displayName}] at 07:01 27 June 2016 Component Ca K Mg Na P Al Ba Co Cr Cu Fe Mn Mo Ni Se Zn 1 2 3 0.485 0.925 0.909 0.560 0.988 0.437 0.253 0.341 0.039 0.929 0.016 0.842 0.236 0.759 0.064 0.916 0.343 0.354 0.310 0.494 0.017 0.798 0.889 0.901 0.839 0.090 0.865 0.129 0.900 0.436 0.550 0.094 0.776 0.034 0.182 0.647 0.092 0.081 0.326 0.158 0.198 0.345 0.478 0.486 0.202 0.385 0.376 0.256 From the nutritional point of view, chocolate is not the preferred source of sodium. Copper introduced in the diet is essential for cellular respiration, to the production of ATP, thermoregulation and it is also involved in antioxidant defense, as well as in the modulation of the immune system (Maggini et al. 2007). The total dietary copper intake by males and females was positively associated with the consumption of chocolate foods (Pennington & Young 1990). Analysis of copper contents in chocolate is also important since copper compounds are widely used as fungicides against black pod disease while farming cocoa. The upper limit to copper is starting from 5 mg/day (EFSA 2016); excess copper intake can cause stomach pain, nausea and diarrhea and can cause tissue damage. For understanding the results of the mineral analysis, we created a rotated factor matrix table (Table 3). Factors are rotated so that they are easier to interpret thus different items are explained or predicted by different underlying factors, and each factor explains more than one item. By factor analysis, we can infer three main components (Table 3): component 1 can be interpreted as the contribution from the cocoa beans, since the mineral content of cocoa reflects the mineral characteristics of the soil in which they have grown (Shittu & Badmus 2009; No€el et al., 2012). Component 2 seems derived mainly from secondary contamination, owing to the manipulation and transformation of the cocoa in chocolate (Beckett 2008); while component 3 represents the milk powder in the milk chocolate, in fact, calcium is part of the component 3 (Table 3). Magnesium, together with copper, zinc and phosphorus is part of component 1, which reflects the mineral characteristics of the soil in which the cocoa beans have grown. Thus, the data partially confirming (for phosphorus, iron and zinc) the significant differences found due to the effect of cocoa origin (TorresMoreno et al. 2015). Iron is a mineral essential for life and deficiency of Fe in the body could result in anemia, of course, the form in which iron is present in food is a major factor of bioavailability. Approximately 20–30% of heme Fe (found only in meats) is absorbed, whereas absorption of nonheme Fe is in the range of 5–10%. Among nutrients important to feed babies and small children, chocolate contained significantly more Fe (25 mg/100 g) than milk, honey and eggs (Falandysz & Kotecka 1994). The iron content of cocoa is higher than the level found in other foods as beef or chicken liver (Paoletti et al. 2012). The bioavailability of iron is quite higher in chocolate with respect to other vegetables, because of low levels of phytic acids therein. The samples of chocolate at 80% and 90% cocoa are an excellent source of Iron confirming the upward trend of the content of iron by increasing the percentage of cocoa. Iron, Chromium, Aluminum, Barium, Cobalt and Molybdenum are part of Component 2; these elements probably may reflect the possible contamination of the cocoa beans or because of human activities such as industrial processes to transform cocoa in chocolate. Selenium is another essential micronutrient due to its presence in the active site of some enzymes involved in thyroid hormone metabolism, modulation of the immune system and defense activity against oxidative stress (Arthur et al. 2003). Based on the significant role of selenium in antioxidant enzymes, it is important to consider the contribution of chocolates containing 80% and 90% cocoa in its diet intake. Zinc has a wide variety of roles in mammalian system, exerts multiple functions in the cell and is the key component of the catalytic site of hundreds of metalloenzymes, playing a central role in the growth and cell differentiation (EFSA NDA Panel 2014). Zinc has a considerable influence on the immune system and the lack of this mineral causes atrophy of lymphoid organs (Tuerk & Fazel 2009). Zinc nutritional deficiency is a global health problem (Osendarp et al. 2003). The content of zinc in the chocolate with 90% cocoa contributes approximately one third of the nutritional requirement by consuming 100 g of such product, while for Manganese this value rises significantly. Chromium(3þ) enhances the action of insulin, a hormone critical for the metabolism and storage of carbohydrate, fat and protein in the body. Due to lack of data, the European Food Safety Authority recommended a maximum tolerable intake level (upper limit) for the Cr3þ equal to 0.25 mg/day. The content of copper, zinc and INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION chromium is always far below the safety limits for human health. The originality of this research is to try whether the different mineral content in cocoa beans and chocolate are explained or predicted by different ‘‘underlying factors’’. By exploratory factor analysis, using the rotated factor matrix table, we inferred three main components: the geographical origin of cocoa beans, the secondary contamination until their transformation into chocolate and the added ingredients. Further tests are ongoing to confirm such preliminary results. Downloaded by [${individualUser.displayName}] at 07:01 27 June 2016 Conclusions Dark chocolates are confirmed as an excellent source of magnesium: in chocolate containing 90% cocoa, it corresponds to 67.0% NRV. Similarly, the high percentage of cocoa is related to iron content: in chocolate containing 80% cocoa, the intake corresponds to about 80.3% NRV. 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