DIFFERENCES IN MACRO- AND MICROELEMENT CONTENTS IN MILK AND YOGHURT

The aim of this study was to determine the concentration of macroelements (Ca, Na, K, Mg) and essential elements (Zn, Fe, Se, Cu) in milk and yoghurt collected from the Croatian market. Inductively coupled plasma-optical emission spectrometry was used as an analytical technique. Mean concentrations measured in milk and yoghurt were (mg/kg): Ca 1406.9 and 1153.8, K 1995.5 and 1526.5, Na 608.3 and 519.2, Mg 136.4 and 108.0, Zn 3.92 and 3.48, Fe 0.332 and 0.231, Cu 0.005 and 0.124. Significantly higher concentrations of Ca, K, Na and Cu (p<0.01-p<0.001) were found in milk than in yoghurt. The element concentrations measured in milk, with the exception of higher K and Na levels, were in agreement with the literature data. Variations in the content of all elements in yoghurt were found in comparison to data from other countries. These results represent the first data on macroand microelements from the Croatian market. The variations found in comparison to other countries suggest that unique processes are utilized in the production of yoghurt products.


INTRODUCTION
Due to the nutritional importance of dairy products, special attention is paid to the physicochemical characteristics of these products.The quality may be influenced by the quantitative content of fats, proteins, vitamins, carbohydrates, organic acids, enzymes and minerals.Their concentrations vary according to various factors, the most important of which are the geographical origin of milk, genetic characteristics of animals, environmental status, type of grazing, stage of lactation, dairy cattle ration and, finally, the technological processes used in manufacturing (Licata et al., 2004;Dobrzarnski et al., 2005;Suhaj and Korenovo, 2008).Milk and dairy products a good source of macrominerals and essential elements, and play an important role in the preservation of health.Minerals in the diet are essential for biological processes, for normal metabolism, growth and development as constituents of complexes with various proteins, lipids, carbohydrates and low molecular weight ligands such as citrate and amino acids (Sola-Larrańaga et al., 2009).The main functions of the most impor-tant macrominerals calcium (Ca), magnesium (Mg), sodium (Na) and potassium (K) are related to the maintenance of pH, osmotic pressure, nerve conduction, muscle contraction, energy production and almost all other aspects of body functions (Institute of Medicine, 2005).In addition, disruption in the optimal physiological levels of essential elements such as iron (Fe), copper (Cu) or zinc (Zn) can initiate the development of pathological processes (Park, 2009).Therefore, health problems can be attributed to inadequate dietary intake of these elements, resulting in deficiency or excess, and the development of specific diseases such as hypertension, osteoporosis, cardiovascular disease, cancer and many others.
In industrialized countries, the current tendency is to develop and produce a wide variety of dairy products, including yoghurts, with different characteristics.Yoghurt is a dairy product from the naturally occurring process of fermentation of milk by bacteria, which leads to the fermentation and acidification of milk into lactic acid.Lowering pH causes changes in the composition, structure and reactivity of casein micelles, thereby modifying the mineral equilibrium.The acidic nature of yoghurt also has positive effects on the gastrointestinal absorption of Ca from milk.This product is considered a good dietary source of essential minerals and could contribute significantly to the recommended daily requirements, especially for Ca and Mg (de la Fuente et al., 2003).
In Croatia, the industrial production of yoghurt has sought out new technological processes to achieve quality and a variety of dairy products.Several dairies produce traditional and innovative dairy products that have become popular in the daily diet of the consumer.Data on the macro-and microelements in dairy foods from Croatia are limited (Sikirić et al., 2010).
The aim of this study was to determine the concentration of macroelements (Ca, Na, K, Mg) and essential elements (Zn, Fe, Se, Cu) in milk and yoghurt collected from the Croatian market.

Sampling
Three basic dairy products produced by different dairies in different marketplaces in the capital of Croatia, Zagreb, were randomly collected: 9 milk, 9 cheese and 9 yoghurt samples.Samples of pasteurized milk (9) and fermented milk products (9) had 2.8% milk fat content (as declared by producers).Samples were labeled and stored at -18°C until analysis.

Sample preparation
Samples (2 g) were weighed in a paraformaldehyde (PFA) digestion vessel and 1 ml of H 2 O 2 and 6 ml HNO 3 were added.A Multiwave 3000 microwave oven (Anton Paar, Ostfildern, Germany) was used for acid digestion of samples.The digestion program was: step 1 power 800 W for 15 min, 800 W for 15 min; step II power 0 W for 15 min.Digested samples were diluted with ultra-pure water to a volume of 50 ml.Analytical batches contained a blank sample and two spiked samples.
The limits of detection levels calculated according to three times the standard deviation of ten blanks were (mg/kg): Ca 0.01, Na 0.01, K 0.025, Mg 0.02, Cu 0.01, Fe 0.005 and Zn 0.005.
For quality control for recovery analysis, certified reference materials of skimmed milk powder (BCR-063, IRMM, Belgium) were used.Results obtained in the comparison of certified and analyzed values showed good agreement and the following recovery results were determined: Ca 98.3%, Na 96.1%, K 98.5%, Mg 93.7%, Se 98.9%, Cu 97.7%, Fe 98.6% and Zn 98.9%.

Analysis of elements
For element measurement, the Optima 8000 (Perkin Elmer, Waltham, Massachusetts, USA) inductively coupled plasma optical emission spectrometer (ICP-OES) was used.Instrumental operating conditions are shown in Table 1.

Data analysis
Statistical analysis was conducted using Statistica 6.1 software (StatSoft® Inc., Tulsa, USA).Concentrations were expressed as mean ± SD.One-way ANOVA was used to test for statistical differences in element levels between dairy products.Results were considered significant at p≤0.05.

RESULTS AND DISCUSSION
In recent decades, there has been a continued increase in the production of fermented dairy products such as yoghurt (Aly et al., 2010).Yoghurt production processes may result in the redistribution of elements, mainly those associated with caseins, and consequent changes may affect nutritional properties.Therefore, element occurrence in yoghurt is of particular concern in view of the demanding quality standards from the nutritional point of view (de la Fuente et al., 2003).
The macro-and microelement contents in milk and yoghurt are presented in Table 2. Copper concen-trations were measured below the limit of detection (<0.01 mg/kg) and were assigned as half the value of limits of detection.Significant differences in the concentrations of Ca, Mg, K and Cu were found in milk and yoghurt.Accordingly, the results showed that production processes affect the contents of Ca, K, Mg and Cu.However, there were no significant differences in Na, Zn and Fe content among the products.
The highest levels of Ca are typically measured in dairy products, especially cheese (González-Martín et al., 2011;Chekri et al., 2012).Milk products such as skimmed milk, dried skimmed milk powder and yoghurt retain essentially all the Ca present in milk prior to processing.The high content in ionic Ca generated in yoghurt during simulated gastro-intestinal digestion could be an index of the high bioavailability of Ca in this dairy product (Delisle et al., 1993).It is suggested that the high content of nonsedimentable Ca in yoghurt may represent a nutritional advantage (de la Fuente, 2003).Over the past two decades, studies have demonstrated the potentially beneficial roles of Ca in a number of disorders or chronic diseases, such as hypertension, colon cancer, breast cancer, kidney stones, premenstrual syndrome, obesity, osteoporosis, polycystic ovary syndrome, insulin resistance syndrome and lead poisoning (Nicklas, 2003).
In the present study, Ca levels in milk and yoghurt ranged from 1024.6-1625.2mg/kg and 1026.7-1337.6 mg/kg, respectively.Significantly higher concentrations of Ca were found in milk than in yoghurt
In the body, Mg is part of more than 300 biochemical reactions, including the maintenance of normal muscle and nerve function and heart rhythm (Durlach, 1998).Because excessive magnesium content in the body is excreted, Mg toxicity, either by dietary intake or parenteral administration, is improbable (Rude, 1998).Magnesium deficiency in animals and humans cause symptoms such as growth retardation, nausea, muscle weakness, tetany and affected cardiac function (Antoine et al., 2012).Also, Mg deficiency is a possible risk factor for osteoporosis in humans (Park, 2009).
In this study, Mg levels ranged from 105.8-190.2mg/kg in milk and 93.3-127.3mg/kg in yoghurt.Significantly higher concentrations of Mg were detected in milk than in yoghurt (p<0.01).Magnesium contents found in milk were in agreement with those presented measured in different countries in the range of 91.8-165.01mg/kg (Lante et al., 2006;Sola-Larrańaga and Navarro-Blasco, 2009;Soares et al., 2010;Navarro-Alarcón et al., 2011;Chekri et al., 2012).The concentrations determined in yoghurt were in line with a study conducted in Brazil (Kira and Maihara, 2007), but lower than those measured in Spain (Martin-Diana et al., 2003;Navarro-Alarcón et al., 2011).High contents of Mg (399 mg/kg) were reported in yoghurt made from goat milk (Güler and Sanal, 2009).
Potassium is essential to the cells as the dominant intracellular cation in the regulation of the acid-base and osmotic balance, muscle contraction and nerve impulse transmission through the movement of K and Na ions during action potential, and as a cofactor of many enzymes involved in protein synthesis, energy transfer and the storage of carbohydrates for use as fuel in muscles (Antoine et al., 2012).It is the third most abundant mineral in the body.
In this study, the K content of milk samples ranged from 1682.9-2215.5 mg/kg, while the range in yoghurt samples was 1257.3-1817.9mg/kg.Therefore, significantly higher K concentrations were determined in milk than in yoghurt (p<0.001).The mean level found in milk was higher than concentrations reported in Italy (1096 mg/kg), Spain (1344 mg/kg) and France (1679 mg/kg) (Lante et al., 2006;Sola-Larrańaga and Navarro-Blasco, 2009;Chekri et al., 2012).There is a lack of data regarding K and Na levels in yoghurt.Mean values measured in yoghurt were higher than those measured in Brazil (1358 mg/kg; Kira and Maihara, 2007).
Sodium is essential for regulating blood pressure and blood volume, and for muscle and nerve function.Excessive intake of Na can lead to hypertension and serious implications in people with congestive heart failure, cirrhosis or renal disease.Sodium is present naturally in most foods but most of it, approximately 75% of dietary Na, is found in processed foods (Antoine et al., 2012).
The Na content measured was similar in milk and yoghurt and ranged from 422.4-851.2mg/kg.These concentrations were in line with previously reported levels in yoghurt (Kira and Maihara, 2007).However, mean levels found in milk (608.3 mg/kg) were higher than those in Italy, France and Spain (411 mg/ kg, Lante et al., 2006;372 mg/kg, Sola-Larrańaga and Navarro-Blasco, 2009;432 mg/kg, Chekri et al., 2012).
As previously stated, concentrations of the microelements Cu, Fe and Zn are low in dairy products (Park, 2009;Reykdal et al., 2011).After Fe, Zn is the second most abundant microelement in the human body, and it is important for different cell processes, including cell growth, homeostasis, development, differentiation, DNA synthesis, RNA transcription, cell division, activation and apoptosis (King, 2011;Chasapis et al., 2012).Zinc is regarded to be a globally deficient microelement.Although a mild Zn deficiency may lead to growth retardation, rough skin, poor appetite, problems with wound healing, mental lethargy, immune dysfunction and neurosensory changes, severe symptoms of Zn deficiency, observed mainly in patients with genetic disorders affecting zinc uptake (acrodermatitis enteropathica), include bullous pustular dermatitis, alopecia, diarrhea, emotional disorders, weight loss, higher sensitivity to infections, hypogonadism, neurosensory disorders and ulcers (Prasad, 2009).
In previously reported studies of different foodstuffs, tested dairy products and meat were found to be the main contributors of Zn (Coudray, 2011;Gaucheron, 2011;Jablonska et al., 2013).In this study, Zn concentrations in milk were in the range 2.59-5.52mg/kg and were in line with previously reported concentrations in different countries (Dobrzanski et al., 2005;Lante et al., 2006;Soares et al., 2010;Navarro-Alarcón et al., 2011;Reykdal et al., 2011;Noël et al., 2012).In addition, Zn concentrations were similar to the values estimated in milk, with mean concentrations of 3.48 mg/kg.The values obtained were in line with the values reported in other countries (de la Fuente et al., 2003;Kira and Maihara, 2007;Navarro-Alarcón et al., 2011;Chekri et al., 2012).
Copper is involved in a variety of biologic reactions, and severely altered homeostasis of Cu may induce oxidative stress and consequently oxidative damage to different proteins, lipids and DNA (Festa and Thiele, 2011).Low dietary intake of Cu during childhood contributes to bone malformation, osteoporosis in later life, impaired melanin synthesis, frequent infections, cardiovascular disorders and altered cholesterol metabolism (Lopez de Romana et al., 2011).Altered Cu metabolism may cause pathologic conditions such as diabetes, cardiovascular disease, malignancies, Alzheimer's disease or other neurodegenerative diseases (Festa and Thiele, 2011).
Cu concentrations in the milk samples in this study were found below the limit of detection (<0.01 mg/kg).Similar levels were found in Spain and Iceland (Martín-Diana et al., 2003;Reykdal et al., 2011), though other studies have reported concentrations in the range 0.091-1.73mg/kg (Dobrzanski et al., 2005;Navarro-Alarcón et al., 2011;Noël et al., 2012).Concentrations of Cu found in yoghurt in our study were significantly higher concentration than those found in milk (p<0.001).Copper levels in yoghurt were in accordance with contents measured in Korea, but lower than those from Brazil (Kira and Maihara, 2007).
Iron plays a major role as an oxygen carrier in haemoglobin in blood, or myoglobin in muscle, and it is also required for many metabolic processes (Gambling et al., 2011).Iron deficiency, i.e. anemia, is still the most prevalent nutritional problem worldwide (Park, 2009).Milk is a poor source of Fe and it cannot be significantly increased by oral administration of Fe salts to lactating humans or animals (Park, 2009).Results of studies conducted to evaluate the potential of Fe fortification in milk and dairy products has been discouraged in some cases, due to certain negative changes in product quality.
Iron levels measured in milk in this study were within the ranges recently reported in Spain, Iceland and France (0.20-0.399 mg/kg) (Sola-Larrańaga and Navarro-Blasco, 2009;Reykdal et al., 2011;Millour et al., 2012).Concentrations of Fe found in yoghurt were in the range 0.141-0.323mg/kg and were similar to milk levels.The iron content was 8-fold lower than that measured in yoghurt from Brazil (Kira and Maihara, 2007).

CONCLUSIONS
Our results represent the first data on macro-and microelements from the Croatian market.Significantly higher concentrations of Ca, K, Na and Cu were determined in milk than in yoghurt samples.For the other elements measured, there were no significant differences found between products.Concentrations of Ca, Mg, Zn and Fe found in milk were in line with contents reported in other countries.However, K and Na content were higher than previously reported concentrations in milk.Variations in element content in yoghurt were demonstrated in comparison with the results for yoghurt samples in other countries.Thus, these variations suggest that unique processes are utilized in the production of yoghurt products.These results found may play a role in the assessment of the nutritional quality of the studied products.

Table 1 .
Operating conditions for measurement of elements with inductively coupled plasma optical emission spectrometer.

Table 2 .
Concentrations of macro and microelements in milk and yogurt samples.