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Food Sci. Technol. Res., 16 (2), 123 – 134, 2010

Determination of Metal Contents in Sugar Beet (Beta vulgaris) and Its Products:

Empirical and Chemometrical Approach

Biljana Škrbić 1* , Nataša ÐuriŠić-MladeNović 1 and Nada MačvaNiN 2

1 Faculty of Technology, University of Novi Sad, Bulevar cara Lazara 1, Novi Sad, Serbia

2 Faculty of Medicine, Institute of Occupational Health, Novi Sad, Serbia

Received August 20, 2009; Accepted November 3, 2009

Multielements K, Na, Ca, Mg, Zn, Fe and Cu were estimated in 25 composite samples of sugar beet, extracted sugar beet pulp, dried sugar beet pulp, molasses, and white sugar collected during 50 days of the 2005 campaign in one beet sugar factory, and in 65 soil samples collected from the fields where beet was grown. Mean total contents of analyzed elements in sugar beet and different sugar beet based products were in the range: 18.11-37510 mg/kg dry matter (d.m.) for K, 6.54-8945 mg/kg d.m. for Na, 14.36-5220 mg/kg d.m. for Ca, 0.09-2550 mg/kg d.m. for Mg, 0.01-10.85 mg/kg d.m. for Zn, 0.42-360.4 mg/kg d.m. for Fe, and 0.07-7.09 mg/kg d.m. for Cu. Mean extractable amounts of these elements in the soils sampled in the fields where beet was cultivated were as follows: 207.6 mg/kg d.m., 60.3 mg/kg d.m., 14.4 mg/kg d.m., 404.7 mg/kg d.m., 1.44 mg/kg d.m., 9.89 mg/kg d.m. and 1.61 mg/ kg d.m., respectively. In order to get a better insight into the metal patterns of the investigated samples, three statistical techniques were used. Principal component analysis (PCA) and cluster analysis (CA) approved to be more powerful than Spearman’s test in revealing the specific correlations among the variables (i.e. metal contents). PCA and CA pointed out the specific metal pattern of molasses on one hand and of sugar beet, extracted and dried sugar beet pulps on the other. Moreover, the chemometrical approach pointed out that main components that classified the metal behavior in the examined samples were the ones correlated with Na and K on one hand, and on the other hand with the remaining metals.

Keywords: sugar beet, metals content, AAS, principal component analysis, cluster analysis

Introduction It is well known fact that the chemical composition of plants reflects in general the elemental composition of the growth media. Higher plants take up metals from air (or water) by the shoots, but the absorption from soil by roots could be regarded as the main pathway of elements to plants (Kabata-Pendias and Pendias, 2000). The concentration of metallic elements in soils is associated with biological and geochemical cycles and is influenced by anthropogenic ac- tivities, such as agricultural practices, transport, industrial activities, waste treatment and disposal (He et al., 2005). Metals exist in soil in immobile (sulphides, phosphates, sili- cates, etc.) and mobile forms, with later being more impor-

*To whom correspondence should be addressed. E-mail: biljana@tf.uns.ac.rs

tant for understanding the migration patterns and the uptake by plants. It has been well documented that the soil contami- nation is likely to result in a corresponding contamination of harvested crops and the food consumers (Fergusson, 1990, Younas et al., 1998, Nan et al., 2002). Furthermore, process- ing of raw materials and the process conditions (additives, pH, etc.) could also influence the quality of the final food products including their metal contents (de Brujin and Bout, 1999, Mohamed, 1999). The elemental composition of sugar beet has been a sub- ject of few previous studies investigating sugar beet quality and its influence on the quality of the final products (Mauch and Farhoudi, 1979/80, Škrbić et al., 2005a, Škrbić and Ðurišić-Mladenović, 2005). Additionaly, the heavy element contents of sugar beet has been investigated to determine the beet uptake as a consequence of either the growth on

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contaminated soils (Bojinova et al., 1996) or different agri- cultural practices (Saleh et al., 1999, Mantovi et al., 2003). The contents of alkaline and alkaline earth metals in sugar beet were also the subject of a sugar beet nutrition study by Wakeel et al. (2009). The main sugar beet product, white sugar, is referred as a very pure food product, although it contains a very small but significant amount of impurities. The control of inorganic impurities in the sugar, i.e. trace elements, is important because of its fundamental nutritional role in the diet of humans and especially in the diet of infants and the elderly. Besides a safety perspective of the sugar quality, the content of elements also determines usefulness of sugar for various industrial applications (de Bruijn and Bout 1999, Wojtczak and Krol, 2002), making the sugar element analysis more important. The main inorganic cations in sugar are calcium (Ca), magnesium (Mg), potassium (K), while there are also some trace elements in sugar such as arsenic (As), mercury (Hg), lead (Pb), cadmium (Cd), iron (Fe), cop- per (Cu) and zinc (Zn). The occurrence of toxic elements in sugar such as As, Hg, Cd, Pb should be in compliance with current legislations on the maximum permissible levels on toxic elements. For instance, in Serbia the maximum permis- sible contents for As, Pb and Cu in white sugar are set to be 1 mg/kg of each. In some EU Member States (Hungarian Regulation, 2004) even more stringent limits has been speci- fied for As and Pb in sugar, i.e. 0.1 and 0.5 mg/kg, respec- tively, while for Hg, Cd, Cu and Zn the limits are 0.02, 0.02, 2.0 and 3.0 mg/kg, respectively. Consequently, the content of toxic elements in sugar is usually very low and generally does not result in any safety concerns for sugar. The content of microelements such as Fe, Cu and Zn in sugar is impor- tant from the technological point of view for the evaluation of the technological process as well as for the evaluation of the quality of sugar. The content of these elements may be treated as an additional criterion of the purification and fil- tration process of beet juice and of the sugar crystallization process (Wojtczak and Krol, 2002). Considering the research studies dealing with elemental analysis of commercial beet sugar there are several published papers generally referring the analysis of heavy elements either essential and/or toxic ones (Leblebici and Volkan, 1998, Ronda et al., 2001, Wojtc- zak and Krol, 2002, Škrbić et al., 2002, Škrbić and Gyura, 2006, Škrbić and Gyura, 2007). The content of impurities is important also to be known for the sugar beet by-products such as molasses and dried sugar beet pulp (fibers) nowa- days, because they are widely used as food supplements due to claimed health benefits. Molasses is a valuable ingredient with high contents of vitamins and minerals, and is ideal in specialty bread and bakery products, crackers, cookies, ce- real bars, cakes, pies, syrups, sweet dressings and as natural

b. Škrbić et al.

colorant, buffering agent and as a raw material for baker’s yeast production (Hickenbottom, 1996). On the other hand, due to high content of natural calcium in sugar beet fibers, their addition to foods and beverages may improve the cal- cium balance without adversely affecting other minerals (Wolever and Jenkins, 1997). Also, sugar beet fibers display many unique functional properties in processed foods result- ing from its combination of soluble and insoluble fibers (Ang and Crosby, 2003). Thus, due to high contents of pectic and cellulosic substances, there is a great potential for dried sugar beet pulp addition into the health promoting foods. The objective of this study was quantitative determina- tion of four macroelements (K, Na, Ca, and Mg) and three microelements (Zn, Fe and Cu) in sugar beet and its products (extracted sugar beet pulp, dried sugar beet pulp, molasses and white sugar) obtained in the 2005 campaign at the lead- ing sugar factory in Serbia, and the mobile forms of the same metals in the samples of soils where beet was grown in order to elucidate the links between metal patterns within the chain field - raw material - products. Furthermore, to obtain more reliable information about relationships among the metals and classification of the samples, these analytical data were evaluated with statistical techniques as Spearman’s test, prin- cipal component analysis (PCA) and cluster analysis (CA). This is the first attempt to get a deeper insight into the metal patterns of materials used and produced in the sugar industry and also to find links with the pattern existing in soils where sugar beet was grown. As this study followed up the contents of 7 metals, including those important for the sugar beet nu- trition (Na, K, Ca, Mg, Cu, Zn), and also for the evaluation of the technological process (Ca, Fe), and their distribution from the sugar beet to the final products, it could be regarded as a contribution to field of agricultural and cultivation sci- ence as well as to a better understanding of the impact of sugar beet processing on the metal content distribution in the by-products and final product (white sugar).

Materials and Methods Sampling Sugar beet (slices) and its products, extracted sugar beet pulp, dried sugar beet pulp, molasses and white sugar were collected during 2005 campaign from the leading Serbian sugar factory accounting for about 17% of the total national white sugar production. The factory is located in Vo- jvodina, the province on the north of Serbia, also known as a main Serbian agricultural region. In the campaign approximately 15 samples were pooled together during a day to obtain test samples for particular day. Ten test samples representing a 10-day period of the campaign were mixed to obtain composite samples for that period, so-called “decade”. As the 2005 campaign lasted for

Metals in Sugar Beet and Its Products

about 50 days, it means that for five “decades” of the cam- paign (i.e. five 10-day periods), five representative composite samples (nos. 1-5) of sugar beet, its by-products and final product (white sugar) were prepared. In this way, the sam- pling procedure provided that the qualities of the composite samples for each particular period (“decade”) were related among each other, e.g. sugar beet sample no. 1 was related with the extracted sugar beet pulp no. 1, etc. Composite soil samples were prepared from 65 sites lo- cated in the arable fields of the Province of Vojvodina where sugar beet was collected during campaign. At each sampling site, four core subsamples (0-50 cm) were collected by a plastic tool from a rectangular area of approximately 20 ×

50 m. Then, the subsamples were combined and mixed to a

composite sample of ~500 g. The samples were air-dried and sieved through a 2-mm polyethylene sieve before analysis. Then, they were ground until fine particles were obtained.

According to a comprehensive survey of the Vojvodina’s ag- ricultural soils (Ubavić et al., 1993), soils examined here be- long to a neutral pH reaction soil type with medium to good provision with humus and total nitrogen, and good provision with available phosphorus. All the prepared samples were stored in polyethylene bags at the temperature about 4to avoid changes in chemi- cal composition. Apparatus Samples were analyzed depending on the type of metals by Perkin-Elmer atomic absorption spectrom- eter (AAS) model 5000. Software HG Graphic II (Perkin Elmer, Norwalk, CT, USA) was employed for spectrum ac- quisition and data processing. Reagents All the chemicals used were of ultrapure grade (Suprapur, Merck). The glassware was cleaned prior to use by soaking overnight in 10% v/v HNO 3 and rinsing with Milli-Q water. Standard stock solutions of elements were purchased from Merck (Germany). Sample preparation and determination of elements The aliquots from the pooled samples of sugar beet slices, ex- tracted sugar beet pulp, and molasses were first evaporated down to a homogeneous state. All the samples from the sugar factory were wet acid – digested. Method of sample preparation was described pre- viously (Škrbić et al., 2005a,b). Briefly, one-gram samples were dissolved with 10 cm 3 of concentrated nitric acid and

10 cm 3 of perchloric acid and heated under the reflux. In or-

der to complete the destruction of the organic matter several portions of 1 cm 3 of nitric acid were added. After obtaining a

clear, colorless solution, it was evaporated to dryness under vacuum and transfered with deionised water Milli-Q TM type (18 MΩ) into a 50 cm 3 flask. Exchangeable (mobile fraction of) macroelements (K,

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Na, Ca, and Mg) were extracted from the air-dried soil by mixing 10 cm 3 of neutral 1 N ammonium acetate with 10 g

of soil and shaken for 5 min. These elements were measured

by analyzing the filtered extract. Extractable (mobile fraction of) zinc, copper, and iron (Zn, Cu, and Fe) were determined

by treating 10 grams of air-dried soil with 20 cm 3 of DTPA

(diethylene triamine pentaacetic acid) extracting solution

(0.005 molar DTPA, 0.1 M TEA (triethanolamine), and 0.01

M CaCl 2 , adjusted to pH 7.3). After shaking for 2 h, the soil

sample was filtered and the extract was analyzed. K, Na, Ca, Mg, Zn, Fe and Cu, concentrations in the samples were determined by direct aspiration of the aqueous solution into air-acetylene flame of AAS at 766.5 nm, 589.0 nm, 422.7 nm, 285.2 nm, 213.9 nm, 248.3 nm and 324.7 nm, respectively. The measurable range for each element was as follows: 0.2-1.6 mg/L for K, 0.10-1.20 mg/L for Na, 0.2-3.2 mg/L for Ca, 0.02-0.60 mg/L for Mg, 0.10-1.20 mg/L for Zn, 1.25-10 mg/L for Fe, and 0.10-3.00 mg/L for Cu. The samples with the overanged contents of elements were di- luted in order to be in the specified range. The exception was the determination of the Zn, Fe and Cu contents in the sugar samples that was performed by AAS with electro-thermal atomization. The measurable ranges here were: 0.1-0.5 μg/L for Zn, 4-10 μg/L for Fe, and 0.7-2.7 μg/L for Cu. The oper-

ating conditions were based on those suggested by the manu- facturer (Beauty, 1988) and were presented elsewhere (Škrbić and Čupić, 2004). Analytical quality control Limits of detection (LOD)

and limits of quantification (LOQ) were calculated on the ba-

sis of three and ten times the standard deviation of the mean

blank determinations, respectively, obtained from 5 measure- ments. The obtained values of the method LOD (LOQ in pa- rentheses) for metals in sugar beet and the products were as follows: 0.2 (0.7) mg/kg for K; 0.02 (0.07) mg/kg for Na; 0.1 (0.3) mg/kg for Ca; 0.001 (0.003) mg/kg for Mg; 1.00 (3.00) mg/kg for Zn, 1.10 (3.60) mg/kg for Fe and 0.9 (3.0) mg/kg for Cu. For the method of Zn, Fe and Cu determination in the sugar samples that was performed by AAS with electro- thermal atomization, LOD (and LOQ) values were 0.05 (0.16) μg/kg, 1.0 (3.3) μg/kg, and 1.0 (3.3) μg/kg, respectively. The LOD (and LOQ) values of the method for soil metal analysis were: 0.02 (0.07) mg/kg for K; 0.002 (0.007) mg/kg for Na; 0.01 (0.03) mg/kg for Ca; 0.0001 (0.0003) mg/kg for Mg; 0.1 (0.3) mg/kg for Zn; 0.11 (0.36) mg/kg for Fe; and 0.09 (0.30) mg/kg for Cu. Quality control consisted of analysis of blank, spiked and duplicate samples. Blank samples were included in every batch of samples to check for possible contamination. They were treated and analyzed with the same method as the ac- tual samples.

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The accuracy of the procedure was checked by the analy- sis of the sugar samples fortified with known quantities of analyzed elements, i.e. 10 mg/kg for K, Na, and Ca; 0.02 mg/ kg for Mg, Zn, and Cu; and 0.5 mg/kg for Fe. The recoveries for investigated metals were in the range of 79-107% with relative standard deviations less than 15%. The obtained data were not adjusted on the basis of these recoveries and were presented as mean value of the duplicates. Statistical data analysis In order to investigate the ele- ments correlation and samples classification, the obtained analytical results were arranged into the input matrix in the following way: the concentrations of the metals (variables) were arranged in the columns, whereas the investigated sam- ples (cases) of sugar beet, molasses, dried sugar beet pulp, extracted sugar beet pulp, and sugar were in the matrix rows. Additionally, the mean values of the elements determined in the soil samples was also put in the input matrix in order to examine links amongst the metal patterns within the chain soil-raw material-products. In this way the 26 × 7 (cases × variables) matrix was obtained. Throughout the study the software packages Excel 2003 (Miscrosoft Corporation) and STATISTICA 6.0 (StatSoft Inc., Tulsa, OK, USA) were used. Spearman’s non-parametric correlation test In order to quantitatively analyze the relationship among micro- and macroelement contents in sugar beet, sugar beet products and soil where the beet was grown, the Spearman’s non- parametric rank correlation coefficient was applied to the obtained data. The non-parametric correlation coefficient is

a common parameter used to quantify the strength of asso-

ciation between the pairs of variables when the presumption

about normality is violated. Definition of the Spearman’s non-parametric correlation coefficient could be found in

standard textbook of statistics (Massarat et al., 1997). A limit value that separates significant from non-significant correla- tions is chosen for the probabilities predefined (at 1 and 5%)

at a given number of observation (n) , i.e. for a given degrees

of freedom (n-2) (Massarat et al., 1997). Principal component analysis (PCA) PCA has suc- cessful application in various field of chemistry, includ- ing environmental analysis and food chemistry (Heberger et al., 1999, Csomos et al., 2002, Heberger et al., 2003, Golobočanin et al., 2004, Škrbić et al., 2005b, Škrbić and Onjia, 2007, Škrbić and Ðurišić-Mladenović, 2007, Škrbić et al., 2009). This is the multivariate analytical tool used to re- duce a set of original variables and to extract a small number of latent factors (principal components, PCs) for analyzing relationships among the observed variables (which are metal contents in this study) and classification of samples. The newly extracted variables, i.e. PCs, are linear combinations of the original ones and are sorted into descending order ac-

b. Škrbić et al.

cording to the amount of variance that they account for in the original set of variables. The loadings express how well the new abstract principal components correlate with the old variables. PCA will show which kinds of metals are similar to each other, that is, carry comparable information, and which ones are unique. Relationships among the samples are evaluated on the score plots, whereas the loading plots show the extent to which each variable contributes to the sample separation (grouping). In this work, the data were firstly mean-centered (column means subtracted from each matrix element). Then each matrix element was divided by the standard deviation of the respective column and the established matrix was submitted to PCA. In this way, we removed scalar differences and the overwhelming influence of high-concentration metals that would otherwise numerically “swamp” major differences in lower concentration metals, placing all variables on equal ba- sis in PCA. Number of PCs extracted from the variables was determined by Kaiser’s rule (Kaiser and Rice, 1974). This criterion retains only PCs with eigenvalues that exceed one. According to Morrison (1967) principal components should account for approximately 75% of the total variance. In order to interpret the significance of retained PCs in terms of the original variables, only those loadings (coefficients) whose absolute value was greater than 60% of the maximum coef- ficient in absolute value for each PC were considered (Jolliffe, 1986). The algorithm of PCA can be found in the standard textbooks (Massart et al., 1997). Cluster analysis (CA) CA is used to classify samples into groups, i.e. clusters. It can be considered to be an alter- native to PCA. An important step in clustering is to select a distance measure, which determines the similarity or dissimi- larities of the samples. The distance between two points is well-defined; the simplest distance measure is the Euclidean distance. However, numerous clustering algorithms exist as to what is considered to be a distance between two groups. It may be defined as the distance of the two closest points or the two farthest points, etc. Weighted schemes are also reliable alternatives. The most popular clustering algorithm is perhaps Ward’s method. Definitions of distance measures and clustering algorithms can be found in standard chemo- metric textbooks (Otto, 1999, Vandeginste et al., 1998). In this study, Ward’s method as an amalgamation rule and the Euclidean distance as a measure of the proximity between samples are used for the cluster analysis of the input matrix previously described.

Results and Discussion Empirical approach The mean total contents and stan- dard deviations of examined metals in samples of sugar

Metals in Sugar Beet and Its Products

127

beet and its products representative for the production of the studied sugar factory are presented in Table 1. This table also gives the mean extractable levels (and standard deviations) for the metals in soil where the beet was grown. The highest concentrations of the alkali metals (K and Na) were determined in molasses followed by sugar beet, ex- tracted sugar beet pulp, dried sugar beet pulp, soil and sugar (Table 1). In fact, K and Na are two very important monova- lent cations in sugar beet nutrition. Both are macronutrients being taken up and utilized in large quantities by sugar beet crops producing optimum yield. Sugar beet is a halophyte, i.e. natrophilic (“sodium-loving”) crop, due to its original shoreline habitat, absorbing and assimilating Na that partly replaces K (Draycott and Christenson, 2003; Wakeel et al., 2009). It has been known that the K and Na contents of sugar beet fluctuate widely and are dependent, in particular, on lo- cation, weather conditions, soil, fertilizer usage, and time of

Škrbić et al., 2002). Considering the alkaline earth metals (Ca and Mg), the highest contents were found in extracted and dried sugar beet pulps, with the Ca/Mg ratio of about 2 (Table 1). In molasses and sugar Ca was also predominant with 25 to 159-times higher content than Mg, respectively (Table 1). However, in sugar beet and soil the opposite was seen as the content of Mg was approximately 2 and 28 times higher than the respective Ca content (Table 1). It is obvious that Ca in sugar beet products predominantly originated from the beet processing, i.e. from the addition of calcium oxide during juice purification. During the course of the manufacturing process, the calcium ions (and magnesium ions present in the sugar beet) are precipitated as carbonate, sulphite, sulphate and eliminated. However, this does not happen completely and depends on the composition of the juices, its alkalin- ity and pH-values. In this study, the mean Ca content of

harvesting (Van der Poel et al., 1998). Regarding Mauch and Farhoudi (1979/80), the sodium content of sugar beet is nor- mally much lower than level of potassium; this was also the case in this study with the mean content of K in sugar beet found to be 3 times higher than Na (Table 1). Similar value of the K/Na ratio was also observed in extracted sugar beet pulp, dried sugar beet pulp and sugar, while in molasses this ratio was even higher (about 4; see Table 1). It is interesting to note that the ratio of mean mobile fraction of K and Na in

the white sugar was high (14.36 mg/kg, Table 1) indicating low effectiveness of the second carbonatation, in which the surplus of Ca, previously added to the process in main lim- ing, should be precitated. Similar result was obtained for the same factory during campaign of 2001 (Škrbić et al., 2002), while the low Ca content (up to 1.2 mg/kg) confirmed the effectiveness of that procedure (de Brujin and Bout, 1999). In the review paper of Mauch and Farhoudi (1979/80), only Ca of the alkaline earth metals in sugar has been detected in

soil samples was also about 3. All these findings confirmed

a

concentration that is worth mentioning. As Mg hydroxide

the fact that K and Na in sugar beet products originated from

is

highly insoluble, most of Mg extracted from beets leaves

sugar beet without notable influence of manufacturing pro- cess on their contents, whereas their content in sugar beet reflected the extractable content of K and Na in soil. Com- parison of the obtained data on K and Na with the published ones was possible only for sugar as for the other investigated samples there were no published data. The mean content of Na and K in white sugar obtained here corresponded to the values previously reported to be in the ranges 6.5-8.5 mg/kg and 9.33-48 mg/kg, respectively (de Brujin and Bout, 1999,

the process with the carbonatation slurry from the first carbo- natation, and therefore, its concentration in the sugar is low. In this study, mean level of Ma in white sugar (0.09 mg/kg) was similar to the data of de Bruijn and Bout (1999) (0.1-0.3 mg/kg). With respect to three investigated microelements, Fe was the most abundant in all investigated samples (Table 1). The mean Fe content in the studied sugar beet (360.4 mg/kg) was in the range from 17.2 to 502.6 mg/kg reported by Mauch

Table 1. The mean total contents and standard deviations of metals in sugar beet and its products (mg/kg d.m.) and mean extractable levels and standard deviations of metals in soil (mg/kg d.m.).

Sample

K

Na

Ca

Mg

Zn

Fe

Cu

Sugar beet

7315±770.8

2370±103.4

970±53.8

2275±80.5

6.76±1.10

360.4 ± 55.9

7.09 ± 0.75

Extracted sugar beet pulp

3600±667.4

1165±285.7

4720±406.7

2550±64.0

5.89±0.31

116.0 ± 11.2

4.69 ± 0.47

Dried sugar beet pulp

2650±369.2

1025±197.2

5220±189.7

2400±122.5

10.10±0.26

156.2 ± 20.2

6.32 ± 0.37

Molasses

37510±1364

8945±1161

3019±432.0

117.6±38.1

10.85±0.93

134.9 ± 28.2

2.30 ± 0.30

Sugar

18.11±2.10

6.54±0.51

14.36±0.71

0.09±0.03

0.01±0.00

0.42 ± 0.02

0.07 ± 0.01

Soil

207.6±70.3

60.3±32.8

14.4±12.3

404.7±164.4

1.44±3.59

9.89 ± 4.7

1.61 ± 1.86

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and Farhoudi (1979/80). The sugar samples also had the content of Fe in the range of concentrations given in the lit- erature (Table 2). It is known that Fe is generally removed during juice purification, while in the case of a low pH-value

it dissolves from the metal surface of the equipment causing

a rise in the iron content in white sugar (de Brujin and Bout,

1999).

Copper mean level in the investigated sugar beet (7.09 mg/kg) was higher than the concentrations given by Mauch and Farhoudi (1979/80) of 1.9 - 3.5 mg/kg and also by Man- tovi et al. (2003) of 1.5-6 mg/kg, but was lower than 11.8 mg/kg determined for beet grown at contaminated area in Bulgaria (Bojinova et al., 1996). As the major part of Cu contained in the press juice is eliminated during juice puri- fication, its mean level in the studied white sugar was 0.07 mg/kg (Table 1). It was in the range of concentrations given in the literature (Table 2) and lower than allowed level of Cu in sugar defined by the Serbian (1992) and by Hungarian Regulations (2004). The mean Zn content in the sugar beet found to be 6.76 mg/kg was similar with the lower value of the range 7-19 mg/kg reported by Mantovi et al. (2003), and much lower than the data given for the beet grown at contaminated area in Bulgaria of 37.1 mg/kg (Bojinova et al.,1996). Mean Zn level in the investigated white sugar was 0.01 mg/kg; it was

b. Škrbić et al.

within the concentrations given in the literature (Table 2) and lower than allowed level of Zn in white sugar (3 mg/kg) de- fined by the Hungarian Regulation (2004). Chemometrical approach Spearman’s test The Spearman’s correlation matrices were computed and the significant correlations obtained for the criterion values of probability p<0.05 and p<0.01 were presented in Table 3 with respect to the metal contents in the analyzed samples. The limit values that separated significant from non-significant correlations were found in the correla- tion coefficient table (Massart et al., 1997) and in our case for n = 26 they were 0.496 and 0.388 at the 1% and 5% sig- nificance levels, respectively. According to the results (Table 3) strong (significant) correlation of positive sign can be seen among majority of the investigated elements: K and Na, K and Zn, K and Fe, Na and Zn, Na and Fe, Ca and Mg, Ca and Zn, Mg and Fe, Mg and Cu, Zn and Fe, and Fe and Cu. Positive correlation coefficient between the contents of two elements indicates that the high concentration of one element in the analyzed samples is associated with the high content of the other and vice versa. The Spearman’s test was also applied on the pairs of sam- ples in order to elucidate the similarities among them based on their element patterns. The obtained diagonal matrix of the correlation coefficients (all were positive i.e. in the range

Table 2. Ranges of Fe, Cu and Zn concentrations (mg/kg) in white sugar samples reported by various studies.

Fe

Cu

Zn

Origin

Reference

0.14-0.23

0.02-0.04

0.02-0.06

The Netherlands

de Brujin and Bout (1999)

0.10-1.01

0.01-0.12

0.01-0.05

Serbia

Škrbić and Gyura (2007)

1.41-1.79

0.02-0.035

Turkey

Leblebici and Volkan (1998)

0.03-1.13

0.01-0.36

0.01-0.30

European sugar beet refineries

Wojtczak and Krol (2002)

Table 3. The Spearman’s non-parametric correlation matrix for metals content in sugar beet, its products and soil where the beet was grown.

 

K

Na

Ca

Mg

Zn

Fe

Cu

K

1.000

Na

0.979**

1.000

Ca

0.198

0.231

1.000

Mg

0.151

0.111

0.734**

1.000

Zn

0.660**

0.676**

0.597**

0.237

1.000

Fe

0.583**

0.591**

0.328

0.394*

0.560*

1.000

Cu

0.361

0.382

0.491*

0.636**

0.373

0.788**

1.000

* Correlation is significant at p < 0.05. ** Correlation is significant at p < 0.01.

Metals in Sugar Beet and Its Products

0-1) was transformed into “color” map that represented the magnitude of correlation by a “color” scale (Fig. 1). The “colors” were scaled from white for the correlations with the coefficients in the range 0 -0.6 to medium grey (referring to the ranges of coefficients 0.6-0.8) and dark grey (with coef- ficients higher than 0.8). In this way, the most similar (cor- related) samples can be seen from the color map through the occurrence of dark shades. Thus, as the strongest correlations existed between the same type of the samples despite of the time (“decade”) of sampling (Fig. 1) it could be concluded that rather constant element patterns among them existed during the campaign. Few more correlations emerged (Fig. 1):

129

among sugar beet and molasses, sugar beet and most of the sugar samples, extracted and dried sugar beet pulps, extract- ed and dried sugar beet pulp with some sugar samples, and molasses and sugar. Based on these observation the follow- ing could be drawn: a) the metal pattern of molasses reflected the one of sugar beet, even though the opposite has been seen after the direct comparison of analytical (empirical) results, particularly concerning the contents of Ca, Mg, Zn and Cu (Table 1); b) pressing and drying of extracted sugar beet pulp did not impair its metal pattern, resulting in similarity be- tween the patterns of extracted and dried pulps; and c) metal pattern of sugar reflected the pattern of sugar beet, extracted

Sugar beet Extracted sugar beet pulp Dried sugar beet pulp Molasses Sugar Soil 1 2
Sugar beet
Extracted sugar beet pulp
Dried sugar beet pulp
Molasses
Sugar
Soil
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
Soil
SPEARMAN’S
COLOR
COEFFICIENT
0 - 0.6
0.6 - 0.8
0.8 - 1
Sugar
Molasses
Dried sugar beet pulp
Extracted sugar beet
pulp
Sugar beet

Fig. 1. Color map of the Spearman’s correlation coefficients between samples based on their element patterns. Numbers 1-5 represent 5 composite samples of each type of material representing 5 “decades” (10-day-periods) of the 2005 campaign in the selected sugar factory.

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and dried pulp, and also of molasses. It is interesting to note that the correlation between soil and sugar beet samples was found to be statistically significant, coinciding with the fact that the metal patterns of plants is largely dependent of the soil composition. Principal component analysis To gain better insight into the latent structure (hidden regularities) of the obtained data and to investigate similarities and dissimilarities of metal contents of the investigated samples PCA was applied

b. Škrbić et al.

and the obtained results are shown in Table 4 and Fig. 2. Three PCs were retained explaining 97.7% of the total variance in the data (Table 4). The first principal component, PC1, explained 45.7% of the data variance and it was signifi- cantly loaded with Cu > Zn > Mg > Fe > Ca (Table 4). The second component, PC2, accounting for 37.3%, correlated markedly with alkali metals, i.e. K > Na, while the third one, PC3, explained 14.7% of the variance and correlated posi- tively with Fe and negatively with Ca (Table 4).

Table 4. Results of principal component analysis for elements in sugar beet and its analyzed products and soil where sugar beet was grown (a) .

Original variables

PC 1

PC 2

PC 3

K

-0.981

Na

-0.963

Ca

-0.678

-0.728

Mg

-0.791

Zn

-0.814

Fe

-0.745

0.652

Cu

-0.910

Eigenvalue

3.20

2.61

1.03

Proportion of total variance

45.7

37.3

14.7

Cumulative

45.7

83.0

97.7

(a) only loadings higher than 60% of the maximum absolute value in each PC are presented.

60% of the maximum absolute value in each PC are presented. Fig. 2. The PCA biplots

Fig. 2. The PCA biplots for variables and cases: a) PC1 vs. PC2, b) PC1 vs. PC3 (sugar beet, extracted sugar beet, dried sugar

beet,

molasses,

sugar, soil).

Metals in Sugar Beet and Its Products

Figires 2a and 2b show the biplots of PC1 vs. PC2 and of PC1 vs. PC3, respectively, displaying graphically the infor- mation obtained on samples and variables by PCA. During the biplot construction, each loading and score values were divided with the respective maximum values obtained for particular PC; thus, the biplot presented the relative posi- tions of the element loadings and the sample scores within the range from –1 to +1. In this way, the interpretation of the variables correlation and the influence on the samples clas- sification could be made more obviously. The correlation between alkali elements (Na/K) as well as between Cu/Mg and Fe/Ca in relation to the data is unambiguous and signifi- cant. It could be also said that the correlated elements carried out the same amount of information and the influence on the samples classification. It is interesting to note that Fe/Ca cor- relation seen by PCA and linked to the influence of the tech- nological process was not seen by the Spearman’s correlation analysis (Table 3). Distribution of the samples along PC1 was influenced primarily by their contents of Cu, Zn and Mg, whereas the content of Na and K governed the sample positioning along PC2 (Fig. 2a). The following groups were observed in the PC1 vs. PC2 biplot: group I consisting of the sugar beet, dried and extracted sugar beet pulps, group II gathering all 5 samples of molasses, and group III rounded the soil and sug- ar samples. Group I had the highest levels of Cu and Mg, and intermediate level of alkali metals. Moreover, slight differen- tiation in the metal patterns could be seen in all five samples of each sample type reflecting slight pattern differences from the first to the fifth “decade” of the campaign. Group II gath- ered samples of molasses that contained the highest content of alkali metals and intermediate content of the remaining elements; again, the difference among the samples composed in different “decades” could be observed and it was more evi- dent than within the sample types in group I. In the group III, the samples with the lowest levels of all of the analyzed met- als were gathered; sugar samples had lower metal contents than the soil. Within this group all five points representing five sugar samples in the biplot (Fig. 2) that were collected during five 10-day period (“decades”) of the 2005 campaign in the factory included in this study were overlaid indicating that they had identical metal patterns. Thus, it could be con- cluded that the final step of sugar production (ash removal by washing procedure in the centrifugals) removed differences observed in the metal patterns of sugar beets from the first to the fifth “decade” of campaign that could be also seen in the patterns of intermediate- and by-products. The biplot of PC1 vs. PC3 showed slightly different groupings of the samples governed by the Fe and Ca con- tents. The sugar beet samples separated clearly from the rest

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of the samples due to the highest content of Fe. Moreover, they contained the lowest Ca content in comparison to the by-products. Thus, separation along PC3 reflected obviously the influence of technological process on Fe and Ca contents:

decreasing of the Fe level from sugar beet to the by-products and white sugar is a result of its removal during juice puri- fication, and increase of the Ca content in the by-products originates in the addition of the technological material (lime) in the beet processing. The separation of sugar and soil samples were influenced again with the lowest levels of all investigated metals. Cluster analysis CA uses less information (distances only) than PCA. Dendrograms (the tree plots) of CA using Ward’s method and the Euclidean distance as a measure of the proximity among the metals in the samples and among the samples, are shown in Figs. 3 and 4, respectively. Considering the CA dendrogram of variables (Fig. 3) two main clusters could be seen, cluster with alkali metals and the one with all remaining metals analyzed in the samples, within which Ca/Zn and Mg/Cu/Fe created two sub-clusters, respectively. Thus, in general CA indicated similar correla- tions among variables as PCA. Nevertheless, CA did not find close correlation of Fe/Ca like PCA was. Dendrogram in Fig. 4 shows 3 main branches (clusters) of samples according to their metal patterns. Within cluster I that corresponded to the PCA group I, two sub-clusters could be seen: one that gathered all sugar beet samples, and another that clearly separated extracted from dried sugar beet samples. Cluster II analogue to the PCA group II rounded all molasses samples. All five sugar samples and soil sample formed the distinct cluster III on the CA dendrogram corre- sponding to the PCA group III.

Conclusion This paper represents the first attempt to elucidate the correlations in metal patterns in the chain soil - sugar beet - sugar beet products and to classify the samples according to their contents of some macro- and micro-elements. In gen- eral, it could be seen that the structure of the data set formed from the analytical results obtained in this study could not be interpreted easily and satisfactory by the simple Spearman’s non-parametric test. The interpretation of the sets was more completely performed by multivariate data analysis tech- niques like principal component analysis, PCA, and cluster analysis, CA, developed for dealing with strongly collinear and numerous variables. However, a color map used for graphical presentation of the correlation coefficients between the samples was the only method that implied association between metal contents in sugar beet and soil. PCA and CA showed the same sample classification, but they indicated

132

b. Škrbić et al.

K Na Ca Zn M g Cu Fe 0 2 4 6 8 1 0
K
Na
Ca
Zn
M
g
Cu
Fe
0
2
4
6
8
1 0
1 2
1 4

Li nkage Dist anc e

Fig. 3. Cluster analysis dendrogram showing association among the metals investigated.

soil 0 5 10 15 20 25 30 dried extracted sugar sugar molasses sugar beet
soil
0 5
10
15
20
25
30
dried
extracted
sugar
sugar
molasses
sugar beet
sugar
beet
pulp
beet pulp

Linkage Distance

Fig. 4. Cluster analysis dendrogram showing association among the investigated samples in sugar beet, its products and soils.

Metals in Sugar Beet and Its Products

slightly different correlations among the metals content in the samples. Only PCA reflected the opposite correlation between Fe and Ca that could be attributed to the influence of technological process. PCA also showed that the samples classification was primarily governed by Cu, Zn and Mg, on one hand and Na and K on the other. The metal pattern of molasses revealed by both multivariate techniques was quite different from that found in sugar beet, extracted and dried sugar beet pulp that formed one group (cluster) with similar patterns. Presence of the investigated metals in sugar was uniform during the whole campaign, regardless of the metal contents fluctuation in sugar beet and its by-products, indicating the efficiency of purification processes. The metal contents of sugar and soil were the lowest and similar be- tween each other, which was the reason of gathering these quite different matrices into the same group (cluster) by PCA and CA. Naturally, all of these statements are valid within the scope of the investigation. Samples from other regions might behave differently.

Acknowledgements This work was supported by the Serbian Min- istry of Science and Technological Development as a part of the Project 152001B: Sources identification and correlations amongst the elements and organic compounds in abiotic and biotic matrices:

risk analysis and a contribution to the monitoring and improvement of the environmental status, coordinated by prof. B. Škrbić.

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