DETECTION OF SORBATE POTASSIUM IN BRAZILIAN COMMERCIAL FERMENTED MILKS Detecção de sorbato de potássio em leites fermentados comercializados no Brasil

This study aimed to determine the sorbic acid and its salts presence in fermented milk marketed in Brazil, as well as to quantify it when the sample was positive. Twenty-six commercial samples were analyzed (n = 3), totaling 78 samples. The samples were divided into two groups: natural (5 samples) and with non-dairy ingredients (21 samples). A total of 61.53% were positive for the sorbic acid and its salts. In these, 3.85% were natural fermented milk, which is fraud, according to Codex Alimentarius and Brazilian legislation. In addition, all samples were outside the Brazilian legislation (0.3 g.kg-1) but not for Codex Alimentarius (1.0 g.kg-1). It can be concluded that the sorbic acid and its salts were detected in fermented milk both natural and with non-dairy ingredients.


INTRODUCTION
Milk presents a high nutritional value that is a source of proteins, essential fatty acids, carbohydrate, vitamins, and minerals (ALBENZIO et al., 2012). Economically, the fermented milks are an essential dairy pro duct. It is considered a product with high poten tial for the development of new products. Besi des, fermented milk consumption is associated with health, which is explored by the dairy industries (COSTA; CONTE JUNIOR, 2013;COSTA et al., 2013). However, some addi tives, as preservatives, can be present in fermented milk. According to Brazilian legis lation and Codex Alimentarius, the additives presence, such as sorbic acid and its salts, is allowed in fermented milks with non-dairy ingredients, which is transferred through the optional ingredients (BRASIL, 2007;CODEX ALIMENTARIUS, 2010). These com pounds may have implications for consu mer health, once these products are consu med daily.
Several studies have evaluated the diffe rent preservative content in foods, including sorbic acid and its salts (CRESSEY;JONES, 2009;AKBARI-ADERGANI et al., 2013;ESFANDIARI et al., 2013). However, there were not found studies which evaluated these compounds presences and concentration in fermented milks. For these reasons, the present study aimed to verify the sorbic acid and its salts presence or absence in fermented milks Brazilian marketed. In addition, in the positive samples, the potassium sorbate was quantified.

Location of the collection and sample storage
The fermented milk samples for this experiment were purchased in Rio de Janeiro and Salvador markets. Immediately afterward, they were transported in an isothermal box to the laboratory and stored at 4 ± 1 ºC until the analysis. Twenty-six fermented milks were subdivided into 18 yogurts (5 natural and 13 with non-dairy ingredients) and 8 fermented milks (all with non-dairy ingredients). From each sample, 3 different lots (n = 3) were collected, totaling 78 samples ( Figure 1).

Sorbic acid and its salts
The sorbic acid and its salts qualitative analysis were used as a screening method in fermented milks. The qualitative analyzes in fermented milks were carried out according to the protocol established by Brasil (2006), with some modifications. Initially, 1 g of the samples was weighed in triplicate, then 20 mL of distilled water and 0.6 mL of sulfochromic solution were added. This mixture was taken to the fire. After boiling, 1mL of the 0.3% 2-thiobarbituric acid solution was added with further reading. Sorbic acid or its salts oxidize to the malonic aldehyde to form a compound of red or pink coloration. In this way, the formation of red to pink color indicates these compounds presences. On the other hand, the yellowish coloration indicates the absence.

Potassium sorbate quantification
In the quantitative analysis, sixteen positive samples (n = 48) were evaluated. The potassium sorbate content was estimated based on the method described by Brasil (2006). The samples were weighed (2 g), and 10 mL of 2 N sulfuric acid was added. Then, 10 g of magnesium sulfate heptahydrate was added to the distiller by steam stripping to obtain a 125 mL of distillate. Distilled water was added to complete the 250 ml volume, and 2 mL of 0.3 N sulfuric acid solution and 2 mL of 0.147% potassium dichromate solution were added to the water bath for 5 minutes. The beaker tubes were immersed in cold water, 4 mL of the 0.5% 2-thiobarbituric acid solution was added and then they stood in the water bath for another 10 minutes before cooling down. Subsequently, the spectrophotometer (SP22 -Biospectro) was calibrated at 532 nm. The potassium sorbate results were expressed in g.kg -1 of sorbic acid.

Statistical analysis
The qualitative data were organized in a Microsoft Office Excel ® spreadsheet, and descriptive statistical analysis tools were used to show the percentage values found. The quantitative results were analyzed by analysis of variance (ANOVA) and reported as means ± standard deviations. All ANOVA results were submitted to the Tukey test at P < 0.05 using the XLSTAT version 2013.2.03 (Addinsoft, Paris, France). In this experiment, three different sample batches (n = 3) were evaluated.

RESULTS AND DISCUSSION
The qualitative results demonstrated that Figure 1 -Study design illustrating the stages involved in the experiment 61.53% of the samples were positive for sorbic acid and its salts presence, and 38.47% of the samples were negative (Figure 2). Moreover, 3.85% of the fermented milks (yogurts) without non-dairy ingredients presented a positive result for these compounds ( Figure  2). Based on Brazilian legislation (BRASIL, 2007) and Codex Alimentarius (CODEX ALIMENTARIUS, 2010), in some fermented milks it is accepted the sorbic acid and its salts presence, including fermented milk with fruit pulp aggregate (or fruit preparation for industrial use) and flavored fermented milks. In both references, the preservative presence, including sorbic acid and its salts, in fermented milks without non-dairy ingredient addition is prohibited. Thus, positive samples in natural fermented milk (Figure 2), that is without non-dairy ingredients, are characterized as fraud. Therefore, 26 fermented milks analyzed (3.85%) disagree with the legislation.
Consequently, the sorbic acid and its salts qualitative analysis is a useful screening method to analyze the fraudulent addition of this preservative in fermented milks without non-dairy ingredients. However, in fermented milks added with non-dairy ingredients, it is relevant to quantify the sorbic acid and its salts levels. Since, its presence is permitted with a maximum concentration established (0.3 g.kg -1 and 1.0 g.kg -1 , Brazilian legislation and Codex Alimentarius, respectively). Table 1 exhibits the quantitative results (mean ± standard deviation) of potassium sorbate, expressed as g.kg -1 sorbic acid. Only natural yogurt, which should be absent from this preservative, showed a potassium sorbate concentration within the maximum per mitted limit in Brazilian legislation. Accor ding to this, only fermented milk with an aggregate of fruit pulp or fruit preparation for in dus trial use can present sorbic acid and its salts at the maximum concentration of 300 mg.kg -1 (0.30 g.kg -1 ) in the final product (BRASIL, 2007). Thus, all samples were outside the standard proposed by the Brazilian legislation, characterizing fraud. On the other hand, for Co dex Alimentarius a maximum concentration of 1000 mg.kg -1 (1.0 g.kg -1 ) as sorbic acid is accepted in flavored fermen ted milks (CODEX ALIMENTARIUS, 2010). In this case, for Codex Alimentarius, all samples were within the maximum permissible limit.
Sorbic acid and its salts are preservatives used to inhibit the formation of molds and yeasts in foods, including dairy products such as dairy drinks and dul ce de leche (JA-VANMARDI et al., 2015). However, regular consumption of these pre ser vatives may favor allergic reactions, hi ves, toxicological and mutagenic damage, and elevated blood pressure. Furthermore, chil dren are the principal consumer group of these products and may develop hyperac tivity and attention deficit due to the con sump-tion of preservatives (POLÔNIO; PERES, 2009). Therefore, it is essential to control the potassium sorbate concentration, respec ting the maximum value allowed. Future studies should be carried out to evaluate the long-term effects of sorbic acid consumption on the health. In addition, it should also look for natural substances that can be used as a substitute for this preservative.

CONCLUSION
It was concluded that the potassium sorbate, a preservative, was detected in fermented milk both natural and with non-dairy ingredients. Moreover, in several cases, the concentration exceeded the maximum permissible by Brazilian legislation. Thus, there is a fraud by potassium sorbate addition in some fermented milks marketed in Brazil.