# Detection of Carrageenanen in Cheese Using
## 3 Results and Discussion
### Lectin Histochemistry
#### 3.2.3 Validation of the Method on Samples from the Retail Market  
Detecting carrageenanan in food is very problematic. Thus, there is no general method for its analysis yet [17; 47]. Several methods for detecting carrageenanan in food have been described in the literature. These include, for example, colorimetric methods [17; 48; 49], [PERSON] [50] detected carrageenanan in milk photometrically with the addition of methylene blue. [PERSON] et al. [51] used photometric titration to detect carrageenanan. Quantitative determination of carrageenanan in infant formula, chocolate milk, and ice cream has been performed using a method based on the degradation of proteins by papain and subsequent precipitation of carrageenanan with Hyamine solution and addition of phenol-sulfuric acid to produce a color change in carrageenanan [52; 53]. Other detection methods have used included potentiometry [54], chromatography [47] or microscopic methods [17; 55]. In our work, carrageenanan was detected in samples of processed cheese purchased in the retail market. The samples were examined by Human Inspection and CIE b *. Human inspection was chosen as the reference method for this comparison. The results are presented in Table 4. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Sample No.**} & \multirow{2}{*}{**Result**} & **Declared** & \multicolumn{2}{c}{**Human**} & \multicolumn{2}{c}{**CIE b***} \\  & & **Ingredient** & **Inspection LH** & **Threshold Comparison** & **ANOVA Comparison** \\ \hline Control & 0/2 0/2 0/2 0/2 & N & N & N & 43.64 \(\pm\) 4.10 a  \\  
N & P & P & 16.53 \(\pm\) 3.26 g  \\ \hline \hline \end{tabular} Note: number of positive sections/negative sections;* after adding the required measurement uncertainty (3 \(\times\) SD), a positive result could not be guaranteed. Mean values denoted by different letters at individual column are significantly different (\(p<0.05\)). Data are expressed as mean value \(\pm\) standard deviation.  
\end{table} 
Table 4: Results comparison of the retail market samples by Human Inspection and by b* of CIE L*a*b*. The results show that the declaration for most samples was identical to the examination of lectin histochemistry. There were no statistical differences demonstrated between the Human Inspection and CIE b* methods by [PERSON]'s test (\(p>0.05\)). On the contrary, the samples (67-19, 68-19, 70-19, 71-19, 146-19, 147-19, 148-19, 153-19, 154-19) that were declared not to contain carrageenanan by the producer returned positive results according to lectin histochemistry. The reason for this may also be the non-inclusion of carrageenanan in the ingredients list of the product. For example, carrageenanan is one of the most commonly used substances for adulteration in meat and meat products [56]. Another reason is product contamination. These theories are supported by sample 154-19, which is shown in Figure 3. The figure contains brown fragments of possible carrageenanan, although this sample should have been negative according to the manufacturer. Products with undeclared addition of carrageenanan were examined by the CIE L*a*b* method as well. With regard to the quantitative result, there were not only differences between the positive and negative groups of samples, but also differences within these groups (Table 4). The reason for this is the different raw materials used as well as the different forms of carrageenanan used. For clarity and interpretation of the variability of individual measurements, the results are also shown in scatter plots (Figure 4). The results confirm that the samples declared as positive (right) differed in the value of b* from the negative samples and, primarily, the negative control (left). Sample 66-19 differed from the negative samples with respect to b*, and was evaluated as negative by a human evaluator. However, according to the set threshold value for b*, this sample was evaluated as positive by the CIE L*a*b* method.