# 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
The essence of the evaluation of the lectin histochemistry results is to find a fragment of the target substance, i.e., carrageenanan. In the case of computer-assisted analysis, evaluation is more difficult, because the b* value may be skewed by non-specific binding of lectin to the matrix. For products with undeclared addition of carrageenanan and a positive result from Human Inspection, the value of the b* parameter is partially intertwined with the negative and positive samples as seen in Figure 4 (samples exceeding the LoD). Sample 68-19 was judged to be negative on the basis of the b* threshold. Samples 67-19 and 153-19 exhibited a large variability in the b* value between measurements (Table 2 and Figure 4). This dispersion could have been caused by an uneven distribution of carrageenanan in the products, or a non-specific LH reaction  
Figure 3: Positive sample of processed cheese, lectin histochemistry, background staining B-Calleja (green—protein, brown—carrageenanan (arrows)). with one of the additives. Even in this case, with regard to the large dispersion, it can be assumed that the substance was unevenly distributed. Samples 68-19, 70-19, 146-19, 147-19, 148-19, and 154-19 confirmed the results of the Human Inspection, and were to be considered positive on the basis of the CIE L*a*b* method. Samples declared positive were also confirmed to be positive by the CIE L* a* b* method. The largest variability was confirmed in sample 72-19 (Table 3, Figure 4). However, for this sample, after adding the required measurement uncertainty (3 \(\times\) SD), a positive measurement result could not be guaranteed. As with false positive samples, uneven distribution of carrageenanan in the cheese matrix can be assumed in this case as well. This may be due to the low concentration of carrageenanan used or the technological processing of adding carrageenanan to the product. Usually, carrageenanan is added to foodstuffs in an amount of 0.005-2.0% by weight [6]. Statistically significant differences were also demonstrated between individual products. Their reason was not verified in the work, we assume that it was mainly a different concentration of the carrageenanan used, as well as different types of carrageenanan. With regard to applicable legal provisions, the manufacturer is not obliged to provide this information. The only difference in labeling is between refined and semi-refined carrageenanan. Refined carrageenanan is designated as E407, while semi-refined is designated as E407a [57]. The differences shown are given in Table 3 and point to the possibility of further discrimination by the CIE L*a*b* method for processed cheese. Validation of the CIE b* method on marketable products confirms that the detection of carrageenanan in the cheese matrix can also be performed by changing the color of the matrix in the b* value. In addition, not only due to the presence of characteristically formed carrageenanan with a positive LH reaction (Figures 3 and 5), but also due to a change in the color of the protein matrix (Figure 3), we assume that the color change of the cheese  
Figure 4: Individual value plot of CIE b* measurement variability of retail market samples. matrix (b*) occurs due to the specific binding of DAB to carrageenan galactose that is bound to the protein network. Carrageenan forms a strong bond with protein [58]. A false negative reaction can also occur with non-specific protein-protein binding, when the carbohydrate is insufficiently recognized by lectin [59]. Even with related methods, a false positive reaction may occur, for example, in the case of immunofluorescence, where a false positive reaction occurs due to autofluorescence or non-specific binding of antibodies [60]. Immunohistochemistry can also give false positive results due to specific protein binding [61].