*3.4. Anthocyanins Composition and Stability of the Colouring Formulations*

The main compounds responsible for the colouring properties of haskap fruits are their anthocyanins; thus, an analysis of these compounds in the juice samples was performed and five of the six anthocyanins detected in the previously described hydroethanolic extract were found in the colouring formulations: cyanidin-*O*-hexoside-*O*-hexoside, cyanidin-3-*O*-glucoside, cyanidin-*O*-rhamnoside-*O*-hexoside, peonidin-3-*O*-glucoside, and peonidin-*O*-rhamnoside-*O*-hexoside. The difference in the detected compounds could be explained by the different kind of extract, which for anthocyanins analysis was hydroethanolic and for the colorant's preparation was the centrifuged fruit juice; or even the temperature to which the juice was subjected in the pasteurization and spray-drying processes.

Regarding the spray-drying process, due to the high concentration of sugars in haskap juice, it was not possible to prepare a control sample without the stabilizing materials, thus, two formulations were successfully prepared with good yields: haskap + maltodextrin (80%) and haskap + maltodextrin (40%) + arabic gum (40%), presenting yields of 46 and 51%, respectively.

To evaluate the stability of the prepared formulations, the total concentration of these compounds was considered, alongside with the colouring parameters (L\*: lightness, white–black; a\*: red colour intensity, red–green; and b\*: yellow colour intensity, yellow–blue), both measured at 4, 8, and 12 weeks for samples stored at room and refrigerated temperature. In terms of lightness, the formulation containing maltodextrin + arabic gum allowed higher values (Figure 2a), as well as for the red (Figure 2b) and yellow (Figure 2c) colours' intensity. Even though this formulation presented better results concerning colour parameters, the anthocyanin levels were very similar for both preparations (Figure 2d). Nevertheless, it presented a higher stability over time, with less notorious variations with time and storage temperature than the colorant containing only maltodextrin, as it can be observed in Figure 2a–c. The slight variation observed in the total anthocyanin concentration confirmed the efficient protection of these compounds over time, making spray-drying a suitable methodology for anthocyanin-based colorants development.

In order to guarantee the safety of the prepared colorants, the samples were submitted to microbial load evaluation considering the microorganisms that could represent a risk for the formulation's stability over the time by causing degradation. These formulations were also evaluated in terms of cytotoxicity in non-tumour cells to assure the safety of their consumption. These analyses were performed after samples preparation (t0) and after 12 weeks of storage. According to the obtained results (data not shown), no microbial counts were possible to detect, meaning that the pasteurization process together with application of the spray-drying technique were able to maintain the stability of the colouring formulations. The spray-drying technique allowed the formulation to be in powder form, which drastically decreases the microbial contamination over time. Regarding cytotoxicity, the samples did not reveal any toxic effects on the non-tumour cells at the maximum tested concentration of 400 μg/mL, corroborating its safety for application in foodstuff, which is a very important result, once the objective was to prepare food colourants.
