**1. Introduction**

With consumer demands shifting towards more natural ingredients and clean labeling, functional vegetable concentrates containing a high carotenoid content present an attractive coloring alternative to natural and artificial color additives. Compared to the use of isolated carotenoids as food colorants, the advantage of "coloring foods" such as carrot concentrates, lies in the recovery without a solvent dependent, selective extraction step of the main coloring components, which is often perceived as more natural by consumers.

However, the replacement of artificial colors with coloring vegetable concentrates comes with certain challenges, which are linked to the oxidative susceptibility and the crystalloid nature of the carotenoids as active ingredient as well as the complex matrix containing multiple carrot derived co-components [1,2]. Carrot carotenoids (e.g., β-carotene, α-carotene, lutein), are lipophilic pigments which tend to form supramolecular aggregates in hydrophilic environments [3]. In raw carrots (*Daucus carota*), most of the carotenoids are present in a crystalline state derived from accumulated carotenoid aggregation in carrot chromoplasts [4]. The presence of crystalline carotenoids as well as a small fraction of monomolecular (dissolved) carotenoids is responsible for the typical orange hue of the carrot, whereas a yellow hue occurs when the carotenoid pigments are completely dissolved in organic solvents or lipids [5]. Thus, when color is a target function of carrot concentrate powders, both the monitoring of this supramolecular conformation as well as the total pigment concentration during processing and storage is of importance.

In some of their physical properties crystalline carotenoids differ from the lipid-dissolved carotenoids and have also shown to react differently to certain processing conditions such as heat [3,6,7]. While emulsions, in which carotenoids are dissolved in oil droplets, are suitable delivery systems for carotenoids, the emulsification process mostly requires dissolving crystals, upon which the characteristics (particulate character and hue) of the crystalline carotenoids are lost. For the preservation of the desired properties, processing and drying requires an approach with a minimal effect upon the crystalline structure. Understanding the impact of formulation and powder processing on the crystalline structure of the carotenoids, as well as their degradation during processing and storage is crucial in order to provide high quality functional food systems. Only limited information on the mechanism and the role of different impact factors for the stabilization of carotenoid in powders made from complex vegetable concentrates is available and the relevance of the crystallinity has never been studied in this context. Additionally, the analysis and identification of crystalline carotenoids within the complex matrix of plant concentrates still presents an analytical challenge, since their concentration within the concentrates is too low to apply methodology which can be used for the determination of crystallinity of pure and highly concentrated components (e.g., X-ray diffraction analysis and differential scanning calorimetry).

Due to a change in the optical properties of carotenoids when they change from the dissolved to the aggregated or crystalline state, their macromolecular conformation can be determined by optical methods such as UV/Vis spectroscopy and circular dichroism spectroscopy [8]. The measurement of the crystalline carotenoids in a hydrophilic dilution is hindered by the high turbidity of the carrot concentrate dispersion. On the other hand, carotenoid extraction through organic solvents, or concentration which is necessary for most carotenoid quantification methods, leads to a crystal dissolution, which prevents a characterization of the physical state. Hence, suitable analytical procedures have to be developed to derive information about the macromolecular conformation and its changes through processing within the turbid sample matrix.

The present study investigated the stability of carotenoid crystals as part of a natural carrot matrix during the preparation of spray dried (SD) and freeze dried (FD) powders and during further storage. Different process and recipe-based approaches to increase the shelf life of functional carrot concentrate powders were evaluated with regard to their effect on carotenoid stability and integrity of their crystalline state. Whereas the general impact of the investigated approaches is already known for many monomolecular active ingredients, the aim of this study was the quantification of the effect of aforementioned factors for the given complex food system containing crystalline carotenoids. Since the shelf life of carrot concentrates is strongly linked to the preservation of the hue provided by the crystalline state of the carotenoids as well as by their concentration, both the stability of the physical state and the pigment concentration during production and storage were monitored. An analytical approach was applied which allowed for the qualitative assessment of the physical state of the carotenoids based on their UV/Vis spectrum. Thereby, it was possible to specifically monitor the impact of the different formulation strategies on the stability of the macromolecular conformation.
