**1. Introduction**

Color is an important sensory characteristic that consumers rely on when making food choices, and even the slightest changes due to processing or during shelf life can deter a potential buyer and alter the consumer's perception of product quality. The loss of color pigment is an important shelf life factor for many food products, especially for those undergoing thermal treatment [1]. Anthocyanins are a large group of natural water-soluble pigments that are responsible for red, blue and purple colors in many fruits, vegetables, flowers, leaves, stems and roots [2,3], and their deterioration is an important factor in loss of color in many food products [1]. The bright colors of the pigments are due to the conjugated double bonds that are responsible for the absorption of visible light [4], and the pH dependent equilibrium allows their utilization as natural food colorants [3,5]. These pigments usually occur in the glycosylated polyhydroxy and polymethoxy form of 2-phenylbenzopyrylium salts (flavylium ion skeleton). Beyond their effect on color, the increasing interest in the research of these molecules stems from their reported various health promoting properties such as anti-inflammatory [6], anti-cancer [7] anti-cardiovascular [8] and other bioactivities [8–11]. As members of the polyphenol group, anthocyanins and anthocyanidins (the aglycone form of anthocyanins) possess antioxidant properties [8,9], the conjugated sugar moiety of the anthocyanins is known to reduce the radical scavenging activity as compared to aglycone [8,12]. It was suggested that attached sugar unit reduces the ability of the anthocyanin radical to delocalize electrons [8].

While anthocyanins are widely occurring in food products, they tend to be unstable during processing and storage and to be degraded and/or decolorized. Some studies explore the chemistry and stability of anthocyanins, mostly in fruit and vegetables and their processed products, but also in some model systems [9,12–16]. The stability is reported to be affected by pH, temperature, chemical structure of the pigment, concentration, solvents, oxygen, light, enzymes, etc. [2,3,9,17,18]. During processing and storage, anthocyanins degradation increases with the rise in temperature [13,19]. At room temperature, color is reported to be stable only in acidic media. In alkaline media, cleavage of the pyrylium ring takes place and decrease in color intensity is noticed [20]. It might be assumed that thermal degradation of anthocyanins begins in the opening of the heterocycle and the formation of the chalcone form [21,22]. Heating shifts the equilibrium towards the chalcone and the reversion of chalcone to flavylium is slow to impossible [22]. Thermal degradation of anthocyanins follows first-order reaction kinetics [23–25] and can be reduced by decreasing the pH [1,25]. Another suggested mechanism excludes the formation of chalcone glycoside form, and by combination of heat and pH levels (2–4) hydrolysis of the glycosidic bond occurs, followed by conversion of the aglycone to chalcone and then to degradation products [22]. At aqueous solution at pH 2–4, temperature elevation leads to the hydrolysis of glycosidic bond, resulting in the loss of sugar moieties of the anthocyanins, leading to further loss of color as the anthocyanidins are much less stable than anthocyanins [20]. The presence of the sugar group is responsible for increased water solubility and stability (compared to aglycone) [4,26], with the number of sugar rings also suggested to influence the stability [22,27,28].

Many food products, especially juices, are fortified with ascorbic acid (AA), a natural antioxidant, to protect against oxidation and to increase the nutritional value [29,30]. AA might have several negative influences on anthocyanins' stability. In the presence of oxygen, AA can accelerate the degradation of anthocyanins and enhance the formation of polymer pigment, which results in anthocyanins pigment bleaching [29–31]. The exact mechanism is still controversial and addition of AA to anthocyanins results in increase of the degradation rate of both molecules. The postulated mechanisms are either direct condensation of AA with anthocyanins or formation of hydrogen peroxide and oxidative cleavage of the pyrylium ring by peroxide [26,29,31]. In previous works, the focus of anthocyanins stability in the presence of AA is explored in food matrixes [32,33] or with a focus on specific molecules [29,31].

The aim of this study was to explore, systematically, in purified model systems, the influence of anthocyanins' structure (by increasing the size of the conjugated sugar), pH (6.5 and 4.5) and the presence of ascorbic acid on their stability and spectral properties during simulated shelf life. The degradation was tested in purified model systems to avoid possible interferences from food matrix. In addition, we aimed to better understand the kinetics of the formation of degradation products for the most sensitive of the tested anthocyanins—cyanidin. To our knowledge, no other work examines the stability of series of three molecules that differ only by the presence and type of sugar moiety, by high performance liquid chromatography system-mass spectrometry (HPLC-MS) analysis and quantification, monitoring of the spectral properties and identification of the degradation products in purified system.

## **2. Materials and Methods**
