Effects of liposome encapsulation, spray drying, and ascorbic acid presence on color stability of black carrot anthocyanins

In the food industry, food colorants determine the food preferences of customers as color of a food is a function of freshness, type, ingredients and production method. In addition, it also affects customers' purchase behavior since an appealing color is highly desired by consumers. In today's world, food products are transported to far away places where they will be consumed. This may result in degradation and appearance loss. In addition, food producers desire to sell their products with an optimum color, and, therefore, food colorants are used extensively. In recent years, the increasing tendency of customers to consume healthy foods has led the food industry to seek natural colorants instead of stable synthetic colorants due to possible harmful effects of synthetic colorants. Black carrot anthocyanins are water soluble and pH sensitive natural pigments. Black carrots are currently grown in the center part of Turkey as a highly nutritive crop. In addition, it is currently used in shalgam production and the canned food industry due to its high anthocyanin content. Furthermore, black carrot (Daucus carota var L.) is a potential source of acylated anthocyanins. Thus, black carrot anthocyanins are relatively stable. Nevertheless, they still suffer from degradation, that is they loose their color over time, especially in systems containing other reacting compounds such as vitamin C. In a food system, encapsulation is generally used to create a barrier between the component and environment. Encapsulation may be a suitable method to enhance their stability to the point where they may be used to replace synthetic colorants. In this research a framework on characterization of black carrot anthocyanins and liposome encapsulation effect on stability of black carrot anthocyanins has been developed. The objectives of this Ph.D. thesis were (i) to optimize black carrot extraction using Response Surface Methodology (RSM) by evaluating anthocyanin concentration, phenolic content, antioxidant activity, and color attributes of black carrot; (ii) to compare the stability of black carrot anthocyanins, which are highly stable due to their large amount of acylated anthocyanins, with and without a liposome system in the presence of ascorbic acid; (iii) to understand the effects of both intramolecular copigmentation and liposome encapsulation on ascorbic acid related anthocyanin degradation; (iv) to investigate physical and chemical stability of liposomes with and without black carrot during storage; (v) to evaluate the stability of liposomes with black carrot extract during the spray drying process. In first part of the thesis, optimum conditions for the extraction of black carrot anthocyanins were determined by response surface methodology. Central composite design of extraction factors (pH 2.5–6.5, temperature 4–72 °C, solvent/solid ratio 5:1–25:1 v/w, ethanol/water ratio 0:100–100:0 v/v) was generated as two replicates. Total phenolic content, total monomeric anthocyanin content, polymeric color, total antioxidant activity, and anthocyanin composition determined by high performance liquid chromatography (HPLC) were used as responses. Except for color values, higher temperature, solvent/solid ratio, and ethanol concentration were observed to increase the extraction yield. However, polymeric color results were found to have minimum values at lower pH and solid/ solvent ratio, lower or moderate temperature, and higher ethanol concentration. Optimum extraction conditions were found as follows: 50 °C, pH 3.5, solvent solid ratio 10:1 (v/w), and ethanol/water ratio 75:25 (v/v) when all responses were considered. The validation of the optimum conditions for black carrot extraction was performed at specified values. Optimum extraction conditions were valuable to protect anthocyanins at the first step for further processes. In the second part, stability of black carrot anthocyanins is evaluated in the presence of L-ascorbic acid. The degradation of vitamin C (L-ascorbic acid) on anthocyanins is a known fact in beverage systems. In this part, the effects of various liposomal systems, including black carrot extract (0.1%, 0.2%, 0.4% w/w) and lecithin (1%, 2%, 4% w/w), on the color and degradation of anthocyanin in different ascorbic acid (0.01%, 0.025%, 0.05%, 0.1% w/w) concentrations were examined via UV/VIS spectroscopy and visual control of the color. The physical characteristics of the liposomal systems resulted in particle diameters of 41–46 nm and zeta-potentials of (−23)–(−20) mV. The encapsulation efficiencies of the liposomal systems increased up to 50% with increasing lecithin concentrations. The encapsulation of black carrot extract in liposomes enhanced the color and stability of the anthocyanins during storage. This study showed that the degradation of anthocyanins due to ascorbic acid can be reduced by liposomes in aqueous solutions. In the third part, the protective effect of the anthocyanin acylation and liposomal encapsulation of black carrot extract (BCE) on anthocyanin degradation was investigated in the presence of ascorbic acid. BCE (0.1%, 0.2%, 0.4% w/w) were encapsulated in liposomes using different lecithin concentrations (1%, 2%, 4% w/w). Physical characterization of liposomes was performed. Particle diameters were found to be less than 50 nm and ζ-potentials of (-23)-(-20) mV. Encapsulation efficiency determined by HPLC indicated that increasing lecithin concentrations raised the efficiency at the same BCE concentration up to 60%. Different concentrations of AA (0.01%, 0.025%, 0.05%, 0.1% w/w) were added to the BCE solutions and liposomes (1%, 2%, 4% lecithin) containing BCE (0.2% w/w). AA related color loss and individual anthocyanin degradation were examined by UV/VIS spectroscopy and HPLC, respectively. This study presented the potential positive effect of liposomal encapsulation on individual particularly acylated anthocyanins (sinapic, ferulic and p-coumaric acid derivates of cyanidin-3-xylosylglucosylgalactoside). In the fourth part, the protection effect of black carrot anthocyanins on the stability of liposome was evaluated during storage. Physical and chemical stability of extracts (0.1%, 0.2%, and 0.4% w/w) including liposomes with various lecithin content (1%, 2%, and 4% w/w) were demonstrated. Z-average particle diameter and zeta potentials of liposomes were evaluated before and after 21 days of storage. Particle diameter of samples were found to be lower than 50 nm after storage and no statistical change was determined (p<0.05). Extract degradation, phenolic content, and antioxidant capacity of liposomes were also evaluated during storage to evaluate the biochemical stability. Hexanal analysis was performed to investigate the lipid oxidation in liposomes within 60 days. According to the results, oxidation of polyunsaturated fatty acids may be inhibited with the addition of extract to liposomes, however, lipid content should be limited according to the extract concentration. The study provided valuable data on the contribution of anthocyanin addition to liposomes to overcome oxidation of unsaturated phospholipids. In the final part, spray drying of black carrot anthocyanin were evaluated in different encapsulation systems (extract only, primary and secondary liposome systems and chitosan extract mixture). BCE included primary and secondary liposomes were successfully spray dried. All samples were found physically stable after spray drying processing. In addition, SEM images of liposomal systems showed powder without any crack that indicates stability of liposome systems to spray drying. However, liposome samples showed lower stability compared to BCE and BCE and chitosan mixtures according to the biochemical analysis of reconstituted powders. To sum up, detailed analyses of colorant potential of black carrot anthocyanins in encapsulation and ascorbic acid conditions were evaluated. Short term storage results showed liposome encapsulation may provide enhancement of color attributes of black carrot in the presence of ascorbic acid. In addition, our liposomal system are clear and transparent and suitable for beverage industry. However, color loss in liposomal system was observed in long term storage. This may be prevented with copigmentation with other phenolics, second layer on liposomes, or using saturated lecithin source. On the other hand, results showed us anthocyanins can be used to reduce the oxidation rate of liposomes. Those studies lead us increase of copigmentation in liposomal system may result in mutual protection. Therefore, encapsulated black carrot anthocyanins can be used as natural colorants stable in process conditions.