Study of bioavailability and bioactivity of black carrot polyphenols using digestion models combined with a novel co-culture model of intestinal and endothelial cell lines

Nowadays, black carrots, originating from Turkey and the Middle and Far East, have gained increasing interest due to their substantial content of polyphenols, especially anthocyanins. Black carrots are often not consumed as such, instead they are either processed into other products or used as food colorants. Processing of black carrots generates large amounts of by-products, which are often discarded as waste or, in the best case valorized in low-value applications such as animal feed. However, the plant processing by-products of black carrots have the potential to be used as relatively cheap but valuable resources of polyphenols, which could be used in the development of functional foods.

Considering the above, a research framework to study the bioavailability and bioactivity of black carrot polyphenols from various sources has been developed. The objectives of this Ph.D. thesis were (i) to determine the effect of food processing and storage on black carrot polyphenols; (ii) to investigate the bioaccessibility and intestinal transport of key polyphenols in black carrots, processed products and their agronomic by-products using various in vitro gastrointestinal digestion and absorption models; (iii) to valorize the by-products from black carrot via enrichment of food products; (iv) to develop an in vitro model that is able to combine absorption effects with changes in endothelial cell metabolism.

To achieve these goals, four different experiments (Chapters 3-6) were conducted. Firstly, black carrots were processed into jams and marmalades and stored in the dark and the changes in polyphenol content were monitored (Chapter 3). Then, black carrots, jams and marmalades, as well as plant processing by-products of black carrot, i.e., peel and pomace, were subjected to in vitro gastrointestinal digestion (Chapters 3, 4). Following that, black carrot pomace was used as a source of polyphenols to enrich cakes (Chapter 5). Finally, an in vitro co-culture of intestinal and endothelial cell model was developed to determine the ability of polyphenols from black carrots and by-products to modulate the inflammatory response in endothelial cells (Chapter 6).

In Chapter 1, research framework and objectives of this Ph.D. thesis are introduced. Following that, in Chapter 2, a comprehensive review on the bioavailability and bioactivity of polyphenols is presented, with a specific focus on black carrot polyphenols. Initially, black carrot polyphenols, i.e., anthocyanins and phenolic acids, and their related health effects have been introduced. Then, the studies investigating the effect of food processing and storage on black carrot polyphenols have been reviewed. Afterwards, factors affecting the bioavailability and the methods used to assess the bioavailability of polyphenols were discussed with an emphasis on black carrot polyphenols. Lastly, the impact of polyphenols on endothelium and the trends and potential applications of cell culture models for polyphenol research were described.

In Chapter 3, the effects of food processing, i.e., jam and marmalade processing, storage conditions and in vitro gastrointestinal digestion on total and individual polyphenol contents and total antioxidant capacity of black carrots were examined. Black carrot jams and marmalades were prepared traditionally using either sugar or sweetener and stored in the dark at two different temperatures (4 °C and 25 °C) over a period of 20 weeks. Total polyphenol contents and total antioxidant capacity were determined using spectrophotometric methods, whereas individual polyphenols were quantified using HPLC–PDA. The major anthocyanins detected in black carrots used in all experimental chapters (Chapter 3–6) were cyanidin-based with different sugar moieties, among them two were non-acylated (cyanidin-3-xylosyl-glucosyl- galactoside and cyanidin-3-xylosyl-galactoside), and three were acylated with sinapic acid (cyanidin-3-xylosyl-sinapoyl-glucosyl-galactoside), ferulic acid (cyanidin-3-xylosyl-feruloyl-glucosyl-galactoside) and coumaric acid (cyanidin-3- xylosyl-coumaroyl-glucosyl-galactoside). The results showed that although processing of black carrots into jams and marmalades resulted in significant decreases in polyphenol content and total antioxidant capacity (49.5–96.7%) (p < 0.05), after digestion polyphenols from processed products were found to be more bioaccessible compared to the ones in raw material (0.8–31.5%). In addition, after 20 weeks of storage the reduction in polyphenol content of samples stored at 25 °C (26.4–92.2%) was higher than samples stored 4 °C (19.0–46.4%). In conclusion, this chapter showed that black carrot jams and marmalades provide considerable polyphenol intake, which are preserved to a certain degree after storage and digestion, and hence these products can serve as novel sources of functional foods. The HPLC method applied in this chapter was used in the succeeding chapters (Chapters 4-6).

Processing of foods of plant origin including black carrot generates large amounts of by-products. These by-products represent a major disposal problem for the industry concerned; however, they are also promising sources of bioactive compounds. Considering that, in Chapter 4, we focused on the changes in polyphenols and total antioxidant capacity from black carrot and its by-products, i.e., peel and pomace, during in vitro gastrointestinal digestion, which consisted of a three-step procedure simulating the digestion in the stomach, small intestine, and colon. The results showed that although the amount of polyphenols decreased significantly as a result of digestion (23−82%) (p < 0.05), the pomace anthocyanins released at all stages of in vitro gastrointestinal digestion were higher than black carrot anthocyanins, suggesting that pomace may be a better source of bioaccessible anthocyanins. Overall, this chapter highlighted black carrot by-products as substantial sources of polyphenols, which may be used to enrich food products. In vitro digestion method developed in this chapter is used later in Chapter 6.

Taking into account the findings of Chapter 4, in Chapter 5, black carrot pomace was used to enrich cake samples. We investigated the digestive stability of polyphenols from black carrot pomace enriched cakes and monitored changes in their total antioxidant capacity using a new standardized static in vitro digestion model. Results showed that for undigested samples enrichment of cakes with black carrot pomace at levels of 5%, 10% and 15% caused a dose-dependent increase in anthocyanins (72 to 267 μg/g dw), phenolic acids (49 to 148 μg/g dw), total phenolics (54 to 202 mg GAE/100 g dw) and total antioxidant capacity (21−129 to 153−478 mg TE/100 g dw). During the in vitro digestion in the mouth and stomach the amount of polyphenols were reduced significantly (35−74%) (p < 0.05), whereas no anthocyanins were detected in the intestine after intestinal digestion. On the other hand, significant increases in total phenolics and total antioxidant capacity were obtained in the stomach and intestine (up to 5− and 12−fold respectively) (p < 0.05). Overall, this chapter demonstrated that black carrot pomace, which can serve as a functional ingredient might be utilized in the baking industry.

In Chapter 6, we determined the potential of polyphenols from black carrot and its by-products to modulate the inflammatory response in tumor necrosis factor α (TNF- α) stimulated endothelial cells co-cultured with differentiated intestinal cells. As the bioactivity of polyphenols depends on their bioavailability, gastrointestinal digestion as well as transepithelial intestinal absorption was also considered while evaluating the anti-inflammatory effects of polyphenols. The results indicated that after 4 h of treatment, the transport of polyphenols was higher for digested samples (1.3−7%) compared to undigested ones (0−3.3%). The transported polyphenols were able to regulate the secretion of pro-inflammatory markers, i.e., interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1), under normal and TNF-α induced inflammatory conditions. The most pronounced effects were observed with digested samples under inflammatory conditions, which significantly decreased the secretion of all markers from 120−203% down to 34−144% (p < 0.001). Eventually, the results of this chapter suggest that polyphenols from black carrot and its by- products may function through an inhibitory regulation of the inflammatory cascade in endothelial cells, which can maintain a sustainable cardiovascular effect under pre-existing low-grade inflammation caused by Western-type diets.

Finally, in Chapter 7, based on the outcomes of the previous chapters, the general discussions and conclusion, as well as the future perspectives on the bioavailability and bioactivity of polyphenols is presented. The status and main outcomes of this thesis were discussed under the headings of characterization of black carrot polyphenols, impact of food processing and storage on black carrot polyphenols, black carrot by-products as sources of polyphenols, bioaccessibility of black carrot polyphenols, intestinal transport of black carrot polyphenols and bioactivity of black carrot polyphenols. During the discussion on the bioaccessibility of black carrot polyphenols, special attention has been paid to comparison of different in vitro gastrointestional digestion models and the effects of processing and other dietary compounds. Similarly, while referring to the bioactivity of black carrot polyphenols, different antioxidant capacity assays were compared and co-culture models to study anti-inflammatory effects of polyphenols were discussed.