Siyah kuş kirazı (Aronia melanocarpa) ilave edilmiş yoğurtlarda antosiyaninlerin in vitro biyoyararlılığının incelenmesi

Milk and other dairy products are consumed all over the world. Among these, yogurt is one of the most popular and unique dairy products, which is being enjoyed for its refreshing taste and beneficial properties. It is easily digestible and has high nutritive and therapeutic properties. The importance of yogurt as part of a balanced and healthy diet is admitted by regulatory authorities and scientific institutions in most countries. An increasing number of epidemiologic and clinical proof suggests that yogurt consumption may act beneficially on weight regulation and metabolic risk factors. Aronia melanocarpa, known as black chokeberry are one of the richest plant sources of phenolic compounds especially anthocyanins, which have beneficial effects on health like prevention of cancer and cardiovascular diseases. It was demonstrated that, aronia berries exhibited different biological effects both in vitro and in vivo such as antioxidant, gastroprotective, hepatoprotective, and antiproliferative activities. Therefore, fruit added yogurts have been gaining more attention by consumers and manufacturers because of their additional health benefits beyond normal nutritional value. However, despite the presence of large amounts of polyphenols present in ingested food, only a very small portion can be actually absorbed. Which may be a result of processing factors or interactions between food components. In this study, in vitro simulated gastrointestinal digestion of anthocyanins from black chokeberry (Aronia melanocarpa) added to homemade yogurts was investigated in terms of bioaccessibility, antioxidant activity and the possible fate of anthocyanins. Two types of black chokeberry yogurts were prepared by adding pulp (1%) and minced fresh berries (10%), aiming to add similar amounts of anthocyanins. The pulp and berries were added to plain yogurt, which were prepared based on full fat cow's milk, after fermentation, or directly to the milk before fermentation. All batches were prepared in duplicate. Methanolic extracts of samples were analyzed for their contents of total monomeric anthocyanins, and total phenolics as well as their antioxidant capacity after 1 day and 8 days of storage. In addition to that, major individual anthocyanin content of juice, pulp, minced berries and chokeberry added undigested yogurts was analyzed by high performance liquid chromatography (HPLC) coupled with photodiode array detector (PDA). The dry matter content of pulp was 25.5 ± 0.4% while it was 17.1 ± 0.7% for fresh berries. Also, the dry matter content of all yogurt batches ranged between 14.8 and 15.3%. The highest total phenolic content and antioxidant capacity measured by DPPH assay were observed in fresh berries, which were found to be 3242±37.4 mg GAE/100 g DM and 22.2±0.49 mM TEAC/100g DM, respectively. In contrast, the anthocyanin content and antioxidant capacity measured by CUPRAC assay of the black chokeberry pulp was higher than the other samples, which were determined as 7564±139 mg cya-3-glu/100 g DM and 42.2±4.23 mM TEAC/100 g DM respectively. However, although the anthocyanin content of pulp was higher than the berries, no real difference was observed between antioxidant activity of pulp and berries. On the other hand, all measured values were greatly lower in chokeberry juice than in berries and pulp. By the HPLC analysis, four individual anthocyanins were observed in the extracts of chokeberry juice, berries and pulp as well as chokeberry added yogurt samples. These anthocyanins can be ordered from abundant to rare one as follow: cya-3-gal, cya-3-ara, cya-3-glu, cya-3-xyl. Cya-3-galactoside was the main anthocyanin in chokeberry juice, berries and pulp. It composed 70%, 68% and 66% of total chokeberry anthocyanins, respectively. No differences in any of the measured parameters was observed for the plain yogurts, indicating that batches were reproducible. In all batches, the highest total phenolics, total anthocyanins and antioxidant capacity were observed in berry added yogurts. On the other hand, the higher the anthocyanin content, the higher the total phenolic content and antioxidant capacity. In undigested yogurts, no effect of storage days on the anthocyanin content was observed (p>0.05). For both yogurts with added berries or pulp, a lower amount of anthocyanins was measured when the berries and pulp (p<0.001) were added before compared to added after fermentation. On the other hand, even when the fruits were added to the yogurt after fermentation, only 70% of the anthocyanins from the berries were measured, and even not 20% of the pulp anthocyanins. A significant effect on the total phenolic content of undigested yogurts was observed depending on the type of fruits added (p < 0.001) in the following order: no0.05). The 2-way interaction of type of fruit x type of fermentation was highly significant on the antioxidant capacity of undigested yogurts (p<0.001), as well as the main effects, type of fermentation and fruit (p<0.001). No significant effect of the 2-way interaction terms (type of fruit x storage days; type of fermentation x storage days) was observed (p>0.05 for each) on the antioxidant activity of undigested yogurts, as well as no influence of the storage days (p>0.05). During the buccal phase of digestion no or slight amount of phenolic compounds were observed in plain yogurts and pulp yogurts. On the other hand, the measured total phenolic compounds in salivary berry samples were much higher (approximately 83-84% of the total phenolic content of undigested yogurts) compared to plain and pulp added yogurts. After gastric digestion and intestinal digestion much higher total phenolic content was observed both in plain yogurts and pulp or berry added yogurts, even values that are higher than what was measured in the undigested samples. A higher antioxidant capacity observed in fruit added yogurts compared to plain and pulp added yogurts. The bioaccessibility of anthocyanins was greatly decreased after the intestinal digestion. Higher amounts of anthocyanins can be recovered in berry added yogurts compared to pulp added yogurts. The highest recoveries were 3.5% and 32.1% (for IN and OUT sample respectively) for pulp added yogurt as well as 6.3% and 34.3% (for IN and OUT sample respectively) for berries added yogurt. Our results show that, anthocyanins are more stable at acidic conditions and highly sensitive to pancreatic digestion. On the antioxidant activity, samples showed higher antioxidant capacity when they were analysed with CUPRAC method in comparison to DPPH method. In all berry added yogurt samples a higher DPPH radical scavenging activity and cupric ion reducing ability were observed compared to plain and pulp added yogurts and this higher antioxidant capacity remains stable during the digestion process. However the antioxidant activity substantially decreased in the dialysed fractions. The degree of hydrolysis increased when the samples were subjected to pancreatic digestion. No differences in the degree of hydrolysis were observed between the yogurt varieties in each digestion step except oral phase, reflecting that the digestion of proteins was not affected by the anthocyanins. According to SDS-PAGE analysis, Bovine serum albumin and caseins found to be the prevailing proteins in yogurt samples. A distinct band over the 66.2 kDa molecular weight area was observed in undigested yogurt samples which might be the BSA (bovine serum albumin). Also, outstanding bands were observed around 31 kDa molecular weight range which are likely to be the caseins. After the addition of saliva, the protein bands remained the same with undigested yogurts. At the end of the gastric phase, a clear degradation of the large molecular weight proteins to low molecular weight proteins was observed and undefined bands appeared in the low molecular weight region (<6.5 kDa) of the gels. Similar trends in gel patterns between BID-OUT samples and completely digested samples (PG-OUT) were observed. When the pancreatic solution was introduced, a further degradation of small molecular weight proteins resulting leaner bands in the low molecular weight range was observed and some new bands in the higher molecular weight area, which were indicated as digestive juice proteins by the other studies. The gel patterns of proteins showed that proteins were not affected by salivary digestion and started to degrade continuously until the digestion finishes. Further analysis of the different digestive fractions are needed to elucidate the fate of the anthocyanins i.e. interaction with major yogurt components, or transformation in new metabolites during digestion.