Determination of the stability of beeswax encapsulated red beetroot extract in kefir

Ayan, Kübra
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Institute of Science and Technology
Red beet is a plant belonging to the family of Amaranthaceae and is among 10 vegetables with high antioxidant activity. The antioxidant effect in the red beetroot is due to its phenolic and betalain compounds. Betalaines are water-soluble and nitrogen-containing color pigments that give a characteristic color of red beetroot. Betalains are separated into two main groups, these are betacyanins and betaxanthins. While betacyanins have red – violet color pigment, betaxanthins have yellow – orange color pigments. The reasons for this difference are due to the different chemical structures and formation pathway. They play an important role in the scavenging of free radicals and the prevention of oxidative stress-related diseases because of their high antioxidant potency. Betalaines can also be used as coloring agents in foods because they are suitable for use at a wide pH range (3 - 7) and can naturally provide the desired color. Especially in recent years, the use of betalaines has become widespread as consumers have preferred natural additives in food products. Taking all these into account, betalaines can be used for functional food production. However, the low stability of these substances limits its use in the food industry. The main factors affecting stability can be listed as follows; temperature, water activity, enzyme activity (polyphenol oxidase, peroxidase, glucosidase enzymes), presence of oxygen, light and metal ions. The most decisive factor is the temperature. Encapsulation is a very effective method to stabilize betalaines because it can protect betalains from environmental factors which make them unstable. In this process, the active substance is trapped in a matrix having a protective structure. In betalain encapsulation, various materials can be used as coating material; these substances are generally classified as carbohydrates, proteins and oils / fats. In selecting appropriate coating material; the encapsulation technique to be applied, the targeted solubility characteristics, the process conditions and the food matrix are effective factors. In this study, the red beetroot extracts were encapsulated into beeswax by using the emulsification method to enhance its stability. The reason of preferring beeswax as coating material is to prevent degradation by protecting the active ingredient from oxidation and hydrolysis. For this purpose, red beetroot extracts were obtained by using a mixture of ethanol and water then it was lyophilized and stored at -20C for further analysis. After that, the encapsulation was carried out and the capsules were prepared using different active ingredient / coating material ratios (1: 1, 1: 2, 1: 3 and 2: 3 w / w). In order to determine the contents of beet extracts and the capsules; total phenolic content, total flavonoid content and betalain amounts were determined by spectrophotometric and chromatographic methods. Antioxidant activity of the extract and capsules was also determined by DPPH and CUPRAC analysis. Finally, color measurements were conducted for the extract and capsules. Particle size analysis, zeta potentials and encapsulation activities of the capsules were determined to select the optimal encapsulation condition. The encapsulation efficiency was determined by using total phenolic content analysis. After the appropriate encapsulation rate was determined, the capsules were incorporated into the kefir formulation to examine the effect of the encapsulation on stability. The kefir samples were collected on 1st, 7th, 14th and 21st day of storage and the contents and antioxidant activities were controlled. Besides that, the color measurement was also conducted in order to detect color change during storage time. In addition to capsules added kefir (KC), plain kefir (K), extract added kefir (KE) and empty wax capsules added kefir (KB) samples were prepared as control samples. All kefir samples were stored at 4C and in the dark during storage. The content of the obtained extract is as follows; total phenolic content is 2448.38  179.84 mg GAE / 100 g extract, total flavonoid content is 4001.45  71.91 mg CE / 100 g extract, betacyanins content is 453.99  4.12 mg BE / 100 g extract and betaxanthin content is 360.08  2.44 mg VE / 100 g extract. Apart from spectrophotometric analysis, phenolic and betacyanin profile were also determined by HPLC, and the results show that the extract includes ferulic acid, hydroxybenzoic acid, vanillic acid, ethyl 3,4 dihydroxybenzoate, gallic acid, catechin, epigallocatechingallate, syringic acid, vanillin, hesperedin, caffeic acid, chlorogenic acid, p-coumaic acid, sinapic acid, rosmarinic acid, luteolin, quercetin, rutin, myricetin, epicatechin, betanin and isobetanin. The antioxidant activity of the extracts was determined as 42.29  0.72 mM TE / g extract in DPPH analysis and 59.72  1.12 mM TE / g extract in CUPRAC analysis. The color values of the extract were measured as L* = 31.30  1.97, a* = 33.13  1.08, b* = 8.11  0.38, C* = 34.10  0.34 and h= 13.76  0.59. Both particle size and encapsulation efficiency were examined in capsules prepared at different ratios to determine the optimal encapsulation condition. According to the results, the particle size of the capsules was found to be between 265.9  5.29 and 414.3  28.87 nm and it was observed that the ratio of different active substance / coating material was significantly effective on the obtained particle size (p < 0.05). In addition to the particle size, the encapsulation efficiency was also determined and the highest efficiency was found in 1: 2 ratio as 67.16% while the lowest efficiency was found in 2:3 ratio as 42.18%. Besides that, it was observed that capsules prepared at different ratios have significant effect on the encapsulation efficiency (p <0.05). When all these are taken into consideration, the 1:2 ratio, in which the highest encapsulation efficiency is detected, has been chosen to carry out further studies. The contents of the prepared capsules are as follows; total phenolic content is 101.68  14.75 mg GAE / 100 g capsules, total flavonoid content is 103.12  7.38 mg CE / 100 g capsules, betacyanins content is 26.95 ± 1.06 mg BE / 100 g capsules and betaxanthin content is 36.03 ± 0.35 mg VE / 100 g capsules. In addition to spectrophotometric assays, phenolic profile and betacyanin profile were also determined by HPLC. According to the results, the capsules include; hydroxybenzoic acid, vanillic acid, epigallocatechingallate, vanillin, caffeic acid, p-coumaric acid, ferulic acid, gallic acid, catechin, epicatechin, syringic acid, chlorogenic acid, betanin and isobetanin. The antioxidant activity of the capsules was determined as 3.05  0.13 mM TE / g capsule in DPPH analysis and 2.40  0.10 mM TE / g capsule in CUPRAC analysis. The color values of the extract were measured as L* = 68.93 0.56, a* = 17.75 0.57, b* = 16.11  0.18, C* = 23.97 0.19 and h = 42.23 0.65. The capsules which are detected for encapsulation efficiency and contents were added to kefir formulation for stability study during storage time (21 days). There was no significant difference in the phenolic content of KC and KE samples after 21 days (p > 0.05) according to phenolic content results obtained. However, KC samples have higher phenolic content during storage period than phenolic content of KE sample (p < 0.05). The same results were also observed in total flavonoid conent of KE and KC samples. Although there are no significant differences in phenolic content of KE sample, the HPLC results showed that some individual phenolic compounds got lost during storage time. On the other hand, phenolic profile in KC sample remained stable during whole storage time (p > 0.05). The main reason of vanishing some phenolic compounds is thought as phenolic – milk protein interactions. In addition to phenolic and flavonoid content of the kefir samples, betacyanin and betaxanthin content were also measured in a specified time intervals. While both of betacyanin and betaxanthin content in KC could be preserved during 21 days, the initial betacyanin and betaxanthin content in KE sample are significantly different in 7th day of storage (p < 0.05). The obtained results were corrected with HPLC analysis of betanin during storage. Therefore, beeswax encapsulation could preserve phenolic compounds and betanins during storage of kefir samples. Apart from phenolic, flavonoid and betalain content analysis, antioxidant activity was also controlled during storage of the kefir samples by DPPH and CUPRAC assays. In DPPH assay, KC sample showed more antioxidant activity than KE sample (p < 0.05) and antioxidant activity in both sample did not show any significant difference during storage. In CUPRAC analysis, KE sample has more antioxidant activity than KC sample but this difference is not significant in 1st and 21st day of storage. The initial antioxidant activity of KE samples could not be protected until 7th day and there is a significant decrease (p < 0.05). On the other hand, antioxidant activity of KC sample remained stable during 21 day. As a result, it appears that beeswax encapsulation can also protect antioxidant activity. Lastly, color measurements were conducted to detect color changes during storage. The results show that there are no significant color change in KE sample but lightness, redness, yellowness and chroma value of KC sample increased significantly during storage. The reason of it may be releasing red beetrrot extract from beeswax into kefir. Taking all these results into consideration, beeswax encapsulation showed positive effect on preserving of betalain and phenolic compounds. Nevertheless, the further stability studies are needed to fully observe the effect of the beeswax encapsulation on the stability.
Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2018
Anahtar kelimeler
red beetroot, kırmızı pancar, fermented foods, mayalanmış gıdalar