INTRODUCTION:

Plant extracts for development of various products including functional drinks have received importance in recent years. The trend is prompted by the ample bioactive compounds in the plant, in which those components are reported to bring health-promoting effects¹. Red dragon fruit, also popular as pitaya², has gained popularity in many countries. The flesh is consumed, while the peel is discarded. And now, the fruit’s peel is extensively researched for its potential use as food ingredients for reasons³.

Dragon fruit, both flesh and peel, is rich in antioxidant compounds such as betacyanin, anthocyanin and cyanidin⁴. Furthermore, dragon fruit peel contains flavonoid, vitamins (A, C and E), polyphenols and betacyanin⁵.

Functionality of drink from red dragon fruit peel could be enhanced by enrichment with gelatin. Poungchawanwong et al (2020)⁶ reported that addition of gelatin could raise content of precipitated tannin in dragon fruit extract, which make it easily excluded during filtration, leading to increase of anthocyanin content. Catfish (Clarias gariepinus sp) bone could be one of the promising alternatives to source of gelatin. Catfish bone gelatin for functional drinks seems to be more prospective over mammalian gelatin due to reasons such as halal concern and contribution to waste disposal.

Dragon fruit peel-based drink relies on color stability and functional properties. Color produced from the plant pigments is conditioned at a stable level, which means that functional properties of the drink are also unchanged. To maintain color stability, some factors are controlled strictly, such as pH, oxidative agents, light and temperature. This present work aimed to formulate a functional drink made from red dragon fruit peel and catfish bone gelatin.

MATERIALS AND METHODS:

Materials:

Sample was made from red dragon fruit peel, catfish bone gelatin and sweetened with cane sugar. The analysis of samples used chemicals ethanol 95% (Merck), DPPH (1,1-diphenyl-picrylhydrazyl) (Merck), methanol (pa) (Merck), citric acid (Sigma Aldrich), FeCl₃ (Merck), citrate buffer (SigmaAldrich), Folin-Ciocalteu (Merck), anhydrate Na₂CO₃ (Merck), hydrogen peroxide (Merck), and PCA medium (SigmaAldrich). The instruments were listed as follows: freeze dryer (Mechatech), spectrophotometer UV-Vis (Shimadzu), cuvette, plastic containers, knife, oven (Memmert), rotary evaporator (IKA), water bath (Memmert), heater (Heidolf), ose needle, incubator Memmert, reaction tubes, petri dish, Erlenmeyer, glass wares.

Preparation of peel extract, catfish bone gelatin and their mixture:

Extraction of red dragon fruit was carried out using maceration. Peels (5kg) were washed using distilled water and air-dried for 1 week. After dried, the dry peel was pulverized to convert it into powder. The peel powder (50g) in a beaker glass was added with 250mL of distilled water and macerated for 1 week. The sample was then filtrated through Buchner filter and evaporated using rotary evaporator. The filtrate was collected for further use.

Catfish bone gelatin was prepared using maceration using HCl 4% for 48 h. After completing maceration, leached bone (ossein) was thoroughly washed to reach pH 6 – 7, then extracted in water bath for 5 h at 80°C. The maceration was replaced with 4% citric acid for 48 h, and the ossein was maintained at pH 6. The filtrate obtained was evaporated using oven at 80°C for 8 h.

Gelatin (3 g) was dissolved in 100mL of distilled water, agitated and heated in magnetic hot plate for 30 min at 60 °C. The solution was added with 3 mL of peel extract solution, homogenized for 15 min at 60°C. After cooled, the mixture was frozen and dried in freeze dryer (Raj and Dash, 2020) to yield peel extract-catfish bone gelatin powder.

Preparation of drinks:

The peel extract-gelatin powder was dissolved in water at following concentration: A1 = 1%, A2 = 3% and A3= 5%. The sugar was added to the solution at B1 = 10% and B2 = 15%, then heated until boiled. Citric acid was also added to the drink at C1 = 0,5%, C = 1% and C3 = 1,5%. The combination of these variables (powder, sugar, citric acid) was presented in Table 1. All drink samples were filled to bottle and sterilized for 15 min.

Table 1: Formulation of functional drinks

Code	Description
1	Peel extract-gelatin powder 1%, sugar 10%, citric acid 0,5%
2	Peel extract-gelatin powder 1%, sugar 10%, citric acid 1%
3	Peel extract-gelatin powder 1%, sugar 10%, citric acid 1,5%
4	Peel extract-gelatin powder 1%, sugar 15%, citric acid 0,5%
5	Peel extract-gelatin powder 1%, sugar 15%, citric acid 1%
6	Peel extract-gelatin powder 1%, sugar 15%, citric acid 1,5%
7	Peel extract-gelatin powder 1%, sugar 10%, citric acid 0,5%
8	Peel extract-gelatin powder 3%, sugar 10%, citric acid 1%
9	Peel extract-gelatin powder 3%, sugar 10%, citric acid 1,5%
10	Peel extract-gelatin powder 3%, sugar 15%, citric acid 0,5%
11	Peel extract-gelatin powder 3%, sugar 15%, citric acid 1%
12	Peel extract-gelatin powder 3%, sugar 15%, citric acid 1,5%
13	Peel extract-gelatin powder 5%, sugar 10%, citric acid 0,5%
14	Peel extract-gelatin powder 5%, sugar 10%, citric acid 1%
15	Peel extract-gelatin powder 5%, sugar 10%, citric acid 1,5%
16	Peel extract-gelatin powder 5%, sugar 15%, citric acid 0,5%
17	Peel extract-gelatin powder 5%, sugar 15%, citric acid 1%
18	Peel extract-gelatin powder 5%, sugar 15%, citric acid 1,5%

Sample Analysis:

a) Analysis of color stability:

Color changes of products were evaluated according to their stability against pH and presence of oxidative agents. Th drink samples (2mL) were adjusted to 3, 4 and 5 by mixing with 100mL of citrate buffer. The absorbance was measured at 510 – 550nm. The stability was also tested in presence of oxidative agent. A total of 10mL of drink sample was mixed with 1mL of H₂O₂ as oxidative agent. The absorbance was observed each 3 h at a wavelength of 510-550nm.

b) Determination of phenol content⁷:

Quantification of phenolic content followed method of Folin-Ciocalteu. Different standard concentrations were prepared: 0, 2, 4 and 8ppm, and added with 0,8mL of Folin reagent, then transferred into a 10mL-volumetric flask. 5% Na₂CO₃ was then added up to the mark. After 60 min standing, the absorbance was measured at 750 nm. For samples, the procedure was similar by taking 1 mL of sample from the 10 mL-volumetric flask.

c) Determination of antioxidant activity⁸:

One mL of DPPH reagent (400μM in methanol) was transferred into a reaction tube. The tube was added with 3 mL of methanol and 0.1mL of sample (concentration previously prepared at 10, 50, 100, 150 and 200ppm). The mixture was vortexed and analyzed for absorbance at 517nm after 25 min of incubation. The violet color of DPPH will turn into yellow while decreasing the intensity⁹.

d) Quantification of microbes¹⁰:

Total microbial count was quantified by using dilution method. One mL of sample from each dilution was aseptically moved to petri dish, then filled with 12-15 mL of Plate Count Agar (PCA, 40 – 45 °C) and homogenized. The blank petri dish containing PCA and diluting solution was also prepared. After filled with PCA, the dish was left to solidify at incubator (37°C) at reverse position for 48 h.

e) Hedonic test¹¹:

The test aimed to evaluate degree of consumer acceptance and satisfaction from 30 panelists regarding product attributes (taste and aroma). Panelists were asked to score sample according to 1-7 hedonic scales: 1 (dislike very much), 2 (dislike moderately), 3 (dislike), 4 (neither like nor dislike), 5 (like), 6 (like moderately) and 7 (like very much).

f) Statistical analysis:

Sample analysis was made at duplicate. The data evaluation followed analysis of variance (ANOVA). Significant difference between means were verified using Tukey test at significance level of p<0,05.

RESULTS:

Figure 3 depicts appearance of 18 formulated products, indicating that red color appears more intense as more dragon fruit peel extract is added.

Figure 1: Decomposition of betacyanin (red) into betalamate acid (yellow)

Figure 2: Deglycolysation of betacyanin (red) which forms betanidin (purple)

Figure 3. Appearance of dragon fruit peel-based functional drinks

Color Stability As Response to pH and Oxidizing Agent:

Color stability is essentially reliant on anthocyanin since it is dominant pigment in dragon fruit peel extract. As shown in Figure 4, variability of absorbance represents color change induced by different levels of pH. Furthermore, stability of product’s color due to presence of oxidizing agent was also evaluated (Figure 5).

Figure 4: Changes in absorbance of dragon fruit peel-based functional drinks as response to citrate buffer enabling to destabilize betalain pigment as indicated by reduced absorbance.

Figure 5. Changes in absorbance of dragon fruit peel-based functional drinks as response to oxidations

Total Phenol:

Figure 6 displays variation of phenol content in formulated drinks, ranging from 53,86 to 139,46 mg GAE/L.

Figure 6: Differences in phenol content (mg GAE/L) of dragon fruit peel-based functional drinks

Antioxidant Activity:

In general, variation of antioxidant activity for all samples tested is depicted in Figure 7 with anthocyanin to form brown color.

Figure 7: Differences in antioxidant activity (expressed as IC 50) of dragon fruit peel-based functional drinks

Total Microbial Count:

As depicted in Figure 8, the microbes ranged from 2,59 to 5,49, which is equal to 4,9 ´ 10¹colony/mL - 1.35 ´ 10⁵colony/mL

Figure 8: Total microbial count in all samples tested

Organoleptic Quality:

As presented in Figure 9, the sample tested receives good score for taste and color (average point of >4).

Figure 9: Results of hedonic test on sample 13 for color, aroma and taste attributes

DISCUSSION:

Betacyanin as a key colorant in dragon fruit peel is more soluble in water than in non-polar solvent¹². betanin content that has potent antioxidant capacity¹³. The pigment in dragon fruit includes cyanidin 3-ramnocyl glucoside 5-glucoside. Under alkaline, anthocyanin pigment is unstable as indicated by color changes from red to yellow. The transformation results from decomposition of betacyanin into betalamate acid and cyclo-DOPA-5-0-glycoside, meanwhile at very low pH, betacyanin changes remarkably into betanidin after deglycosylation. Figure 1 demonstrates the decomposition of betacyanin into betalamate acid.

In acid condition, betacyanin shifts into betanidin (purple) via deglycosylation (Figure 2). The betacyanin-glycoside bonds are acetal binding which is prone to degradation induced by strong acids. Therefore, the breakdown of glycoside bond of betacyanin occurs in very low pH condition.

Regarding chemical structure of betacyanin, it originates from an aromatic amino acid 3,4-dihydroxylphenylalanine (DOPA), which produces purplish red color for leaves and fruits. It is a water-soluble and nitrogen-containing pigment, and its synthesis is assisted by light. Betacyanin belongs to member of betalains which have two groups, i.e. betacyanin (purplish red) with λmax of 534 – 555 nm and betaxanthin (yellow) with λmax of 480 nm. Compared to anthocyanin, betacyanin is weaker against acid hydrolysis. Besides pH, stability of betacyanin is affected by water activity, temperature, light, oxygen and peroxide¹⁴.

Betacyanin occurs in dragon fruit peel, which make it red-purple in color. Roriz et al. (2017)¹⁵ reported that betacyanin color could be stronger up to 3 times in comparison with red-purple color from anthocyanin. F

Color Stability As Response to pH and Oxidizing Agent:

Color stability is essentially reliant on anthocyanin since it is dominant pigment in dragon fruit peel extract.

The results show that absorbance varies greatly depending on pH. Herbach et al. (2005)¹⁶ stated that betalain was found at pH of 4 – 6 either in presence of oxygen or anaerobic. Sample 13 shows the highest absorbance in all levels of pH. However, absorbance for sample 14 and 15 tends to decrease as increase in concentration of citric acid, which represents reduction of pigment stability. In higher content of sugar as shown in sample 16 – 18, the absorbance raises remarkably which may link to increment of sugar added to the product. The increase of sugar reduces water activity and improves pigment stability.

The results indicate that higher proportion of peel extract-gelatin powder in formulated drinks (3% and 5%) produces better stability than those added with lower proportion (1%). The stability is reduced up to 36 – 50%.

Red dragon fruit peel is rich in polyphenols; thus, it is source of antioxidant. Antioxidant activity of red dragon fruit peel was reported higher than that of flesh¹⁷. The importance of antioxidant compounds in foods is based on their capability in blocking oxidation, which in turn, improves color stability. Hydrogen peroxide is an oxidizing agent, enabling to destabilize betalain pigment as indicated by reduced absorbance.

Total Phenol:

The highest level of phenol was found in sample 16, as the sample was made from the highest proportion of peel powder-gelatin powder (5%). In other cases, phenol content decreased as more sugar was added to products. Phenolic compounds such as quercetin, kaempferol and myricetin are easily bound with sugar molecules. Phenolic compound are secondary metabolites synthesized in plants¹⁸. As a consequence, more sugar in product formulation enables to raise presence of flavanol and more sugar is bound. This increases the formation of sugar-flavanol complex, thereby reducing solubility of polyphenols¹⁹. Quantity of total phenol can be affected by changes in pH, which may result in differences in phenol concentration²⁰.

Antioxidant Activity:

It is important to note that antioxidant activity is weak as represented by IC 50 of 3100-6760 ppm (>500ppm)²¹. Main factor contributing to low antioxidant activity is the impurity of extract containing flavonoid compounds or phenolic compound²². In this case, flavonoid may still bind with glycoside groups. Statistically, ANOVA reveals that formulation affect significantly antioxidant activity (p<0,05), despite no significant difference among treatments according to Tukey test. Ascorbic acid were taken as standard antioxidant²³.

Addition of citric acid to formulated drink could improve antioxidant activity as revealed by reduction of IC 50²⁴, in contrast, presence of sugar added to sample reduced antioxidant activity of sample²⁵. Depletion of anthocyanin level due to sugar breakdown into furfural and 5-hydroxymethyl-furfural which then react with anthocyanin to form brown color.

Total Microbial Count:

Total microbes on drinks (prepared with hot water, without boiled) were carried out. The prepared drinks have been stored for 1 year in glass bottles prior to analysis. The lowest microbial count occurs in samples added with citric acid of 1%. The acid is effective in deactivate bacteria (bactericidal effects) by collapsing cellular membranes, leading to loss of cellular enzymes²⁶. Antimicrobial effects of organic acids are based on combination between dissociated and non-dissociated molecules, which extremely reduced pH of cytoplasm, destroying cellular membrane and nutrient transport activity. These effects severely destabilize the cellular functions.

The highest microbial count was observed in samples added with peel extract-gelatin powder of 3%. This may relate to high nutrients, especially protein, from the added ingredients, providing nutrients for microbes to grow.

Organoleptic Quality:

Hedonic test was carried out only for sample 13 (added with peel extract-gelatin powder 5%; sugar 10%; citric acid concentration of 0.5%). Such formula results in stable phenolic content and produces high absorbance at pH of 4 and 5.

Functional drinks made from dragon fruit peel extract and catfish bone gelatin (at dose of 5%) could be developed as a promising product, with addition of sugar (10%) and citric acid (concentration of 0.5%). Hedonic tests revealed good acceptance in color and taste.

CONCLUSION:

Dragon fruit peel extract and catfish bone gelatin can used for functional drinks with good acceptance for color and taste

REFERENCE:

1. Tran DH. Yen CR. Chen YK. Effects of bagging on fruit characterictics and physical fruit protection in red pitaya (Hylocereus spp.). Biological Agriculture & Horticulture. 2015; 31(3):158-166. doi.org/10.1080/01448765.2014.991939

2. Temak Y. Cholke P. Mule A. Ahingade A. Narote S. Kagde A. Lagad R. Sake V. In vivo and in vitro evaluation of antimicrobial activity of peel extract of Red Dragon Fruit ( Hylocereus polyrhizus). Research Journal of Pharmacognosy and Phyrochemistry. 2019; 11(1):23-26. doi: 10.5958/0975-4385.2019.00005.0

3. Maran JP. Jeevitha KSP. Jayalakshmi J. Ashvini G. Multiple response analysis and optimization of microwave-assisted extraction of antioxidant phenolic compounds of waste Mangifera Indica L peel. Journal of Food Processing and Preservation. 2015; 39:2276-2285. doi:10.1111/jfpp.12473

4. Magalhaes DS. da Silva DM. Ramos JD. Pio LAS. Pasqual M. Boas EVBV. Galvao E.C. de Melo ET. Changes in the physical and physic-chemical characterictics of red-pulp dragon fruit during its development. Scientia Horticulturae. 2015; 253:180-186. doi.org/10.1016/j.scienta.2019.04.050

5. Xu L. Zhang Y. Wang L. Structure characteristics of a water-soluble polysaccharide purified from dragon fruit (Hylocereus undatus) pulp. Carbohydrate Polimers. 2016;1(146):224-230. doi:10.1016/j.carbpol.2016.03.060

6. Poungchawanwong S. Klaypradit W. Li Q. Wang J. Hou H. Interaction effect of phenolic compounds on Alaska Pollock skin gelatin and associated changes. LWT-Food Science and Technology. 2020; 133:110018. doi.org/10.1016/j.lwt.2020.110018

7. Gopalasatheeskumar K. Ariharasiva KG. SEngottuvel T. Devan S. Srividhya. Quantification of total phenolic and flavonoid content in leaves of Cucumismelo var agrestis using UV-spechtrophotometer. 2019; 12(6):335-337. doi: 10.5958/0974-4150.2019.00062.2

8. Kondragunta K. Karuppuraj V. Perumal V. Antioxidant activity and folic acid content in indigenous isolates of Ganoderma lucidum. Asian Journal of Pharmaceutical Analysis. 2016; 6 (4):213-215.

9. Messaoudi A. Dekmouche M. Rahmani Z. Bensaci C. Phenolic profile, antuoxidant potential of date (Phoenix dactylifera Var. Degla Baidha and Deglet-Nour) seeds from Debila region (Oued Souf, Algeria). 2021; 14(1):1-5.doi:10.5958/0974-4150.2021.00006.7

10. Naidu N. Kumar GS. Sivakrishna K. Anjinaik K. Kumar LP. Sneha G. Anti microbial and antioxidant evolution of aqueous extract of Terminalia chebula using disc diffusion, H2O2 scavening methods. Asian Journal of Research in Pharmaceutical Sciences. 2017; 7(2):112-114.doi:10.5958/2231-5659.2017.00017.0

11. Yardi S. Ashtekar AB. Bhattacharya P. Kalaichelvan C. Development of novel ready-to-drink coconut based beverage and studies on its sensory, nutritional properties and shelf life. Research Journal of Pharmacy and Technology. 2018; 11(6):2357-2359.doi:10.5958/0974-36OX.2018.00437.7

12. Priatni S. Pradita A. Stability study of betacyanin extract from red dragon fruit (Hylocereus polyrhizus) peels. Procedia Chemistry. 2015; 16:438-444. doi: 10.1016/j.proche.2015.12.076

13. Cherian P. Sheela D. LCMS spectral analysis of Betacyanin pigments in Amaranthus L. Research Journal of Pharmacognosy and Phytochemistry. 2017; 9(4):219-222.doi:10.5958/0975-4385.2017.00040.1

14. Herbach KM. Stintzing FC. Carle R. Betalain stability and degradation structural and chromatic aspects. Journal of Food Science. 2006; 71(4). doi: 10.1111/j.1750-3841.2006.00022.x

15. Roriz CL. Barros L. Prieto MA. Morales P. Ferreira ICFR. Floral parts of Gomphrena globosa L as a novel alternative source of betacyanins : Optimatozation of the extraction using response surface methodology. Fod chemistry. 2017; 229:223-234. http://dx.doi.org/10.1016/j.foodchem.2017.02.073

16. Herbach KM. Stintzing FC. Carle R. Identification of heat-induced degradation products from purified betanin, phyllocactin and hylocerenin by high-performance liquid chromatography/electrospray ionization mass spectrometry. Rapid Commum Mass Sp. 2005; 19(18):2603-2616.doi:10.1002/rcm.2103

17. Wu LC. Antioksidan and Antiproliferative Activities of Red Pitaya. Journal of Chemistry. 2006; 95(2) :319-327. https://doi.org/10.1016/j.foodchem.2005.01.002

18. Shlini P. Murthy S. Extraction of phenolic, proteins and antioxidant activity from defatted tamarind kernel powder. Asian Journal of Research in Chemistry. 2011; 4(6):936-941

19. Sharma V. Kumar HV. Rao LJM. Influence of milk and sugar on antioxidant potential of black tea. Food Research International. 2008; 41: 124-129. doi:10.1016/j.foodres.2007.10.009

20. Foegeding EA. Pludrich N. Schneider M. Campbell C. Lila MA. Protein-polyphenol particles for delivering structural and health functionality. Food Hydrocolloids. 2017; 72:163-173. https://doi.org/10.1016/j.foodhyd.2017.05.024

21. Jun M. Fu HY. Hong J. Wang X. Yang CS. Ho CT. Comparison of antioxidant activities of isoflavones from kudzu root (Pueraria lobate ohwi). J of Food Science. 2006;68(6): 2117- 2122.doi:10.1111/j.1365-2621.2003.tb07029.x

22. Ashfaq MH. Siddique A. Shahid S. Antioxidant activity of Cinnamon zelanicum (A review). Asian Journal of Phamraceutical Research. 2021; 11(2):106-112.doi:10.52711/2231-5691.2021.0021

23. Prabakaran M. Thennarasu V. Panneerselvam A. Screening of antioxidant, antimutagenic, antimicrobial activities and phytochemical studies on Sphaeranthus amaranthoides (Burm). 2011;(4):125-129

24. Skowron MJ. Krawczyk M. Grzeskowiak AZ. Determination of antioxidant activity, rutin,quarcetin,phenolic acid and trace elements in the infusions: influence of citric acid addition on extraction of metals. Journal of Food Composition and Analysis. 2015; 40:70-77. doi.org/10.1016/j.jfca.2014.12.015 0889-1575/ 2015

25. Shalaby EA. Mahmoud GI. Shanab SM. Suggested mechanism for the effect of sweeteners on radical scavenging activity of phenolic compounds in black and green tea. Frontiers in Life Science. 2016;7(1):74-81. http://dx.doi.org/10.1080/21553769.2016.1233909

26. Jeon MJ. Ha JW. Synergistic bactericidal effect and mechanism of X-ray irradiation and citric acid combination against food-borne pathogens on spinach leaves. Food Microbiology. 2020; 91:103543. https://doi.org/10.1016/j.fm.2020.103543

Received on 30.08.2021 Modified on 28.07.2022

Research J. Pharm. and Tech 2023; 16(7):3207-3212.

DOI: 10.52711/0974-360X.2023.00527

MATERIALS AND METHODS:

Materials:

d) Quantification of microbes10:

e) Hedonic test11:

d) Quantification of microbes¹⁰:

e) Hedonic test¹¹: