Application of mixture design methodology for development of high antioxidant fruity functional beverage

Abstract Three red color fruit juice (pomegranate (PJ), barberry (BJ), and grape juice (GJ)) and three plant extracts (cardamom essential oil (CE), ginger extract (GE), and hibiscus solution (HS)) were used for the development of different functional beverages. Organoleptic analysis was done to detect the most acceptable fruit juice blend. The physicochemical properties of the samples including total phenols, 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) inhibition percent, anthocyanin, flavonoid, and vitamin C content of optimum fruit juice blend (60% PJ/20% BJ/20% GJ) were 121.57 µg gallic acid equivalent (GAE)/ml, 80.28%, 4.03 mg/L, 64.87 mg/100 ml, and 51.10 mg/100 ml, respectively. To determine the optimum level of extracts and essential oil (GE, CE, and HS) in fruit juice blends, the mixture design method was used and 14 runs (formulations) were obtained. In all formulations, samples containing HS had the highest content of antioxidant and active components and the statistical analysis indicated that the sample containing 0.5 CE/0.5 GE/1 HS (ml/100 ml) had the optimum content of antioxidant components. Thus, the results of this study introduce a functional drink possessing high polyphenols, antioxidants, anthocyanin, and vitamin C content.

Pomegranate, barberry, and red grape are popular red color fruits and all of them are rich in polyphenols (especially anthocyanin) and can be applied for the development of antioxidant-enriched functional fruit juices and drinks (Berenji & Akhavan, 2018;Derakhshan et al., 2018;Jaddi et al., 2020). Several studies have investigated the health-promoting properties of pomegranate, barberry, and grape (Dimitrijevic et al., 2020;Mphahlele et al., 2016;Xu et al., 2010). Fahmy et al. (2020) reported that pomegranate juice is rich in dietary polyphenols like tannins, anthocyanins, and flavonoids which are effective in obesity and diabetes management. Also, grapes include phenolic compounds, anthocyanins, flavanols, stilbenes, catechins, and proanthocyanins (Nadeem et al., 2018). Nadeem et al. (2018) developed a grape-carrot juice blend because of the functional properties of these juices such as antioxidant, anticancer, and antianemic properties of carrot juice and reducing atherosclerosis threat of heart diseases of grape juice. In the case of barberry juice, its predominant organic acid is malic acid and it is rich in polyphenols like gallic acid, catechin, chlorogenic acid, and quercetin (Gundogdu, 2013). On the other hand, in recent years, the addition of herbal extracts and essential oils such as ginger extract (GE), cardamom essential oil (CE), and hibiscus solution (HS) to functional foods has been considered. These materials are of interest due to their high antioxidant, anticancer, and antimutagenic properties. Imran et al. (2021) developed a functional drink containing mint and ginger extract because of their high phenolic compounds and antioxidant activity.
Studies showed that Hibiscus sabdariffa L. (HS) calyx extracts are effective in treating hypertension, liver dysfunctions, and diabetes which is due to their high phytochemical, anthocyanins, phenolic compounds, and organic acids (Salem et al., 2021). Ogundele et al. (2016) studied the effect of blending pineapple, orange juice, carrot, and Hibiscus sabdariffa extracts on antioxidant properties of beverages. In all blends, juices containing HS showed high antioxidant activity. Ogori et al. (2021) investigated the functional and quality characterization of ginger, pineapple, and turmeric juice blends. They concluded that increasing the turmeric juice proportion increased the Mg, Ca, and vitamin C content of blends. Also, turmeric and ginger extracts are very effective on the antimicrobial properties of the juices.
The mixture design is a practical design for optimization and choosing the best formulation of ingredients in a combination (Ogundele et al., 2016). This research work aimed to develop a new antioxidant functional beverage by blending red color fruit juices (pomegranate, grape, and barberry juices) with three plant extracts and essential oils (hibiscus extract, ginger extract, and cardamom essential oil) based on a mixture design method.

| Chemicals
Pomegranate, barberry, and grape concentrates were purchased from Takdaneh Co., Tabriz, Iran. Hibiscus powder, cardamom essential oil (CE), and ginger extract (GE) were purchased from Darou Gostar Barij Essence Co., Kashan, Iran. All chemicals applied in this study were of analytical grade and provided from Sigma-Aldrich, Oakville, ON, Canada.

| Preparation of blends of fruit juices
Seven fruit juice formulations with different proportions of grape, pomegranate, and barberry juices were blended (Table 1) and pasteurized at 92 ± 2°C for 3 min (the Brix of all blended juices was 12°).
Juices were hot filled in the bottles, cooled, and stored at 4°C.

| Initial sensory evaluation
To determine the best proportions of fruit juices in the mixture and then, the maximum acceptable level of GE and CE, which could be added to the formulations, two steps of sensory evaluation were performed.
In the first stage, 30 ml of each seven juice blends (Table 1) was served in transparent plastic cups. Sensory evaluation of the beverages was done through a 9-point hedonic scale where 1 equals extremely dislike and 9 equals extremely like (Gunathilake et al., 2013).
The panelists consisted of 15 members (semi-trained men and women students aged 23 to 30 years) who evaluated the desirability of taste, sourness, sweetness, stringiness, and overall acceptability of different juice blends.
In the second stage, to determine the maximum level of extracts and essence which can be added to the juice from the consumer point of view, 1 ml of three concentrations of CE (0.05, 0.01, 0.005 v/v) or GE (0.1, 0.01, 0.001 v/v) was added to 99 ml of the chosen juice blend (the amount of hibiscus did not lead to undesirable taste and odor up to high levels in the juice, so it was not sensory evaluated). Again, as described above, a 9-point hedonic test was conducted to estimate the maximum amount of plant extracts acceptable as consumer sensory perception. Taste, odor, and overall acceptability were evaluated for the samples. Statistical analysis of the data was carried out through analysis of variance (ANOVA) and significant differences were estimated by the Duncan test.  The pH of beverage samples was analyzed using a pH meter (Fan Azma Gostar, Iran). A handheld refractometer was used for estimating the total soluble solids (g/100 g) of juices. The total acidity of the samples was measured using the titration method (Cassani et al., 2016). Five milliliters of samples was transferred to a 250 ml beaker and 50 ml of boiled distilled water was mixed with the sample. This solution was titrated by NaOH (0.1 N) up to the pH of 8.2 ± 0.1. Total acidity was calculated according to citric acid (g/100 g) using Equation (1): where V is the volume of NaOH and S is the volume of the juice sample.
(1) Acidity = V × 0.1 × 0.064 × 10 S TA B L E 2 Experimental ranges of independent variables applied in D-optimal mixture design for the formulation of functional fruit juice blend (0-100 µg/ml). One milliliter of diluted samples (1/10) or gallic acid was mixed with 5 ml of FCR (10%). Then 4 ml of saturated sodium carbonate was added to the solution and kept for 2 h at room temperature. The absorbance of samples was determined at 765 nm using a ultraviolet-visible (UV-Vis) spectrophotometer (UV-1700, Shimadzu, Japan) and the total phenol (TP) was estimated as mg of gallic acid equivalent (GAE) per 100 ml of sample.

| Total flavonoid content
Total flavonoid content was investigated according to (Orellana-Palma et al., 2021) with some modifications. As much as 0.2 ml of diluted samples (1/10) was added to 1.28 ml of distilled water and 60 µl of NaNO 2 (50 g/L) and kept for 5 min at ambient temperature. Sixty microliters of AlCl 3 (100 g/L) was mixed with the solution and after 6 min, 0.4 ml of NaOH (40 g/L) was added to the solution.
After stirring the mixture, the absorbance was determined at 510 nm applying a UV-Vis spectrophotometer. Total flavonoid content was stated as mg of quercetin equivalent/100 ml of the sample according to the standard curve prepared in the range of 0.05-1.2 g/L.

| DPPH radical scavenging activity
The antioxidant property of samples was studied through DPPH radical scavenging activity according to the method of Viacava and Roura (2015) with some modifications. As much as 0.1 ml of diluted samples (1/10) was mixed with 3.9 ml of DPPH solution (100 µM).
After shaking the solution, it was kept in the dark place at ambient temperature for 30 min. Ethanol (96%) was used instead of the sample in the above procedure to determine DPPH initial absorbance.
Antioxidant activity of the samples was reported as the inhibition percent of the DPPH radical according to Equation (2).
Where A c is the absorbance of the control (methanol and DPPH solution) and A s is the absorbance of the sample solution (sample and DPPH solution).

| Anthocyanin content of samples
Polymeric anthocyanins are resistant to pH change and color degradation and therefore do not participate in adsorption by spectrophotometry. In contrast, the monomeric pigments of anthocyanins are unstable against pH changes. Oxonium forms of anthocyanins become colorful at pH = 1 and hemiketals become colorless at pH = 4.5. Therefore, sample absorbance was measured at pH = 1 to determine the total anthocyanin content and at pH = 4.5 for quantification of polymeric anthocyanins. Finally, total monomeric anthocyanins are determined from differences in absorbance at 520 and 700 nm. The results were expressed as cyanidin 3-glucoside equivalents calculating with Equations (3) and (4): Where Df is the dilution factor, MW is cyanidin 3-glucoside molecular weight (449.2 g/mol), A is the absorbance of the sample, ɛ is the molar extinction coefficient (26,900 L/cm mol), and L is the cell length (1 cm; Monteiro et al., 2016).

| Vitamin C content
Vitamin C content of fruit juices was measured according to the method described by Tareen et al. (2015). In this method, 5 ml of juices was transferred to a 125 ml Erlenmeyer flask and diluted with 45 ml of distilled water. Then, 10 drops of indicator (1% starch solution) were added to the juice. This solution was titrated by iodine solution (0.005 mol/L) until the color of the solution changed to blue-black.
The iodine solution was prepared as follows: 2 g of potassium iodide (KI) was dissolved in 100 ml of distilled water. Then, 1.3 g of iodine was added to the solution. Finally, the solution was diluted with distilled water until it reached the 1000 ml volume (Tareen et al., 2015).
Vitamin c concentration is calculated according to Equation (5): Where V is the volume of iodine solution used in the titration.

| Statistical analysis
All experiments were repeated four times and the data were subjected to statistical analysis using Design-Expert version 11.0.3.0 by Stat-Ease Inc., Minneapolis, MN, USA. A predictive model that can accurately describe the response was selected based on the quality of fit estimated by ANOVA.

| Physicochemical properties of different blends of fruit juices
The results of physicochemical analysis of different fruit juice blends are shown in Table 4. The total soluble solids of all of the seven juice blends were approximately 12° Brix with no significant difference Vitamin C(mg∕100 ml) = 10 × 17.6 × V (p < .05). Blend 4 (25% PJ, 25% GJ, and 50 BJ) and blend 7 (20% PJ, 20% GJ, and 60% BJ) had the lowest pH value and highest acidity.
In terms of active compounds' content, blends 7 and 5 (60% PJ, 20% GJ, and 20% BJ) had higher phenolic content and DPPH radical scavenging capacity (p < .05). On the other hand, blends 3 and 6, which had higher grape juice portions, showed lower phenolic content and antioxidant property than other blends. Significantly higher vitamin C content belonged to blends 2 and 5 having higher portions of PJ, while blend 7 had lower vitamin C than other blends. Blends 4, 5, and 7 had significantly higher total flavonoid and anthocyanin content than other blends and had no significant difference between each other. Blend 6 showed the lowest amount of flavonoid and anthocyanin content.

| Evaluation of sensory properties of fruit juice blends
In this section, panelists scored fruit juice blends for their acceptability of sourness, sweetness, stringiness, and overall acceptability (Table 5).
Blend 7 gains the lowest score for its sourness, sweetness, and overall acceptability. According to the results, blends 4 and 7 were too sour to be acceptable. Probably, it is because of the high proportion of barberry juice in the blend. The highest sweetness was recorded in blends 3 and 6, which had more grape juice. In terms of overall acceptability, blends 2 and 5 were similar and gained the highest scores in comparison with other blends. So, according to physicochemical properties and sensory evaluations, blend 5 with 60% pomegranate juice, 20% grape juice, and 20% barberry juice was chosen as the final blend to be added CE, GE, and HS.

| Determination of the maximum acceptable level of extracts
Sensory evaluation of fruit juice blends containing different concentrations of GE (0.1, 0.01, 0.001 v/v) or CE (0.05, 0.01, 0.005 v/v) was performed to investigate the maximum acceptable amount of extracts that could be added to the fruit juices (Table 6). According to the results, blends containing higher concentrations than 0.001 (v/v) of GE gain lower scores of taste and odor, significantly. In this regard, the overall acceptability of blend containing 0.001 (v/v) GE was higher than the other ones. Also, according to the panelists' scores, the maximum acceptable concentration of cardamom essential oil (CE) in terms of taste and odor in the fruit juice blends was 0.005 (v/v).

| Optimization of antioxidant functional beverage
In the first stage of this research work, selecting optimum fruit juice blend based on physicochemical and sensory properties and determining the maximum level of ginger extract and cardamom essential TA B L E 4 Physicochemical properties of different fruit juice blends

| TP and DPPH radical scavenging activity of beverage formulations
Analysis of variance (ANOVA) was used with the purpose to find out how three plant extract levels affected the total phenol (TP) and DPPH values. It was observed that the model had a very high F-value (F = 91.77) for TP response, implying that the quadratic regression equation (Equation (6)) was extremely significant and incorporation of extracts into fruit blends had a significant effect on the total phenol content at p ≤ .05 (insignificant lack of fit = 0.42).
Where A is CE, B is GE, and C is HS.
The high determination coefficient (R 2 ) and adjusted R 2 values (0.9932 and 0.9824, respectively) indicated that the three independent parameters could well describe the majority of changes in the dependent variable (TP). According to the table of ANOVA for the special quartic model, the interaction effect of AB, BC, and AB 2 C was insignificant in Equation (6), and so these factors were deleted.
The contour diagram (Figure 1a) showed that TP content was mainly dependent on the ginger and hibiscus concentrations in fruit juices (S2, S9, and S13 samples). The response plots for TP indicate that a higher amount of TP is detected in juices with a higher concentration of ginger and hibiscus (maximum TP was 170.9 µg/ml for 1 ml GE/1 ml HS/0 CE juice blend and minimum TP was 132.11 µg/ml for 1 ml GE/1 ml CE/0 hibiscus). There were high amounts of TP in juices even at low concentrations of carda-  (7) Different superscript in each row shows significant difference between values (p < .05).
Higher values of sourness, sweetness, stringiness, and overall acceptability indicate more liking of the characteristic.

Sensory scores
Taste Odor
Values are presented as mean ± SD.
Where A = CE, B = GE, and C = HS.
The R 2 value = 0.9897 and adjusted R 2 value = 0.9732 indicated that this quadratic model had high efficiency.
The contour diagram for DPPH response has been presented in have shown that the addition of HS increased the vitamin C content of juices. While the incorporation of CE and GE did not affect vitamin C content. Tyagi and Tyagi (2018) reported that vitamin C content of Hibiscus rosa-sinensis flower extract is 26.98 mg/100 g. Therefore, high content of the vitamin C of HS can effectively increase the vitamin C content of functional beverages. As it is expected, vitamin C degrades at 70°C and this temperature is less than the one needed for the steam distillation process which is used for the extraction of essential oil and herbal extracts. So, it cannot be found in GE and CE. Therefore, the addition of GE and CE cannot increase the vitamin C content of juices.
However, studies have shown that incorporating extract and essential oil can decrease vitamin C degradation rate in juices during storage time (Kapoor et al., 2011).  (Table 7).

| CON CLUS ION
According to the antioxidant properties, total phenol, anthocyanin, flavonoid, vitamin C content, and sensory analysis, the best fruit juice blend to produce a functional drink was 60% pomegranate juice/20% grape juice/20% barberry juice. It was decided that up to a maximum of 1 ml of GE (0.001 v/v) and 1 ml of CE (0.005 v/v) can be added to 99 ml of juices based on the overall acceptability of sensory properties, but the amount of addition of hibiscus had no limitation according to customer acceptability. According to the mixture design, the best model that could describe the total phenols, DPPH radical scavenging property, anthocyanin, flavonoid, and vitamin C content of juice formulations was the special quartic model. Whereas, linear and special quadratic models qualified better anthocyanin and vitamin C content variations, respectively. The optimum composition of juice formulation was selected based on each desired active component's content and antioxidant responses. Sample 10:0.5 ml CE/0.5 ml GE/1 ml HS was the optimum formulation. The TP content of the formulations was mainly influenced by GE content while CE content was effective on anthocyanin and flavonoid content. On the other hand, HS content was impressive on all of the functional properties of formulations. The optimum fruit juice formulation of this study can be used to produce a functional drink with high consumer acceptability and health benefits.

ACK N OWLED G M ENTS
The authors are very grateful and appreciative of the Food Biophysics and Engineering Laboratory of the University of Tabriz, which technically and financially supported this study.

CO N FLI C T O F I NTE R E S T
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

R E FE R E N C E S
This reference was important because of important methods mentioned in it. We analyzed the acidity and total phenol of juices according to this article. This reference was important because it gave rise to the idea of applying red color fruit juices to produce functional beverages. This reference was important because it reviewed the pomegranate juice properties very well and it was helpful to choose a proper fruit juice for developing functional drink. This reference was important because of its similar aim to produce the functional beverage. We used this article to determine methods for analyzing the physicochemical properties of beverages. This reference was used because it has applied the mixture design in order to select the optimum formulation of additives in the functional drink.