Antioxidant Activity and Consumer Preference of Brown Algae Sargassum hystrix Juice as a Functional Drink

Sargassum hystrix juice is a functional beverage made from S. hystrix through the immersion process, crushing, filtering, and pasteurization. Therefore, this study aims to determine the effect of S. hystrix concentration on antioxidant activity, level of consumer acceptance, and chemical composition of the juice. The fresh S. hystrix used were washed, immersed for three days, and blended at a concentration of 10, 20, 30, 40, and 50%, respectively. Meanwhile, the qualities of juice that were observed include yield, antioxidant activity, total phenol, water, ash, protein, and fiber content, formol number, total sugar, total plate count, and sensory analysis. The results showed that the S. hystrix juice had a value of yield 43.00-85.67%, pH 7.5, antioxidant activity 20.5044.57%, total phenolic 32.78-293.11 mg GAE/g, water 93.03-95.39%, ash 0.36-6.34%, protein 0.88-4.96%, crude fiber 0.41-5.04%, formol number 15-21.67 mL N NaOH/100 mL, total sugar 3.10-4.61%, total plate count 2.3x103-4.3x103CFU/mL, sensory of color 1.83-2.95, flavor 2.49-3.29, and taste 2.29-3.50.


INTRODUCTION
The consumption patterns of Indonesians, especially in urban communities, have a change of eating habits from natural into fast foods that are rich in fat and free radicals (Fagbemi et al., 2013). This condition was one of the factors that caused an increase in the prevalence of degenerative diseases such as coronary heart disease, diabetes mellitus, hypertension, heart disease, stroke, and some types of cancer (Barhe and Tchouya, 2016). Meanwhile, the presence of free radicals in the body leads to degenerative diseases such as diabetes mellitus, narrowing of blood vessels, coronary heart disease, stroke, and cancer (Kang et al., 2010).
Disease prevention is carried out by setting nutrient consumption such as natural antioxidant that is capable to fight degenerative diseases. These antioxidants delay or prevent the occurrence of free radical autooxidation reactions (Farag et al., 2003). Currently, the antioxidant activity of various food is one of the most widely studied topics due to its implications on health, especially the potential as a functional ingredient (Balboa et al., 2013). Therefore, the attention on the use of natural antioxidants is increasing and one source of these is algae. Budhiyanti et al. (2011) have studied the antioxidant activity of the cytoplasm and membrane fractions of Sargassum hystrix. However, the utilization of antioxidant compounds from brown algae S.hystrix in food is limited, therefore, more studies are required on its application in food.
Functional beverages contain ingredients that improve health status or prevent certain diseases (Tangkeallo and Widyaningsih, 2014). Juice is a beverage derived from fruit or vegetables and smoothies (Caswell, 2009), the ones from algae are very rare. Furthermore, Juice is usually served cold, hence, it is easy to maintain and minimize the damage of the antioxidant compounds contained in S. hystrix. Therefore, this study was conducted to produce functional drinks such as S. hystrix juice that contains natural antioxidants.

Sargassum hystrix Preparation and Immersion
The samples of brown algae S. hystrix were collected at Pantai Sepanjang, Gunung Kidul, from January to June 2016 and washed using clean water to remove impurities and residual seawater. Meanwhile, the immersion method based on Supirman et al., (2013) with modification was conducted by soaking 250 g of S. hystrix into 750 mL of water and adding lemon juice until a pH value of 3 is reached. Furthermore, 100g of fresh pandan leaves were for three days to eliminate the fishy smell and every 12 hours, water used was changed to avoid saturation of dirt and salt. In addition, the fresh and soaked samples were tested for antioxidant activity, total phenol content, and proximate analysis.

Sargassum hystrix Juice Processing
The materials required needed to make the juice were brown alga S. hystrix, sugar, and water. After immersion, the sample was blended with water and sugar by the concentration based on threshold test and S. hystrix juice was stored in a freezer (-18°C) before further analysis. Meanwhile, the juice was processed based on Iriani et al. (2005) with modifications. The formula of S. hystrix juice is shown in Table 1.

Sample Analysis
The sample was analyzed to obtain the value of yield (Magro et al., 2015), pH (Zainoldin, 2009), antioxidant activity (Zubia et al., 2009), total phenol (Kang et al., 2010), the content of water, ash, protein, crude fiber, total sugar, formol number, total plate count (AOAC, 2019), and the level of consumer acceptance (Setyaningsih et al., 2010). Meanwhile, a consumer acceptance analysis was conducted by 80 untrained panelists to assess the parameter of color, flavor, and taste of each juice treatment. This assessment was conducted by giving a score from 1 to 5 (1 = strongly dislike, 2 = dislike, 3 = neutral, 4 = like, and 5 = strongly like).

Statistical Analysis
Chemical analysis data were analyzed using ANOVA with a 95% confidence level and the results showed a significant difference which was followed by the Tukey-HSD test. Meanwhile, consumer acceptance data were analyzed using Kruskal-Wallis and when there was a significant difference, it was followed by Mann-Whitney test. This data analysis was carried out with SPSS 20.0.

Yield
The yield of S. hystrix juice is shown in Table 2 and the result showed that its higher concentration had a lower value of the yield. Meanwhile, Magro et al. (2016) stated that the grape juice with and without the addition of enzyme pectinase produced 69.85 and 75.83% of yield, respectively. Iriani et al. (2005) also stated that the yield of kuini mango juice with the addition of Tabel 1. Formulation ratio of materials for making Sargassum hystrix juice. pectinase enzymes had a value of 78.10 to 84.92%. Therefore, the high-water content in the processing of the juice made S. hystrix more soluble in water which led to higher yield, while more solvent produced a higher volume of extract (Kamaluddin et al., 2014).

pH
The pH of each S. hystrix concentration had the same value which was 7.5 (Table 2). This showed that the concentration of S. hystrix in the juice did not affect the pH which tends to be neutral. In this study, the destruction process and pasteurization time, as well as the amount of sugar used in each treatment were the same, therefore, there was no real effect on the juice produced. Moreover, Harjantini and Rustanti (2015) stated that the functional beverage of srikaya yogurt jelly with the addition of carrageenan has a pH of 4.33 to 5.74. Furthermore, Pamungkas et al. (2014) stated that jelly drinks of hantap leaf extract have a pH value of 7.87.

Antioxidant Activity
The antioxidant activity of this juice depends on the concentration of S. hystrix as shown in Table 2, where the highest activity was 43.91% for 40% concentration. Moreover, Husni et al. (2015b) stated that fast drinks with the addition of S. polycystum ethanolic extract have an antioxidant activity of 25.7 to 65.41% while yogurt has 72.34 ± 0.01% at a concentration of 6000 ppm (Husni et al., 2015a). Similarly, Pamungkas et al. (2014) stated that jelly drinks of hantap leaf extract have an antioxidant activity of 20.93%. Furthermore, the heat process applied to the food processing and pH value of food also influenced the stability of the product's antioxidant activity (Tensiska et al., 2003).

Total Phenol Content
Total phenol content as shown in Table 2 indicated that S. hystrix concentrations influenced its content in the juice. Meanwhile, the concentration of 50% had the highest value of total phenol content, while the lowest was at 10 and 20% concentrations. The correlation analysis showed that there was a strong and positive correlation between total antioxidant activity and total phenol content. Moreover, Husni et al. (2015b) stated that the greater the addition of S. polycystum extract in fast drinks, the more the antioxidant activity and positively correlated with total phenol. The relationships between total phenol and antioxidant activity of S. hystrix juice are shown in Figure 1.
Brown macroalgae compounds have potential as antioxidants, namely, polysaccharides, phenolic compounds, lipids, pigments, vitamins, terpenoids, and sterols (Balboa et al., 2013). Meanwhile, fast drinks with the addition of S. polycystum ethanolic extract had a total phenol content from 27.74 to 100.36 mg GAE/g (Husni et al., 2015b). Similarly, Pamungkas et al. (2014) stated that jelly drinks of hantap leaf extract had a total phenol content of 10.6 GAE/100 g. When compared to some of these studies, the total phenolic content of the S. hystrix juice was significantly high.

Proximate Analysis
The water content of S. hystrix juice varied from 93.03 to 95.39% as shown in Table 2. Meanwhile, Pamungkas et al. (2014) stated that the water content of jelly drinks from hantap leaf extract was 87.37% and yogurt with the addition of S. polycystum ethanolic extract varied from 77.69 to 85.76% (Husni et al., 2015a). Therefore, S. hystrix juice had a higher water content because it was liquid. Table 2 showed that the concentration of S. hystrix juice affected ash content, therefore, a greater concentration of S. hystrix produced higher ash content. Moreover, Husni et al. (2015b) stated that fast drinks with the addition of S. polycystum ethanolic extract had ash content from 0.06 to 11.54%. Furthermore, Wibowo and Fitriyani (2012) stated that the ash content of fast drinks Eucheuma cottonii were18 to 25%, while yogurt with the addition of S. polycystum ethanolic extract had an ash content of 0.84 to 1.17% (Husni et al., 2015a). When compared to previous studies, the ash content values of S. hystrix juice were fairly small ranging from 0.36 to 6.34 ppm.
The protein content of the juice was affected by the concentration of S. hystrix and the range was 0.88 to 4.96% as shown in Table 2. A previous study by Pamungkas et al. (2014) stated that jelly drinks of hantap leaf extract had a protein content of 0.10%, while yogurt with the addition of S. polycystum ethanolic extract had 3.22 to 6.53% (Husni et al., 2015a). Therefore, the protein content of S. hystrix juice was not significantly different from a previous study which stated that a higher concentration of S. hystrix in the juice leads to more protein content.

Crude Fiber Content
The crude fiber content of S. hystrix juice was 0.41 to 4.97% as shown in Table 2. and is not significantly different from Wibowo and Fitriyani (2012) which stated that the Eucheuma cottonii fast drinks had a fiber content of 5.25 to 11.83%. Similarly, Harjantini and Rustanti (2015) stated that the functional beverage srikaya yogurt jelly with the addition of carrageenan had a fiber content of 2.14 to 3.40%.

Total Sugar Content
The effect of S. hystrix concentration on the total sugar content of juice is shown in Table 2. Meanwhile, the amount of sugar used in juicing was 10%, however, not all was soluble, some were filtered or lost during the process. There was an insignificant difference between the total sugar content of S. hystrix juice of 10%, 20%, and 30%, and also between 40% and 50% concentrations. The total sugar content was affected by the amount of sugar in the product. Furthermore, the total sugar content of S. hystrix juice (3.10 to 4.61%) was very small compared to the fast drinks with the addition of S. polycystum ethanolic extract (76.13 to 87.18%) (Husni et al., 2015b), due to the 10% (w/v) that was used. Therefore, a higher concentration of S. hystrix in juice reduces the total sugar content of the juice. Table 2 showed that there was no influence of S. hystrix concentration in the juice on the total plate count. The total plates count at 10, 20, 30, 40, and 50% were 2.3x10 3 , 3.0x10 3 , 2.8x10 3 , 4.3x10 3 , and 3.1x10 3 CFU/mL, respectively. This value was not significantly different when compared to pineapple juice fortified by chitosan. Meanwhile, Husniati and Oktarina (2012) stated that the addition of chitosan on pineapple juice had a total plate count of 8.40x10 3 -9.19x10 3 CFU/mL.

Formol Number
The influence of S. hystrix concentration against the formol number of juice is shown in Table 2 which indicated that its concentration affected the formol number of juice (p <0.05). This showed that a higher concentration of S. hystrix increased the formol number of juice which ranges from 15.00 to 21.67 mL N NaOH/100 mL. Meanwhile, increased formol numbers indicated their proteolytic activity in degrading proteins into amino acids, peptides, and ammonia. This number had fulfilled SNI 01-3719-1995, where the minimum requirement formol number of fruit juice was 15 mL N NaOH/100 mL. Furthermore, Su et al. (2005) stated that the ratio of formol number to total protein contributes to the delicious flavor.

Sensory Properties of S. hystrix Juice
Color Table 2 showed that there was an insignificant difference between the juice color and S. hystrix at a concentration of 10, 20, 30, and 40%, respectively. Meanwhile, the S. hystrix concentration of 50% was significantly different from other treatments, therefore, higher concentration reduced the levels of juice color that was dominated by the green and ranges from 1.83 to 2.95. Husni et al. (2015b) stated that fast drinks with the addition of S. polycystum ethanolic extract had color values from 2.4 to 3.2. Similarly, the color value of yogurt with the addition of S. polycystum ethanolic extract was 2.67 to 3.08 (Husni et al., 2015a).

Flavor
The flavor of juice was affected by S. hystrix concentration as shown in Table 2. Meanwhile, the flavor of S. hystrix juice at the concentration of 10, 20, 30, 40, and 50 were 2.83, 3.29, 3.01, 2.89, and 2.49, respectively. This value is approximately equal to instant drink with the addition of S. polycystum ethanolic extract that had flavor values from 1.29 to 3.66 (Husni et al., 2015b), while yogurt had flavor values from 2.42 to 3.10 (Husni et al., 2015a).

Taste
The taste of the juice was affected by S. hystrix concentration as shown in Table 2. Meanwhile, the taste of S. hystrix juice for the concentration of 10, 20, 30, 40, and 50 were 3.50, 3.09, 3.34, 3.15, and 2.29, respectively. The taste of S. hystrix juice was higher compared to fast drinks of S. polycystum ethanolic extract that had a taste of 1.32 to 3.59 (Husni et al., 2015b). Similarly, yogurt with the addition of S. polycystum ethanolic extract had a taste of 2.15 to 2.76 (Husni et al., 2015a). Meanwhile, Supirman et al. (2013) stated that the addition of lime in S. filipendula drink can not eliminate the fishy smell, therefore, preference level panelists to the drink are low.

CONCLUSION
Based on the results, the selection of appropriate formulation for S. hystrix juice depends on treatment with a high value of the antioxidant activity, total phenol content, and high sensory hedonic value. In this study, the best formulation was the treatment of S. hystrix juice concentration by 40% that has high antioxidant activity and sensory value. Therefore, a higher concentration of S. hystrix in the juice leads to higher antioxidant activity and positively correlated with total phenol content.