OPTIMIZATION OF MAKING PROCESS OF API-API ( Avicennia marina) MANGROVE LEAVES STEW WITH RESPONSE SURFACE METHODOLOGY ANALYSIS

, The leaves of the A. marina have the potential to be developed into functional foods such as tea. It contains alkaloids, saponins, tannins, phenolics, flavonoids, triterpenoids and glycosides, with antioxidants, antimicrobials, antifungals and antibiotics potential. This study want to examine the effect of oven duration and stewing duration on the stewing quality of A. marina leaves and to determine the optimum oven duration and stewing duration using analysis of Response Surface Methodology (RSM). The lower limit of the oven time used is 2 hours and the upper limit is 6 hours, while the lower limit for stewing is 5 minutes and the upper limit is 15 minutes. Analysis of the data carried out is a test for tannin levels, antioxidant activity test and hedonic test. Data of tannins and antioxidant activity processed using Software Design Expert 11. The progam suggests formulate with oven duration is 4 hours and stewing duration is10 inutes. The result of the tannin test are prdictd to produce 2.8676% and antioxidant activity 1.282 ppm. The desirability value is 0.961. that means the resulting formula is matchs.


INTRODUCTION
Avicenna marina or mangrove api-api is one of mangrove with abundant availability in Indonesia. Behbahani et al. (2018) stated that A. marina is a potential source of functional food because it has metabolite compounds such as alkaloids, flavonoids, steroids, terpenoids, tannins, and saponins. Its metabolites has potential as antioxidants, antimicrobials, antifungals and antibiotics (Rise et al.,2012). A. marina has a high potential as a component of functional food like herbal tea (Hardoko et al., 2019).
Tea is a functional drink which is generally made from the shoots of Camelia sinensis (Noriko, 2008). The researchers stated that the secondary metabolites in tea leaves include alkaloids, flavonoids, steroids, saponins, triterpenoids (Damayanti et al., 2021). Over time, tea raw materials have developed. Many green plants with high levels of antioxidants are used as raw materials for tea making. Tea processing such as drying and stewing resulted in a decrease of tannin levels and antioxidant activity because these bioactive compounds are unstable in heat. A'yunin et al. (2019) stated that phenolic or polyphenolic compounds are relatively unstable to heat. The length of heating time on the tannin level of areca nut has an effect on the tannin level produced (Karina et al., 2016).
High temperatures and longer drying reduce antioxidant activity (Sari, 2015). Different temperatures and stewing times have an effect on the level of metabolites dissolved in steeping water, one of which is antioxidant activity. Ajisaka (2012) stated that the stewing process aims to maintain the quality of the desired compounds, so that there is no degradation of the chemical content of tea. The short stewing time and the low temperature cause lower tannin compounds (Nindyasari, 2012). The length of time for oven and stewing affect the content of bioactive compounds (tannin content), antioxidant activity, and the level of preference such as color, aroma and taste. It is necessary to optimize using the Response Surface Methodology (RSM) with Central Composite Design (CCD) using two factors, namely oven time and stewing time.
The purpose of this study was to determine the effect of different drying method in A. marina's antioxidant activity, tannin levels and hedonic tests. This study also want determine the optimum oven and steeping time on antioxidant activity and tannin levels using Response Surface Methodology (RSM) which is proven to be effective in optimizing and monitoring food processes CRiyadi et al., 2019).

Subjects
This study used A. marina leaves obtained from Mangrove Park, Kandang Panjang, Pekalongan, Central Java, Indonesia (Figure 1).

Place and time of research
The research was conducted at the Processing Laboratory in the Department of Fishery Processing Technology, Diponegoro University. Tannin levels were carried out by the Chem-Mix Pratama Laboratory, Yogyakarta from September to October 2021.

Tools and materials
The tools used are beaker glass, blender, oven, thermometer and scales. The main material used in this study was A. marina leaves with a leaf length of ± 6 cm.

Research procedures
The drying process was carried out using an oven at 50℃, referring to Aziz (2019).
The stewing process is in accordance with SNI 01-1902-1995 at 100℃. The oven and stewing times were modified according to the Central Composite Design (CCD). The optimization stage was carried out with a CCD design with the Response Surface Methodology (RSM) method using 5 center points and producing 13 experimental treatment points, then each experiment was carried out by response analysis. Parameters tested for each response were tannin levels, antioxidant activity and hedonic tests (SNI 2346(SNI :2015.

Tannin Level Test (Adrianar et al., 2015)
A 80 ml of distilled water were added to 1 g of powdered sample. A 0.5 ml solution was then taken and 0.5 ml of Follin Calcioteu was added. The solution was incubated for 5 minutes. A 1 ml of sodium carbonate was added to the solution, allowed with 30 minutes incubation. The absorbance was read on a spectrophotometer 715 nm.

Radical scavengin activity Test (Burda and Orzek, 2001)
Samples were taken as much as 2 ml with a concentration of 50 mg/L, 100 mg/L, 150 mg/L and 200 mg/L added each 1.5 ml of DPPH solution in ethanol and vortexed for 2 minutes. The purple color change into yellow indicates the efficiency of free radical scavengers. The absorbance was measured on a UV-Vis spectrophotometer on 517 nm after being incubated for 30 minutes. Then the comparison is observed. After the absorbance is obtained, the free radical scavenging activity (percent inhibition) is calculated as the percentage of DPPH color reduction.

Optimization of RSM
Stages of process optimization with RSM using CCD. software used is Design Expert 11. The process of stewing A.marina carried out according to the variation of the combination of CCD determined by RSM with 2 factors are oven time and stewing time ( method and the centralized composite design or CCD. Data processing using Software Design Expert 11. The best result or response from the optimal solution is verified by making a steeping of A. marina in accordance with the optimal treatment of the predicted response surface and testing for tannin levels and antioxidant activity. Furthermore, the predicted value of the response surface is compared with the actual value.

RESULTS AND DISCUSSION
The content of tannins level in the stewing leaves A. marina has an attachment to antioxsidant activity. Data on the response of tannin levels and antioxidant activity are presented in (Table 2).

Tannin level
The desired optimum response result is that the greater the tannin level produced, the greater the free radical scavenging. Tannin levels increased with 4 hours of oven time with 10 minutes of stewing (2,97%). Too long an oven will cause a decrease in tannin levels in the steeping of A. marina. The longer the heating, the tannin level tends to decrease (Karina et al., 2016). Heating for 3 hours resulted in higher tannin levels than 2 and 4 hours. If the heating is too short, the tannin levels are less than optimal.
Tannin level is also affected by the stewing time. The longer the stewing, the more the compound is extracted. In the stewing process, an extraction process occurs, namely the withdrawal of soluble chemical compounds so that they are separated from the material with a liquid solvent. The longer the stewing time, the lower the tannin level due to a decrease in temperature in line with the stewing time (Mutmainnah et al., 2018).
Tannins are not extracted maximally at low temperatures. Sasmito et al. (2020) stated that the longer stewing at high temperatures resulted in a decrease in tannin levels, such as the breakdown of tannins into simpler phenols (Kim et al., 2010).  The influence of factors on the response of steeped tannin levels of A. marina is indicated by a horizontal line parallel to the X axis. The response to tannin levels will increase with increasing oven and stewing time. After reaching the optimal response, the tannin levels will decrease. This is because the longer the heating, the lower the tannin levels in the A.marina. Karina et al. (2016), also stated that the heating time on the tannin level of areca nut has an effect on the tannin level produced. The highest tannin level was found in the 3-hour heating period, which was 10.88%, compared to the 2-hour and 4-hour heating period of 10.79% and 10.87%, respectively.

The Radical Scavenging Activity
Antioxidant activity (Table 1)  the increase in total phenolics, flavonoids, and total tannins with free radical scavenging activity. According to Rafsanjani and Putri (2015) stated that the more tannin level, the greater the antioxidant activity because tannins are composed of polyphenolic compounds that have free radical scavenging antioxidant activity. Thus, the best contributors to antioxidant activity were tannins, total phenolics, and total flavonoids, respectively. Oven and stewing time affect the process of this model. The lack of fit is shown to be insignificant. The parameter that affects the discrepancy of the model is the p-value.
The p-value of lack of fi is 0.447 which means greater than 0.05 and not significant ( Table   4). Lack of fit does not indicate that the model is good. In order to have a significant effect, the p-value must be less than 5% (<0.05) (Fathur et al., 2018).
The R² coefficient value is 0.99 which indicates that 99% of the A.marina influenced by independent variables and only 1% of all variables that cannot be explained by the model. The relationship between the predicted R² 0.98 and Adjusted R² 0.94 values is appropriate and interacts well because it has a difference of less than 0.2. The adeq precision must have a ratio of more than 4, the adeq precisionis 30.30 which means that it indicates an adequate signal (Table 4)

Optimizing steeping response of A. marina leaves
Oven and stewing time are optimized components with a range of upper and lower limits (Table 5). Both can affect the levels of tannins and antioxidant activity of the A.
marina leaves. The response of tannin levels was chosen with a maximum target because the goal to be achieved was to get the maximum antioxidant activity and antioxidant activity with a minimal target tho obtain the effectiveness of counteracting free radicals.
The Design Expert 11 program provides a solution that is close to the target. The optimum results produced by the software design expert 11, with an oven time of 4 hours and a stewing time of 10 minutes, produced a tannin level of 2.885% and an antioxidant activity of 147.181 with a desirability 0.961 (Table 6). The formula solution is recommended as the optimal formula solution and then used as prediction validation because it has a desirability 0.961. According to Ramadhani et al. (2017), the optimal formula is a formula with a desirability value close to 1, indicating the ability of the program to produce the desired product is more perfect.

Verification of optimum conditions for prediction results model
Verification is the process of examining the difference between the prediction results from the software and the analysis at the optimum point. The purpose of this verification is to confirm the actual optimal condition of the response of tannin levels and antioxidant activity. The optimal conditions for steeping A. marina were obtained at a long oven time of 3,680 hours and a stewing time of 10,702 minutes (Table 7). The results of the response of tannin levels were lower and the value of antioxidant activity was higher than the predicted value (Table 7). If the verification value is still in the range of 95% CI low and 95% CI high, then the solution recommended by Design Expert 11 software is considered good. According to Kusnandar et al. (2020)

Color
The colour is dark red-brown to light yellowish brown. The drying process causes the green color of chlorophyll in the leaves to be oxidized to brown due to the browning event (Hermani, 2004). The highest average yield was sample A10 (6 hours of oven time, 10 minutes of brewing) with an average hedonic value of 8.80 (very favored by panelists) and light yellowish brown characteristics. This is influenced by the length of the oven and brewing time. This study is in accordance with Yamin et al. (2017), the fading of the color of the Chinese ketepeng leaf herbal tea is due to the degradation of the pigments in the Chinese ketepeng leaf, especially the chlorophyll pigment which is degraded to pheophytin causing the brown color to turn yellow.

Aroma
The highest aroma value was in sample A10 (6 hours oven, 10 minutes brewing) with an average hedonic value of 8.80 (very favored by panelists) and the characteristic aroma of A. marina faded. According to Sribudiani (2011), the longer the drying time, the less aroma of rosella herbal tea produced. This is due to the destruction of aromatic compounds in the drying process. According to Fellow (1988), the aroma in foodstuffs can be caused by volatile components, but these volatile components can be lost during the processing, especially heat.

Taste
The highest taste value in the A10 steeping sample (oven time 6 hours, brewing 10 minutes) was 7.93 (preferred by panelists) with a slightly bland characteristic. Irina and Mohamed (2012), stated that the bitter taste in food is usually caused by tannins. Tannins are one of the members of polyphenol compounds, namely compounds with a phenol group in their chemical structure found in plants, so they are often called plant polyphenols. Yang et al. (2007). The brewing process is the stage of the process of extracting aroma and flavor compounds by hot water.

CONCLUSION
The results of the analysis on Design Expert 11 software with CCD showed that the oven time and stewing time of A.marina had a significant effect on all responses. The validation results under optimal conditions for the stewing process of A. marina contained in the oven for 4 hours and a stewing time for 10 minutes, the tannin level of 2.86276% and antioxidant activity of 151.282 ppm with a desirability 0.961 were obtained.