Optimization Using Response Surface Methodology (RSM) of NaCl, NaOH, and Activated Charcoal Concentration for the Purification Process of Crude Oil of Common Ponyfish

. Crude oil of common ponyfish ( Leiognathus equulus ) is a by-product of fish protein hydrolysate manufacturing at PT. Berikan Teknologi Indonesia. The quality of the crude fish oil produced falls below the International Fishmeal and Oil Manufacturers Association Standard. Therefore, to improve its quality, a purification process comprising of three stages was employed, degumming with NaCl (5-10%), neutralization with NaOH (10-18° 𝐵𝑒 ), and bleaching with activated charcoal (6-10%). The purification process was optimized using RSM with the Box-Behnken Design. The 15 concentration formulations were obtained with response variables taken based on chemical test parameters (free fatty acids (FFA), acid value, peroxide value, and iodine value). The optimal point of NaCl, NaOH, and activated charcoal concentration was determined at 10%, 18° 𝐵𝑒 , and 9.4%, respectively with a desirability of 0.744. The purified common ponyfish oil exhibited FFA at 9.52±0.21%, acid value at 3.42±0.17 mg KOH/g, peroxide value at 1.25±0.03 mEq/Kg, and iodine value at 4.12±0.13 mEq/Kg. Pure common ponyfish oil resulting from the optimal conditions has been verified. All parameters indicate that the quality of common ponyfish oil is improved by the refining process. This indicates that the optimized process may be applied in real production in the field with a high degree of confidence.


Introduction
Common ponyfish (locally known as Petek) is a demersal fish (bottom fish) that is widely caught in the Banten Sea, the North Coast of Java, and East Lampung [1].According to data from the Ministry of Marine Affairs and Fisheries, common ponyfish production in Indonesia reached 1,444.55 tonnes.The level of utilization of these fish in the community differs from the amount produced since these fish are included in the category of small size and low economic value [2].However, common ponyfish contain approximately 66.58% protein and omega-3 in the form of EPA and DHA of 5.35% and 5.8% of total fat content, respectively [3] [4].One of the utilizations of common ponyfish is used as a basic ingredient in the fish protein hydrolysates production by PT.Berikan Teknologi Indonesia.Fish protein hydrolysate is a product obtained from the decomposition of fish protein by enzymes through a hydrolysis process that will produce simple peptide compounds and amino acids.Making fish protein hydrolysate will produce a by-product of crude fish oil.The crude fish oil produced still contains water fraction, oil fraction, and suspended solids [5].According to research conducted by [6], crude fish oil obtained from the process of making fish protein hydrolysate made from common ponyfish has physical characteristics of reddish yellow colour, acid value 17.00 mg KOH/g, free fatty acids 8.46% and peroxide value 38.95 mEq/Kg.Therefore, these results need to meet the quality standards set by IFOMA (International Fish Meal and Oil Manufacturers Association) regarding the quality of crude fish oil [7].The purification process is required to obtain fish oil that meets the quality standards [8].
The quality standard of crude fish oil refers to the standards set by IFOMA [5].According to Estiasih [5], refining fish oil can be done in three stages, i.e. degumming, which aims to remove phosphatides and slimy compounds, neutralization to reduce the content of free fatty acids in the oil using alkali so that the free fatty acids will be saponified and bleaching to improve the colour of the fish oil, reduce phosphatide levels, and remove any remaining soap residue.One of the factors that can affect the fish oil refining process is the concentration of the compound used.Process optimization is needed to determine the optimum or best concentration of the compound used.Based on this background, the process optimization is carried out by using RSM [9].

Material and research methods
The fish oil purification materials include crude common ponyfish oil, NaCl, NaOH, activated charcoal, and chemicals for analysis.The equipment used is a chromameter, UV-Vis spectrophotometer, etc.The research was conducted in two stages.The first stage of research was conducted to determine the initial characteristics of crude common ponyfish oil before the purification process, such as colour analysis, water content, free fatty acid, acid value, peroxide value, anisidine value, iodine value, and total oxidation.The second stage of the research was used to optimize the formula of NaCl, NaOH, and Activated Charcoal.The concentration of compounds used is NaCl in the range of 5-10%, NaOH in the range of 10-18°, and activated charcoal in the range of 6-10%.The degumming, neutralization, and bleaching process were carried out at a temperature of 25 o C for 30 minutes.The combination of concentrations used was according to the recommendations suggested by the design expert 13 application using the response surface methodology, Box-Behnken design method.The optimized common ponyfish oil will be tested for free fatty acid, acid value, peroxide value, and iodine value.

Results and discussion
First stage research was conducted to test the characteristics and quality of crude fish oil of common ponyfish before the fish oil refining process, including moisture content, colour, free fatty acids, acid value, peroxide value, iodine value, anisidine value, and total oxidation.The colour of common ponyfish oil can be seen from the L*, a*, and b* values.The colour characteristics possessed by common ponyfish crude fish oil are the L* value of 40.07±0.02which indicates that the fish oil is cloudy, the a* value of -0.75±0.03which indicates that it tends to be green, and the b value of 3.55±0.04which indicates that it tends to be yellow, so the colour of the fish oil is greenish yellow.It can occur since it is influenced by the raw materials, such as fish guts and heads, which are naturally red to brown [5].In addition, the colour of common ponyfish oil is influenced by natural pigments, such as xanthophyll, carotene, and anthocyanins.It can also be caused by compounds resulting from the degradation of natural dyes, resulting in yellow or brownish-yellow fish oil [10].Table 1 shows the characteristics of crude common ponyfish oil.Water content analysis was done to determine the water content contained in crude common ponyfish oil and whether it is under the standards set by IFOMA [7] which is 0.5% to 1%.The value of water content obtained from crude fish oil of common ponyfish is 76.8±0.19% .The separation process between water and oil in fish oil production is not yet optimized, resulting in a relatively high water content in the final product.Excess water content contained in crude fish oil can reduce the quality of the fish oil.Due to the ability of water to hydrolyze the oil, free fatty acids will be formed, which leads to the rancidity of fish oil.The high water content in fish oil production due to the inefficient separation process leads to high levels of free fatty acids in the final product.The free fatty acid value contained in crude fish oil of common ponyfish is 18.07±0.10%.According to IFOMA [7], the free fatty acid content that should be contained in crude fish oil is 1% to 7%.The excess free fatty acids are formed and increased due to the reaction between carbon chains in unsaturated fatty acids with heat and air [11].Acid value analysis was conducted to determine the amount of free fatty acids.The acid value obtained was 30.86±0.66 mg KOH/g.Acid value can be used as an indicator that shows the quality level of fish oil caused by processing or damage to fish oil during storage [5].
Peroxide is one of the indicators to determine the level of damage to fish oil due to the presence of hydroperoxide compounds that cause rancidity in fish oil during storage.According to IFOMA [7] standards, good crude fish oil has a peroxide content of 3 mEq/kg to 20 mEq/kg.The peroxide value contained in crude common ponyfish oil is 3.93 ± 0.03 mEq/kg.Iodine value analysis is performed to determine the degree of unsaturation of fish oil [8].The unsaturated fatty acids can increase the amount of iodine and form saturated compounds.The higher the iodine value of the oil, the higher the degree of unsaturation of the oil.The higher the level of unsaturation, the better it is consumed by the human body since oils high in unsaturated fat do not increase the body's cholesterol.The result obtained from the iodine value test was 7.25±0.13mEq/kg.Anisidine value analysis is used to measure oxidation products.The oxidation process begins with primary oxidation to produce peroxide compounds.Then, the compound is decomposed, caused by further oxidation, which will produce aldehydes and ketones.The results obtained in the anisidine value test were 28.22±0.56mEq/kg.These results follow the standards set by IFOMA [7], which are in the range of 4-60 mEq/kg.Total oxidation (TOTOX) is the sum of the primary and secondary oxidation results, namely twice the peroxide value plus the anisidine value [5].The total oxidation value is used to measure hydroperoxides and their derivative products so that the oxidation process that occurs in fish oil can be known.The results obtained in the total oxidation test were 36.08±0.55mEq/kg.These results follow the standards set by IFOMA [7], which are in the range of 10-60 mEq/kg.
RSM was used to optimize the formula of NaCl, NaOH, and activated charcoal in the second stage of the research.The Design Expert 13 application was utilized to determine the optimum formula.Table 2 shows the process involved conducting 15 research runs and analyzing the results obtained through RSM.Refer to Table 3, the optimization requirements can be seen from the P-value of the model which is less than 0.05.A degree of desirability that is close to number one is the optimum formula.

Free fatty acids
Free fatty acids are formed due to triglyceride hydrolysis reactions that break fatty acid and glycerol bonds and are caused by the splitting and oxidation of fatty acid double bonds [12,13].From the data that has been analyzed, the P-value is 0.0134 (P<0.05) in the quadratic model.It shows that the model is significant and illustrates the relationship between the independent variables and the response.Contour plot graphs are needed to see the interaction between variables and the response of free fatty acid values.The graph (Fig. 1) was developed from the quadratic regression equation model.The following is a graph of the relationship between variables and the response of free fatty acid value.Figure 1 shows the relationship between NaCl concentration (%) and NaOH concentration (°) on the value of free fatty acids.The optimum point suggested by Design Expert 13 Software on the response of free fatty acids is on Run 4 with a combination of variable concentrations of NaCl 10%, NaOH 10 °, and activated charcoal 8%.Therefore, 8.1% of free fatty acid levels can be produced.NaOH is the variable that has a major effect on reducing free fatty acid levels in common ponyfish oil.It happens because of the saponification reaction.The reaction occurs because the alkali used in refining can react with free fatty acids to form soap [5].NaOH concentration must be appropriate because if excessive NaOH is used, it will cause triglyceride hydrolysis reactions and form excessive soap to reduce the amount of oil yield from refining [5].

Acid value
The high acid value indicates the amount of free fatty acids formed due to the oil hydrolysis reaction.From the data that has been analyzed, the P-value is <0.0001 (P<0.05) in the linear model.It indicates that the model is significant and illustrates the relationship between the independent variable and the response.The acid value response for the R² value obtained is 0.90, the data variation the model can capture.However, the difference between Adjusted R² and Predicted R² obtained is 0.066, indicating that the model is good.Contour plot graphs (Fig. 2) are needed to see the interaction between variables and the acid value response.The graph is developed from the linear regression equation model.The following graph shows the relationship between variables and the acid value response.Fig. 2 shows the relationship to the acid value between NaCl concentration (%) and NaOH concentration (°).NaCl concentration (%) with NaOH concentration (°) has a directly proportional relationship.The higher the NaCl concentration and NaOH concentration used, the smaller the acid value produced.The optimum point suggested by Design Expert 13 Software on the acid value response is in Run 2 with a combination of variable concentrations of 10% NaCl, 18° NaOH, and 8% activated charcoal, resulting in a contained acid value level of 2.88 mg KOH/g.The acid value is used to measure the parameter of the amount of free fatty acids formed due to oil hydrolysis.It is in line with the results obtained.The basic principle of degumming is the hydration of phosphatides and slimy components.The NaCl solution can bind impurities in fish oil [8].The fatty acids hydrolyzed in the degumming stage will react with NaOH in the neutralization stage, which will cause soap formation.It will cause a decrease in the acid value.

Peroxide value
The active peroxide produced during the oxidation process acts as an oxidizing agent so that it can cause undesirable changes to non-fats, such as damaging carotenoid pigments.Therefore, the lower the oxidation value, the better the quality of the fish oil.From the data that has been analyzed, the P-value is 0.0366 (P<0.05) in the quadratic model.It shows that the model is significant and illustrates the relationship between the independent variables and the response.In the peroxide value response for the R² value obtained of 0.91, the model can capture the amount of data variation.However, the difference between Adjusted R² and Predicted R² obtained is 1.2, indicating an insignificant component in the model.Contour plot graphs are needed to see the interaction between variables and the response of peroxide values.The graph (Fig. 3) was developed from a quadratic regression equation model.The following is a graph of the relationship between variables and the response of the peroxide value.Fig. 3 shows the relationship between NaCl concentration (%) and NaOH concentration (°) on the peroxide value.The lower the NaCl concentration and the higher the NaOH concentration used, the smaller the peroxide value produced.The optimum point suggested by Design Expert 13 Software on the peroxide value response is in Run 2 with a combination of variable concentrations of 10% NaCl, 18°Be NaOH, and 8% activated charcoal, resulting in a peroxide value containing 0.97 mEq/kg.Peroxide value is used to measure the parameters of the formation of hydroperoxide compounds due to the number of oxidized fatty acids [8].Therefore, if you see this in harmony with the results obtained, the free fatty acids contained in fish oil will bind with NaOH to form soap. Thus minimizing the occurrence of autooxidation reactions in fish oil.In addition, the soap formed will bind the peroxide compounds in the oil because these compounds are more polar than oil [8].

Iodine value
Unsaturated fatty acids in fish oil can absorb some iodine and form saturated compounds.The amount of iodine absorbed indicates the value of double bonds in the oil [8].From the data that has been analyzed, the P-value is 0.0198 (P<0.05) in the Linear model.So, it shows that the model is significant and describes the relationship between the independent variable and the response.Contour plot graphs are needed to see the interaction between variables and the iodine value response.The graph (Fig. 4) is developed from the linear regression equation model.The following is a graph of the relationship between variables and the response of iodine value.Figure 4 shows the relationship between NaCl concentration (%) and NaOH concentration (°) on the iodine value.NaCl concentration (%) and NaOH concentration (°) are inversely proportional.The higher the NaCl concentration and the lower the NaOH concentration used, the higher the iodine value produced.The optimum point suggested by Design Expert 13 Software on the iodine value response is Run 14 with a combination of variable concentrations of 5% NaCl, 18% NaOH, and 8% activated charcoal, resulting in a contained iodine value level of 4.55 mEq/kg.The iodine number is used to measure an oil's unsaturation degree.So, if you see this, it aligns with the results obtained.The NaOH solution will bind the free fatty acids in fish oil by forming soap [5] so that the soapiness of free fatty acids and unwanted compounds will minimize damage to common ponyfish oil.

Determination of optimum conditions
Response optimization is done by combining response and independent variables.Optimization is carried out to obtain the desired results, minimizing the response parameters of common ponyfish oil refining (free fatty acids, acid number, peroxide number, and iodine number).Overlay plots optimize multiple responses that work well when only a few independent variables are considered, it show in Fig. 5.The Design Expert 13 program selected the optimum region of each response shown [9].Goal and importance for optimum conditions are shown in Table 4.According to Table 5, Design Expert 13 software offers a solution at 10% NaCl concentration, 18° NaOH concentration and 9.40% activated charcoal concentration.The predicted results of each response are free fatty acid at 9.27%, acid value at 2.79 mg KOH/g, peroxide value at 1.00 mEq/kg, and iodine value at 4.37 mEq/kg with a desirability of 0.744 or 74.4% (Fig. 5).

Validation of optimum results
Based on the optimum point verification research conducted with a NaCl concentration of 10%, NaOH concentration of 18° and activated charcoal concentration of 9.40%.Based on these results (Table 6), it can be concluded that the model obtained is appropriate.Suppose the verification results are still in the Confident Interval (CI) or Prediction Interval (PI) range of 95%.In that case, the model follows what the software shows and can be applied to real production in the field.

Characteristics and quality of purified cammon ponyfish oil using optimal purification conditions
Common ponyfish oil produced after the refining process has clear visual characteristics because the oil has passed three stages in the refining process.These stages are degumming, neutralisation and bleaching.

Conclusion
Optimisation of NaCl, NaOH, and activated charcoal using Response Surface Methodology obtained optimal purification conditions for crude common ponyfish oil: 10% NaCl, 18° NaOH, and 9.40% activated charcoal concentration, with a desirability of 0.744 or 74.4%.The purified common ponyfish oil exhibited FFA at 9.52±0.21%,acid value at 3.42±0.17mg KOH/g, peroxide value at 1.25±0.03mEq/kg, and iodine value at 4.12±0.13mEq/kg.All parameters indicate that the quality of common ponyfish oil is improved by the refining process.

Table 1 .
Characteristic of Crude Common Ponyfish Oil

Table 2 .
Response to treatment concentration NaCl, NaOH, and activated charcoal

Table 3 .
Analysis of mathematical models

Table 6 .
Result validation of optimum results Table 7 is a comparison table of common ponyfish oil, before and after purification.The quality standard of pure fish oil can be referred to WHO Standard [12].

Table 7 .
Characteritics of common ponyfish oil, before and after purification Thank you for the Directorate of Research and Community Service, Directorate General of Research and Development, Ministry of Research, Technology, and Higher Education for research funding through a doctoral dissertation grant in 2023.