Optimization of Enzymatic Hydrolysis of Mackerel (Rastrelliger sp.) Muscle Protein Hydrolysate Using Response Surface Methodology

Mackerel (Rastrelliger sp.) is a widely distributed epipelagic species in South East Asia. Mackerel has a high amount nutrient such as protein (20.83 %) and fat (1.03 %). The high amount of protein and low amount of fat will allow it to be used as a material to produce a good protein hydrolysate. The aim of this study is to determine the optimal enzymatic hydrolysis conditions (time, temperature, and pH) using Response Surface Methodology (RSM). Mackerel Protein Hydrolysate (MPH) was prepared using commercial Flavourzyme. Optimization of MPH was performed by employing Box Behnken Design method of RSM. SN-TCA method was used to calculate the degree of hydrolysis (DH) which is the key parameter in hydrolysis reaction. Optimum hydrolysis conditions were obtained at pH 7, temperature 55°C and 60 min of process. Under these conditions the DH obtained was 17.7293 % with 4% enzyme to substrate ratio. The suggested model for the hydrolysis process is quadratic with the desirability factor of 1. The MPH was further assessed for its amino acid composition using High Performance Liquid Chromatography (HPLC). The hydrolysis process increases the amino acid amounts namely L-Glutamic Acid (19.77%), L-Valin (14.20%), L-Aspartic Acid (11.42%), Glycine (11.04%), L-Alanin (14.20%), L-Prolin (16.80%), and L-Histidin (27.06%). The study suggested that mackerel muscle can be considered to be utilized as fish protein hydrolysis materials.

IOP Publishing doi: 10.1088/1755-1315/1224/1/012038 2 Protein hydrolysis is the process of hydrolyzing proteins into more basic chemical components. Proteins consist of peptide chains, which are made up of an array of amino acids. The hydrolysis process can break the peptide chain with the addition of water molecules so that the protein returns to its constituent components. This process can be accelerated by treatments such as the addition of enzyme catalysts and or by giving acid or base treatments. One of the most effective methods in protein extraction is by making protein through enzymatic hydrolysis process. This process is widely practiced to improve the functional and nutritional characteristics of fish proteins [3]. The enzyme catalyst that is widely used in the hydrolysis process is catalase enzyme, but there are not many studies that optimize the hydrolysis process to produce products with optimal results, one method that can be used is Response Surface Methodology (RSM). RSM is a collection of mathematical and statistical calculations made for experimental design, mathematical modeling, evaluating the effects of several factors, and obtaining the optimum condition of the response with a limited number of trials. Currently, RSM is widely applied in various fields such as electronics, biotech, aviation, etc [4].
Mackerel is a medium economic fish and most of the processing of mackerel production is still traditional. The protein content of mackerel can be utilized as raw material for fish protein hydrolysate to provide added value. Mackerel has a very high nutritional value; for example, every 100 grams of mackerel meat contains 76% water, 22 grams of protein, 1 gram of fat, 20 milligrams of calcium, 200 milligrams of phosphorus, 1 gram of iron, 30 micrograms of vitamin A, and 0.05 milligrams of vitamin B1 [5]. Fish protein hydrolysate is produced through a hydrolysis process to break down protein components into their constituent amino acids. This hydrolysis process can be accelerated by acid and/or alkaline treatment, as well as the addition of catalyzing enzymes. The catalyzing enzymes that can be used in this process are bromealin enzyme, papain enzyme, and catalase enzyme. Various enzymes such as alkalse, bromealin, flavorzyme, and protamex have been used to produce hydrolysates that have functional characteristics and antioxidant properties [6]. In Indonesia, there are three species of mackerel: R. kanagurta, R. branchysoma, and R. faughni. Compared to other species, R. kanagurta has a high mortality rate. In the Indo-Pacific, R. kanagurta is a common fish that is usually collected with gillnets [7]. A small pelagic fish of moderate economic value, mackerel is considered a valuable resource by local fishers. A major product in smallscale fisheries, mackerel is a commercially important fish species often found in coastal waters (neritic zone) [8].
Flavorzyme is one of the protease enzymes that can hydrolyze proteins into amino acid components so that the functional and antioxidant characteristics of fish protein hydrolysis will increase. Treatment conditions such as acidic and alkaline pH will affect the hydrolysis rate of fish protein hydrolysis and the resulting functional characteristics. In addition, the optimal temperature and time of hydrolysis will also affect the final product of hydrolysis. During hydrolysis, the optimum pH is the pH where S/t is always greater than other pHs. Higher temperatures accelerate chemical processes, but also cause enzyme denaturation (at 70 °C, most enzymes become inactive) [9].
Proteolytic enzymes are added to fish to accelerate the controlled hydrolysis process, resulting in a liquid product called fish protein hydrolysate (FPH), which contains various protein components. To increase protein consumption, fish protein hydrolysate can be consumed either as a dietary supplement or in its natural form. This is because fish protein hydrolysate can break down fish protein into smaller peptides, which usually consist of 2-20 amino acids [10].

Material
The raw material used in the preparation of protein hydrolysate is mackerel fish obtained from the Kobong Fish Market, Semarang. Additional ingredients to process protein hydrolysate are mackerel, flavorzyme, distilled water, NaOH 1N, HCL 1N, TCA 20%, and H2SO4.

Research Methodology
The research method used in the processing of mackerel protein hydrolysate with the addition of flavorzyme enzyme is using experimental laboratories method, which is a method to obtain data by conducting experiments in the laboratory. In this study there are dependent variables and independent variables that have been determined. The dependent variables in this study are time, temperature, and pH of the hydrolysis process, while the independent variables are the percentage of hydrolysis degree, amino acid profile content, and proximate value of the sample. RSM analysis was carried out using Design Expert 13. There are 3 independent variables used in this study, namely pH, temperature, and time. The range for each factor is determined by the rule that the distance between the midpoint (0) and the lower limit (-1) must have the same difference as the distance between the midpoint (0) and the upper limit (+1).

Mackerel Meat Preparation
The mackerel used in the preparation of protein hydrolysate was mackerel that weighs 100-200 g. Mackerel was purchased from Kobong Fish Market, Semarang. Frozen mackerel was thawed with water and washed thoroughly. The mackerel was then de-boned using the single fillet technique and cut into smaller sizes. Meat weight ranges from 70-80 g per fish. The clean mackerel meat can be used for the protein hydrolysate manufacturing process.

Mackerel Fish Protein Hydrolysate production
The preparation of protein hydrolysate using enzymatic hydrolysis method which was modified using mackerel [11]. The first step in making protein hydrolysate was adding mackerel meat to distilled water in a ratio of 1:4 and adding flavorzyme with a concentration of 4% of the total volume of the mixture of mackerel meat and distilled water. The mixture of mackerel meat, distilled water, and flavorzyme were then blended until smooth and homogeneous. The hydrolysis process used a water bath with a temperature of 45-65°C for 30-90 minutes and pH 6-8. To inactivates the enzyme, the hydrolysate was also heated at 80°C for 20 min. The sample was then centrifuged for 20 min at 3000 rpm in order to separate the supernatant and natant. The hydrolyzed liquid fish protein was frozen for shipment to the degree of hydrolysis (DH) testing site and the results with the best DH were tested for proximate analysis and amino acid profile. 20.83 Based on the proximate analysis that has been carried out, it can be seen that the sample mackerel has a protein content of 20.83% and a low lipids content of 1.03%. Based on the low lipid content, the sample was not necessarily pre-treated because the low lipid content will not significantly affect the protein hydrolysate obtained. According to [10], high lipids content can affect the hydrolysis process so that component removal is required. The removal of lipids components is intended to optimize the hydrolysis process and maintain product stability during storage. This is also confirmed by research conducted by [12] which showed that of the 3 types of fish (milkfish, tilapia, and milk shark) tested, the fish with the highest protein content produced fish protein hydrolysate with the highest protein content as well.

Degree of Hydrolysis Response Analysis
The Box-Behnken Design experiment resulted in 17 experimental treatments. Treatments with different temperature, time, and pH factors will affect the response results of the degree of hydrolysis of fish protein hydrolysate formed. The response of the degree of hydrolysis of 17 treatments is presented below. 4 how much protein has been successfully hydrolyzed. The highest DH value was seen in the treatment with pH 7, temperature 55 o C, and hydrolysis time for 1 hour with a DH value of 17.7293%. Differences in hydrolysis condition factors such as pH, time, and temperature will greatly affect the DH value produced because these environmental conditions will affect enzyme activity. each protease has certain environmental conditions to achieve an optimal hydrolysis process. When the temperature increases, the denaturation process will occur and the protease enzyme will become inactive. In addition, proteases are also sensitive to pH [13].
The lowest degree of hydrolysis value was found in the treatment with a pH value of 6, a temperature of 45°C and a treatment time of 1 hour with a DH value of 13.3292%. Apart from environmental factors, the use of different enzymes will also affect the DH value. The findings of [14] found that mackerel hydrolysate (Rastrelliger kanagurta) with pepsin enzyme produced DH values in the range of 14.3% -25.9%, while DH hydrolysate with the addition of papain enzyme produced DH values between 11.8% -19.9%. In addition to the difference in enzymes used, the study used a longer process time of up to 6 hours which also affected the DH value of mackerel hydrolysate [14]. Parameters that affect the quality and function of a hydrolysate include enzyme type, enzyme concentration, pH, and temperature are important to optimize [15]. In this study, the enzyme that acts as a catalyst is flavorzyme that originates from Aspergillus oryzae, this enzyme had good characteristics in making amino acids, breaking peptides, and was proven to affect the bioactive properties of compounds such as antioxidant and antimicrobial content. Flavorzyme was a combination of exo and endopeptidase that could break down peptidic chains contained in protein molecules [16].

Degree of Hydrolysis Response Model
The DH response model analysis was carried out with the Design Expert 13 program. The selection of the DH response model consists of 3 stages of statistical calculations, namely Sequential model of square, lack of fit test, and model summary statistics. This model was chosen because of the smallest p-value (<0.0001) and the largest F-value, which means that the independent factors/variables in the experiment have an effect on the dependent variable of the experiment, if the value of F>1 and the value of P<0.05 then the model used is valid [17].  5 (temperature, pH, and time) on the dependent variable (DH) and the greater the R2 the more significant the influence of the independent variable. R2 value has a range between 0 and 1 and is usually expressed in percentage form. The R2 value with a range of 0.3 < r < 0.5 is considered low, the range of 0.5 < r < 0.7 is considered moderate, and r> 0.7 is considered high [19]. From table 3.5, the Quadratic model value is 4.10 in the PRESS column/ PRESS is used to calculate error variation. Generally, a small PRESS value will indicate the most suitable regression model and a high press value will indicate an unsuitable regression model [20].

Degree of Hydrolysis Analysis of Variance
Based on previous statistical testing, it is known that the model used is the Quadratic model. The results of the analysis of variance are presented below. The lack of fit value in the ANOVA table is 0.0606, which indicates that the value is not significantly different (> 0.05) and the model used is correct. The desired lack of fit parameter is insignificant with a value above 0.05 [21]. Based on Table 7. It can be seen that the value of R2 is high (0.9894) because the value of 0.9894 is close to 1. The difference between Adjusted R2 (0.9758) and Predicted R2 (0.8589) is smaller than 0.2 (0.1169) which shows good response results. A difference value of 0.2 between adjusted R 2 and Predicted R 2 indicates the model is fit [22].
Based on Adeq Precision Value on the table, the model can be used because it has a value of more than 4 (25.03) which indicates a high level of precision. Adeq Precision measures the signal to noise ratio. In general, if the Adeq Precision value is more than 4, it is accurate [23] 6 that an increase in the independent variable up to the optimal point will give an increase to the dependent variable. Based on Noviyanti et al. [24], found that temperature has an impact on enzyme activity. Enzymatic reactions occur slowly at low temperatures; as the temperature rises, they move faster until the reaction rate reaches a maximum at the ideal temperature. Enzymes will be denatured and the rate of enzymatic processes will slow down if the temperature is raised above the ideal temperature .

Figure 1. Normal Plot of Residuals Graphic
From the graph above, it can be seen that most of the dots are close to the red straight line. This indicates normal residuals. The normal plot of residuals graph is one of the techniques used to determine whether the data is normally distributed or not. Data will be normally distributed if it is close to a straight line [25].

Model Graph
The relationship between factors and responses can be seen in the contour graph and response surface graph (3D Surface). The relationship between pH and temperature are shown as follows: Contour plot graph is a graph that shows the interaction between factors on the response that can be seen on the lines of the graph and the color shows how much interaction between factors on the response. The various colors of the contour plot graph show the reaction value. The red color displays the highest reaction, while the blue color displays the lowest reaction [26]. Based on the two graphs, it can also be seen that pH and temperature will affect the value of the Degree of Hydrolysis. This is proven by [27] who examined the enzymatic activity of lipase enzymes where the activity will increase with increasing temperature and pH, but if it exceeds the optimum point the enzymatic activity will decrease. Flavorzyme enzyme is a protease enzyme derived from microorganisms that is effective in breaking the peptide chain in a protein. There are several protease enzymes derived from microbes such as Alcalase, Neutrase, Protamex, and Flavorzyme which will produce different degrees of hydrolysis [28]. This is reinforced by research conducted by [29] on duck meat hydrolysis to break down protein compounds into peptide compounds that have bioactive components as antioxidants. Based on the research that has been done, meat hydrolyzed with flavorzyme has better antioxidant properties compared to duck meat that is not hydrolyzed. The relationship between pH and time to the degree of hydrolysis response is as follows: a. b. Figure 3. a. Contour graph and b. Response surface graph (3D Surface) of pH and time factors on the degree of hydrolysis response. Based on the two graphs, it can also be seen that pH and time will affect the value of the Degree of Hydrolysis. Based on the two graphs, it can also be seen that pH and temperature will affect the value of the Degree of Hydrolysis because the enzymatic process is very dependent on environmental conditions such as pH and temperature.

Determination of Optimum Point of Response of Degree of Hydrolysis
Controlled factors in the optimization of the degree of hydrolysis response include pH, time, and temperature. The criteria of each factor and the desired response can be seen in table 8.  8. the factors that can be controlled are between the limits and the lower limit limits that have been determined. The lower and upper limits for the pH factor are 6 and 8, the lower and upper limits for the temperature factor are 45 and 65, the lower limits for the time factor are 0.5 and 1.5. The desired response of hydrolysis degree is maximum. The Design Expert 13 program provides 100 optimization solutions which can be seen in table 9. 1.000 Based on the prediction of the Design Expert 13 program, the selected solution is the treatment with pH 6.936, temperature 57.929cC and time 1.463 hours. This solution is predicted to produce DH of 17.743%. This solution was chosen because it has a desirability of 1 which indicates a good optimal function.if the desirability value is close to 1, then the condition of these factors is the most appropriate to achieve optimal DH [30]. 27.06% Amino acid analysis was performed on the sample with the best DH value. Amino acids are the building blocks of proteins. Amino acids are organic compounds that contain amino groups (-NH2) and carboxyl groups (-COOH) in their structure, and can be linked together by peptide bonds to form polypeptides, which are the precursors of proteins. There are 20 types of amino acids commonly found in proteins, each with a unique combination of properties. Some of these amino acids can be synthesized by the body or are also referred to as non-essential amino acids, while others must be obtained from the diet and are therefore referred to as essential amino acids. Essential amino acids are amino acids that are not made by the body and must be obtained from food sources of protein. Non-essential amino acids are amino acids that can be made by the human body. Protein quality is assessed by the ratio of amino acids contained in the protein [31]. Based on the results of the amino acid analysis carried out, the non-essential amino acid with the highest increase was Glutamic Acid with a value of 4832.103537 mg/kg. The most prevalent amino acid in the human diet, L-glutamic acid (also known as glutamate), is also a major excitatory neurotransmitter in the brain. This amino acid, which is part of proteins and neurotransmitters, is essential for maintaining normal biological processes. However, when present in excess outside of proteins as a single amino acid, glutamic acid becomes excitotoxic (can damage nerve cells) [32]. Glutamic acid is under the main group of neurotransmitters. This amino acid is the main working neurotransmitter of the brain. This compound improves brain function and mental activity by detoxifying the brain from ammonia by attaching itself to nitrogen atoms in the brain and also helps in the transportation of potassium across the blood brain barrier. It can be concluded that glutamate is involved in cognitive functions such as learning and memory in the brain, although excessive amounts can cause neuronal damage associated with diseases such as amyotrophic lateral sclerosis, lathyrism, and Alzheimer's disease [33].

Amino Acid Profile Analysis
Glutamic acid has a use as a flavoring ingredient, the taste caused by glutamic acid is savory or better known as umami. Food additives that provide the umami effect are categorized as flavorings, including glutamate salt compounds such as monosodium glutamate and monopotassium glutamate. Excessive use of glutamate does not make food more delicious and can even spoil the taste. Generally, the use of glutamic acid has a positive reaction to the taste of salty and sour foods, and the optimum addition of glutamic acid to food generally ranges from 0.1-0.8% [34].

Conclusion
The conclusions that can be obtained from the results of the research that has been done are (a) based on the analysis of Response Surface Methodology with Box-Behnken Method, it can be seen that the factors of temperature, time, and pH at the time of hydrolysis have a significant effect on the degree of hydrolysis of mackerel protein hydrolysate samples, and the optimum conditions can be achieved at pH 6.936; temperature 57.929 °C; time 1.463 hours with a desirability value of 1.00 and (b) According to the findings of the amino acid study, there was a shift in the amount of 15 different types of amino acids, with glutamic acid experiencing the largest increase at 4832. 103537 mg/kg. Suggestions that can be given in this research are further research on other factors such as enzyme concentration, the ratio of enzymes to substrates to the results of the degree of hydrolysis to be obtained and the preparation of hydrolysates from other parts of mackerel that have lower economic value such as offal and skin to determine the further potential of mackerel hydrolysates.