OPTIMIZATION OF ENZYMATIC HYDROLYSIS OF MACKEREL (Rastrelliger sp.

) MUSCLE
PROTEIN HYDROLYSATE USING RESPONSE SURFACE METHODOLOGY

P H Riyadi1, Romadhon1, L Bramantyo1

Department of Fish Products Technology, Faculty of Fisheries and Marine Science,

1

Universitas Diponegoro, Jl. Prof. Soedarto S. H., Semarang,

Central Java, Indonesia, 1269

putut.riyadi@live.undip.ac.id

Abstract. 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 55oC and 60 minutes 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 material.
Keywords: Optimization, Mackerel, Protein Hydrolysate, Response Surface Methodology.
1. Introduction
Mackerel (Rastrelliger sp.) is a fish belonging to the Scrombridae family that has a small, slender, and flat
body unlike its relatives such as tuna and skipjack which have a larger size. Mackerel is a fish with medium
economic value because many fishermen catch this type of fish for sale locally and in general the processing of
products from this fish still tends to be simple such as being used as raw material for pindang and salted fish.
Based on statistics from the Ministry of Maritime Affairs and Fisheries (KKP) (2018), sectoral statistics of
annual mackerel production reached 360,676.96 tons per year with the highest province being South Kalimantan
at 31,732 tons per year and Central Java ranked 7th largest with annual production reaching 23,740.85 tons per
year [1]. This large amount of mackerel can be used as a potential for development as a product with more added
value than traditional processing such as making fish protein hydrolysate.
Protein hydrolysate is the end product of protein hydrolysis to take place naturally, as a result. Protein
hydrolysis results in the breakdown of proteins into short-chain molecules such as peptides and amino acids. Due
to the antibacterial and antioxidant properties of hydrolyzed amino acids, protein hydrolysates in the food
industry are commonly used as supplements to provide nutrients to foods, as emulsifiers, and can also serve as
preservatives. Peptide antioxidants in hydrolysates are also increased by the presence of amino acids with strong
antioxidant activity, such as methionine, cysteine, lysine, and leucine. The process of making hydrolysates is
done by hydrolysis of the protein component of the product [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 small-scale 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 oC, 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].

Materials and Methods

1.1. Material and Instruments

The instruments used for the manufacture of mackerel protein hydrolysate are analytical scales, Kjedahl,
water bath, glass jar, destructor, funnel, blender, measuring flask, dropper pipette, gas stove, centrifuge, and
High Performance Liquid Chromatographer. 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.

1.2. 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.

1.3. Mackerel Meat Preparation

The mackerel used in the preparation of protein hydrolysate is mackerel that weighs 100-200 grams.
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 grams per fish. The clean mackerel meat can be used for the protein hydrolysate
manufacturing process.

1.4. Mackerel Fish Protein Hydrolysate

The preparation of protein hydrolysate using enzymatic hydrolysis method refers to Roslan et al. (2014).
which was modified using mackerel [11]. The first step in making protein hydrolysate is 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 was
then blended until smooth and homogeneous. The hydrolysis process used a water bath with a temperature of 45-
650C for 30-90 minutes and pH 6-8. To inactivate the enzyme, the hydrolysate was also heated at 80 0C for 20
min. The sample was then centrifuged for 20 min at 3000 rpm in order to separate the supernatant and natan. 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.

2. Result and Discussion

3.1 Mackerel Meat Proximate Analysis
Table 3.1. Mackerel Meat Proximate Analysis
Proximate Water Fat Protein
Percentage
53,55 1,03 20,83
(%)
Based on the proximate analysis that has been carried out, it can be seen that the sample mackerel has
a high protein content of 20.83% and a low fat content of 1.03% so that there is no need for pre-treatment of the
sample and can produce protein hydrolysates with high content as well. According to Nurhayati et al. (2014),
high fat content can affect the hydrolysis process so that component removal is required. The removal of fat
components is intended to optimize the hydrolysis process and maintain product stability during storage. This is
also confirmed by research conducted by Annisa et al. (2017) 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 [12].

3.2 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.
Table 3.2 Degree of Hydrolysis Response
Factor Respon
No
pH Temperature (oC) Time (Hour) Degree of Hydrolysis (%)
1 7 65 1,5 17,3737
2 6 65 1 14,9129
3 7 55 1 17,7293
4 8 45 1 15,5132
5 8 65 1 14,7373
6 7 45 1,5 16,8132
7 6 55 0,5 14,4912
8 6 55 1,5 15,5192
9 8 55 0,5 15,3812
10 6 45 1 13,3292
11 7 45 0,5 16,2313
12 7 55 1 17,5112
13 7 65 0,5 16,6717
14 7 55 1 17,6681
15 7 55 1 17,4321
16 8 55 1,5 15,2813
17 7 55 1 17,6123
The degree of hydrolysis (DH) is a metric of measurement that indicates how easily a protein can be
hydrolyzed into smaller peptides or amino acids. A large or small value of the degree of hydrolysis will indicate
how much protein has been successfully hydrolyzed. The highest DH value was seen in the treatment with pH 7,
temperature 55oC, 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 45oC 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 Sheriff et al. (2014) 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. Flavorzyme is an enzyme derived from the fungus
Aspergillus oryzae, this enzyme has good characteristics in making amino acids, breaking peptides, and is
proven to affect the bioactive properties of compounds such as antioxidant and antimicrobial content.
Flavorzyme is a combination of exo and endopeptidase that can 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..
Table 3.3 Sequential Model of Sum Square
Mean
Source Sum of Squares df F-Value p-Value
Square
Mean vs Total 4422,95 1 4422,95
Linear Vs Mean 1,91 3 0,6351 0,3044 0,8217
2 FI vs Linear 1,71 3 0,5712 0,2248 0,8770
Quadratic vs
25,10 3 8,37 190,43 <0,0001 Suggested
2FI
Cubic vs
0,2504 3 0,0835 5,84 0,606 Aliased
Quadratic
Residual 0,0571 4 0,0143
Total 4451,98 17 261,88
Based on the Sequential Model of Sum Square, the recommended model to choose is Quadratic vs
2FI. 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].
Table 3.4. Lack of Fit Test
Sum of
Source df Mean Square F-Value p-Value
Squares
Linear 27,06 9 3,01 210,51 <0,0001
<0,,000
2FI 25,35 6 4,23 295,77
1
Quadratic 0,2504 3 0,0835 5,84 0,0606 Suggested
Cubic 0,0000 0 Aliased
Pure Error 0,0571 4 0,0143
The Lack of Fit Test table shows that the model that has minimal unfitness is the Quadratic model
with a value of 0.0606. This can be seen from the p-value which is greater than 0.005 which indicates that the
Quadratic model does not have a large misfit. A fit that is not significantly different p-value>0.05 and the fit of
other parameters that are significantly different (p-value<0.05) indicates the suitability of the model used [18].
Table 3.5. Summary of Statistic
Source Std. Dev R2 Adjusted R2 Predicted R2 PRESS
0,065
Linear 1,44 -0,1500 -0,5995 46,43
6
0,124
2FI 0,0296 -0,4005 -2,0160 87,55
7
Quadrati 0,989
0,2096 0,9758 0,8589 4,10 Suggested
c 4
0,998
Cubic 0,1195 0,9921 * Aliased
0
 Based on the Summary of Statistic indicates that the model chosen is the Quadratic model. This is
indicated from the R2 value of 0.9894 which shows the diversity of data is the influence of the independent
variable (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].

3.3 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.
Table 3.6 Degree of Hydrolysis ANOVA
Sum of
Source Df Mean Square F-Value p-Value
Squares
Model 28,72 9 3,19 72,63 <0,0001 Significant
A-pH 0,8847 1 0,8847 2,014 0,0028
B-Temperatur 0,4089 1 0,4089 9,31 0,0186
C-Waktu 0,6116 1 0,6116 13,92 0,0073
AB 1,39 1 1,39 31,68 0,0008
AC 0,3180 1 0,3180 7,24 0,0311
BC 0,0036 1 0,036 0,0821 0,7828
A2 22,00 1 22,00 500,75 0,0001
B2 1,96 1 1,96 44,52 0,0003
C2 0,0785 1 0,0785 1,79 0,2232
Residual 0,3076 7 0,0439
Lack Of Fit 0,2504 3 0,00835 5,84 0,0606 Not Significant
Pure Error 0,0571 4 0,0143
Cor Total 29,03 16
The results of Analysis of Variance (ANOVA) of the Quadratic model are significantly different, this
can be seen from the p-value of <0.0001 and smaller than 0.05. Variable A (pH), Variable B (temperature), and
Variable C (time) all three have p-values smaller than 0.05 so it can be stated that the three variables have a
significant influence on the dependent variable (DH).
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].
Tablel 3.7. Degree of Hydrolysis Fit Statistics
Category Value Category Value
Std. Dev. 0,2096 R2 0,9894
Mean 16,13 Adjusted R2 0,9758
C.V. % 1,30 Predicted R2 0,8589
Adeq Precision 25,6032

Based on Table 4.7. 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 R2 and Predicted R2 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]. The regression model obtained is as
follows:
2 2 2
DH =17,59+0,0326 × A+ 0,02261× B+ 0,2765× C−0,5899× AB−0,2820 × AC +0,0300 × BC −A −B −C
DH : Degree of Hydrolysis (%)
A : pH
B : Temperature (oC)
C : Time (Hours)
 The equation shows a significant positive relationship between the independent variables (pH,
temperature, and time) and the dependent variable (degree of hydrolysis). Based on the equation, it can be
indicated that an increase in the independent variable up to the optimal point will give an increase to the
dependent variable. Noviyanti et al. (2012) 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 [24].

3.4 Normality of Distribution of Degree of Hydrolysis Response Data

Figure 3.1. 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].

3.5 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:

a. b.
Figure 3.2 a. Contour graph and b. Response surface graph (3D Surface) of pH and temperature factors on
degree of hydrolysis response.
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 Amalia et al. (2013) 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 [27].
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 Alkalase,
Neutrase, Protamex, and Flavorzyme which will produce different degrees of hydrolysis [28]. This is reinforced
by research conducted by Wang et al. (2018) 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
[29]. The relationship between pH and time to the degree of hydrolysis response is as follows:

a. b.
Figure 3.3 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.

(a) (b)
Figure 3.4. (a) Contour Graph (b) Response surface graph (3D Surface) pH and Temperature Factors on
Hydrolysis Degree Response.

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.
3.6 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 3.8.
Table 3.8. Desired Factor and Response Criteria
Upper Lower Upper Importanc
Name Goal Lower Limit
Limit Weight Weight e
pH In Range 6 8 1 1 3
Temperature In Range 45 65 1 1 3
Time In Range 0,5 1,5 1 1 3
Degree of
Maximize 13,3292 17,7293 1 1 3
Hydrolysis
StdErr(DH) None 0,09937402 0,181527 1 1 3
Based on table 3.8. 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 3.9.
 Table 3.9. Optimization Solution
Waktu
No pH Temperatur (oC) DH (%) StdErr Desirability
(Jam)
1 6,936 57,929 1,463 17,743 0,119 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].

3.7 Amino Acid Profile Analysis

Table 3.10. Amino Acid Profile of Mackerel Meat and Protein Hydrolysate
Fish Protein
Mackerel Meat Hydrolysate Difference
Amino Acid (mg/kg) (mg/kg) (mg/kg) Percentage
L-Serin 7210,76 6375,470989 -835,2890105 -11,58%
L-Asam Glutamat 24437,16 29269,26354 4832,103537 19,77%
L-Fenialanin 6122,58 6286,625558 164,0455579 2,68%
L-Isoleusin 8287,73 7903,296674 -384,4333263 -4,64%
L-Valin 9095,69 9142,791305 47,10130526 0,52%
L-Alanin 9974,98 11391,02802 1416,048021 14,20%
L-Arginin 8426,89 7179,289726 -1247,600274 -14,80%
Glisin 7336,14 8146,371811 810,2318105 11,04%
L-Lisin 17991,88 17340,42684 -651,4531579 -3,62%
L-Asam Aspartat 15584,36 17364,32653 1779,966526 11,42%
L-Leusin 13647,97 14013,45924 365,4892421 2,68%
L-Tirosin 4416,75 3894,552211 -522,1977895 -11,82%
L-Prolin 4830,82 5642,474253 811,6542526 16,80%
L-Treonin 9123,2 7630,796421 -1492,403579 -16,36%
L-Histidin 6019,67 7648,469032 1628,799032 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].
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].

4 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 Suraface Methodolgy 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 oC; 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.

5 Acknowledgement
The authors would like to thank the Department of Fishery Products Technology, Faculty of Fisheries and
Marine Science, Diponegoro University.

6 References

[1] Kementerian Kelautan and Perikanan (KKP). 2018. Statistik KKP. Online at
https://statistik.kkp.go.id/home.php?m=total_ikan&i=2#panel-footer. Accessed on 8 Februari 2023.

[2] Kusumaningtyas, E., R. Widiastuti, H. D. Kusumaningrum, and M. T. Suhartono. 2015 Aktivitas

Antibakteri dan Antioksidan Hidrolisat Hasil Hidrolisis Protein Susu Kambing dengan Ekstrak Kasar
Bromelin. J. Teknol dan Industri Pangan. 26(2):179-188.

[3] See, S. F., L. L. Hoo, and A. S. Babji. 2011. Optimization of Enzymatic Hydrolysis of Salmon (Salmo
salar) Skin by Alcalase. International Food Research Journal. 18(4):1359-1364.

[4] Nair, A. T., A. R. Makwana, and M. M. Ahammed. 2014. The Use of Response Surface Methodology for
Modelling and Analysis of Water and Wastewater Treatment Processes : a Review. Water Science &
Technology 69(3):464-478.

[5] Thariq, A. S., F. Swastawati, and . Surti. 2014. Pengaruh Perbedaan Konsentrasi Garam pada Peda Ikan
Kembung (Rastreliger neglectus) terhadap Kandungan Asam Glutamat Pemberi Rasa Gurih (Umami).
2014. Jurnal Pengolahan dan Bioteknologi Hasil Perikanan. 3(3):104-111

[6] Elvarasan K., V. N. Kumar, and B. A. Shamasundar. 2013. Antioxidant and Functional Properties of Fish
Protein Hydrolysates from Fresh Water Carp (Catla catla) as Influenced by The Nature of Enzyme.
Journal of Food Processing and Preservation. 2013. 1-8.

[7] Sampaga, L. O. T., A. I. Nur, and M. Tajuddah. 2019. Kajian Ekologi dan Pengelolan Ikan Kembung
(Ratreliger kanaguarta) di Selat Tiworo. 3(2):52-59

[8] Anggreini, A. P., S. S. Astuti, I. Miftahudin, P. I. Novita, and D. G. R. Wiadnya. 2017. Uji Selektivitas
Alat Tangkap Gillnet Millenium terhadap Hasil Tangkapan Ikan Kembung (Rastrelliger Brachysoma).
Journal of Fisheries and Marine Science. 1(1):24-30.

[9] Koesoemawardani, D., F. Nuarainy, and S. Hidayati. 2011. Proses Pembuatan Hidrolisat Protein Ikan
Rucah. Jurnal Natur Indonesia. 13(3):256-261.
[10] Nurilmala, M. T. Nurhayati, and R. Roskananda. 2018. Limabh Industri Filet Ikan Patin untuk Hidrolisat
Protein. JPHPI. 21(2):287-293.

[11] Roslan, J., S. M. M. Kamal, K. F. M. Yunos, and N. Abdullah. 2014. Optimization of Enzymatic
Hydrolysis of Tilapia Muscle (Orechromis niloticus) using Response Surface Methodology (RSM).
Sains Malaysiana. 43(11):1715-1723.

[12] Annisa, S., Y. S. Darmanto, and U. Amalia. 2017. Pengaruh Perbedaan Spesies Ikan Terhdap Hidrolisat
Portein Ikan dengan Penambahan Enzim Papain. IJFST. 13(13):24-30.

[13] O’Dwyer, M. V., A. W. Sahin, E. K. Arendt, E. Zannini. 2022. Enzymatic Hydrolysis of Pulse Proteins as
a Toll to Improve Techno-Functional Properties. Foods. 11(9):1-25.

[14] Sheriff, A. S., S. Balasubramanian, R. Barnitharan, and P. Ponmurugan. 2014. Synthesis and in vitro
Antioxidant Functions of Protein Hydrolysate from Backbones of Rastrelliger Kangurta by
Proteolytic Enzymes. Saudi Journal of Biological Sciences. 21 (19-26).

[15] Siddik, M. A. B., J. Howieson, R. Fotedar, and G. J. Partridge. 2021. Enzymatic Fish Protein
Hydrolysates in Finfish Aquaculture: a review. Reviews in Aquaculture. 13:406-430.

[16] Alahmad, K., A. Noman, W. Xia, Q. Jiang, and Y. Xu. 2023. Influence of the Enzymatic Hydrolysis
Using Flavourzyme Enzyme on Functional, Secondary Structure, and Antioxidant Characteristics of
Protein Hydrolysates Produced from Bighead Carp (Hypothalmichthys nobilis). Molecules. 28 (1-21).

[17] Peng, Y. U. Khaled, A. A. A. A. Al-Rashed, R. Meer, M. Goodarzi, and M. M. Sarafraz. 2020. Potential
Appllication of Response Surface Methodology (RSM) for the Prediction and Optimization of
Thermal Conductivity of Aqueous CUO (II) nanofluid: A Statistical Approach and Experimental
Validation. Physica A. 554:1-9.

[18] Ruangmee, A. and C. Sangwichien. 2013. Response Surface Optimization of Enzymatic Hydrolysis of
Narrow-Leaf Cattail for Bioethanol Production. Energy Conversion and Management. 73:381-388.

[19] Saleem, A., A. Hussain, A. Chaudhary, Q. Ahmad, M Iqtedar, A. Javid, and A. M. Akram. 2020. Acid
Hydrolysis Optimization of Pomegranate Peels Waste Using Response Surface Methodolgy for
Ethanol Production. Biomass Conversion and Biorefinery. 1-12

[20] Madondo, I. N., S. Rathilal, and B. F. Bakare. 2022. Utilization of Response Surface Methodology in
Optimization and Modelling of a Microbial Electrolysis Cell for Wastewater Treatment Using Box-
Behnken Design Method. Catalyst. 12(9):1-20.

[21] Mojiono dan D. N. Sholehah. 2020. Optimasi Ekstraksi Pati Jagung Madura-3 Berdasarkan Lama
Perendaman dan Konsentrasi NaOH. REKAYASA. 13(2):118-124.

[22] Igwilo, C. N., C. N. Ude, and M. I. Onoh. 2022. Evaluation of RSM and ANFIS Modeling Perfomance in
Fermentable Sugar Production from Enzymatic Hydrolysis of Colocynthis vulgaris shard Seeds Shell.
Egyptian Journal of Petroleum. 31:31-36.

[23] Ganapathy, T., R. O. Gakhar, and K. Murugesan. 2011. Optimzation of Peformance Parameter of Diesel
Engine with Jatropha Biodiesel Using Response Surface Methodology. International Journal of
Sustainable Energy. 1-15.

[24] Noviyanti, T., P. Ardiningsih, and W. Rahmalia. 2012. Pengaruh Temeperatur terhadap Aktivitas Enzim
Protease dari Daun Sansakng (Pycnarrhena caulifora Diels). JKK. 1(1):45-48.

[25] Radhwan, H., Z. Syahfull, M. R. Farizuan, M. S. M. Effendi, and A. R. Irfan. 2019. Optimization
Parameter Effects on the Quality Surface Finish of the Three Dimensional Printing (3D Printing)
Fused Deposition Modeling Using RSM. AIP Conference Proceedings. 2129:1-7.
[26] Nurmiah, S., R. Syarief, Sukarno, R. Peranginangin, and B. Nurtama. 2013. Aplikasi Response Surafce
Methodolgy pada Optimalisasi Kondisi Pengolahan Alkali Treated Cottonii (ATC). JPB Kelautan dan
Perikanan. 8(1):9-22.

[27] Amalia, R. N., R Bulan, and F. Sebayang. 2013. Penentuan pH dan Suhu Optimum untuk Aktivitas
Ekstrak Kasar Enzim Lipase dari Kecambah Biji Karet (Heva brasiliensis) terhadap Hidrolisi PKO
(Palm Kernel Oil). Jurnal Saintia Kimia. 1(2): 1-7.

[28] Yuniarti, T., A. Prayudi, L. Supenti, H. Suhrawardan, dan P. Martosuyono. 2022. Produksi dan Profil
Kimia Hidrolisat Protein dari Hasil Samping Pengolahan Udang Segar. Jurnal Perikanan Universitas
Gajah Mada. 23(1):63-69.

[29] Wang, D., M. Zhang, Ye Zou, Z. Sun, dan Weimin Xu. 2018. Optimization of Flavourzyme Hydrolysis
Condition for the Preparation of Antioxidant Peptides from Duck Meat Using Response Surface
Methodology. JPSA. 55(3):217-223.

[30] Roslan, J., S. M. M. Kamal, K. F. M. Yunos, and N. Abdullah. 2014. Optimization of Enzymatic
Hydrolysis of Tilapia Muscle (Orechromis niloticus) using Response Surface Methodology (RSM).
Sains Malaysiana. 43(11):1715-1723.

[31] Sari, E. M., M. Nurimala, dan A. Abdullah. 2017. Profil Asam Amino dan Senyawa Bioaktif Kuda Laut
Hippocampus comes. Jurnal Ilmu dan Teknologi Kelautan Tropis. 9(2):605-617.

[32] Samuels, A. 2020. Dose Dependent Toxicity of Glutamic Acid: A Review. International Journal of Food
Properties. 23(1):412-419.

[33] Dutta, S., S. Ray, K. Nagarajan. 2013. Glutamic Acid as Anticanacer Agent: An Overview. Saudi
Pharmaceutical Journal. 21:337-343.

[34] Jinap, S. dan P. Hajep. 2010. Glutamate. Its Applications in Food and Contribution to Health. Appetite.
55:1-10.