Screening on halal gelatin extraction from spotted oceanic triggerfish (Chanthidermis maculata) skin using response surface methodology

Spotted oceanic triggerfish fish is one type of fish that has the potential to be a source of halal gelatin. Almost all parts of his body can become gelatin. In the process of processing the fish, many parts are wasted such as skin. Though this skin can be used for gelatin extract. From the results of the study, gelatin yield ranged from 2.7 – 9.3% with an average value of 5.50%. This gelatin has a viscosity value ranging from 6.21 – 7.57 cP which was analyzed using the Rapid Visco Analyzer (RVA) instrument and gel strength analysis using a Texture Analyzer and found results of 80.33 – 97.67 g / cm2. Based on the probability value < 0.05 yield, viscosity, and gel strength of gelatin, it is known that there are several independent variables that have a real effect on yield, namely variables B (concentration of H2SO4), D (soaking duration), F (ratio of water/extraction skin) and G (extraction temperature). In viscosity, the influential variables are C (citric acid concentration), D (soaking duration), E (soaking water/skin ratio) and G (extraction temperature). While on gel strength, the influential variables are A (NaOH concentration) and G (extraction temperature). The influence of factors on response can be sorted sequentially, namely the order in yield is G>F>B>D>A>H>C>E, in viscosity is C>E>D>G>H>A>B>F and in gel strength is G>A>E>H>B>F>C>D. This indicates that the G factor (extraction temperature) was selected as the most important factor, and the 2nd and 3rd most important factors can be selected from A (NaOH concentration), B (H 2SO4 concentration), D (extraction duration) and E (soaking water/skin ratio), and H (extraction duration).


Introduction
Spotted oceanic triggerfish (Chanthidermis maculata) is one type of fish that produces a lot of waste from its cutting.Usually, this fish is sold in the form of meat that has been separated from other parts (skin, bones, head) to be processed into buckets, meatballs, somai, and so on, while the remaining parts (other than meat) are discarded, causing a buildup of fishery waste that has no selling value.This is very unfortunate considering that all parts of the Spotted oceanic triggerfish can be used like skin.Skin waste from Spotted oceanic triggerfish is wasted unused in Mahirah Market, Banda Aceh.Fish skin waste 1297 (2024) 012082 IOP Publishing doi:10.1088/1755-1315/1297/1/012082 2 which covers about 9% of the total body weight of fish is very potential to be used as a source of raw materials in making gelatin [1].
The need for gelatin in Indonesia is increasing, but industries that specifically produce gelatin are not yet available, so this condition forces the government to continue importing gelatin.This gelatin requirement is imported from several countries such as China, Australia, and the European Union.Industrial development to produce gelatin commercially is needed as an effort to reduce dependence on imported products.Gelatin can be extracted from hard bones, cartilage as well as from animal skin.Gelatin on the market is generally non-halal gelatin because it comes from pig skin and bones.The issue of non-halal gelatin has become important for Muslims, causing many developments in the development of alternatives to making gelatin from halal sources.A very potential halal source to be used as raw material for gelatin is collagen derived from fish including waste.
Gelatin is a protein derivative resulting from collagen hydrolysis from the skin, muscles, organs, and bones.Gelatin is multifunctional because it can be applied to various industrial fields such as food, pharmaceuticals, cosmetics and photography.In the food industry, many use the gelatin industry, including in products that require foam formation (whipping agent), stabilizers, increase viscosity and also function as binders.It can also be used in the chocolate industry, jelly candy and dairy products.In the pharmaceutical industry, gelatin is used as an ingredient for the manufacture of hard capsules and soft capsules [2].
Based on the words of the Prophet SAW about the halality of two carcasses, namely, It has been lawful for us two carcasses and two blood.The two carcasses are fish and grasshoppers.The basis for the lawfulness is fish carcasses in all forms of circumstances.Either die by looking afloat or otherwise.Therefore, gelatin sourced from fish and its waste can be guaranteed halal [3].
The amount and physicochemical properties of gelatin depend on several factors, such as the method of preparation (pre-treatment) and the intrinsic properties of collagen.Pre-treatment is a preliminary treatment carried out on raw materials before gelatin extraction.The main production process of gelatin can be divided into three stages, namely the preparation of raw materials, the conversion of collagen into gelatin and the last is the acquisition of gelatin in dry form [2].In this study, Response Surface Methodology (RSM) was used to determine the optimal conditions for gelatin extraction of Spotted oceanic triggerfish skin.RSM is a method that combines mathematical techniques with statistical techniques that aims to analyze a response or Y factor such as yield, viscosity, and gel strength on the influence of several independent variables (free) or X factor [4].

Material
The materials used in this study were spotted oceanic triggerfish skin waste obtained from the Mahirah Market Fish Processing Site in Lampulo Banda Aceh, Aceh Province.The skin of the goats is cleaned and frozen until research will be carried out.The chemicals used in the study were NaOH, H2SO4, citric acid and aquadest.

Research Design
This research was conducted to obtain halal gelatin from goat-goat skin waste through the Response Surface Methodology (RSM) approach using Design Expert v.12 software.Response Surface Methodology (RSM) is used to obtain some optimum points of gelatin extraction from Spotted oceanic triggerfish skin using screening stages.At this stage, screening of 8 independent variables was carried out with a fractional factorial design of 28-4.The independent variables selected were (A) NaOH concentration (%), (B) H 2SO4 concentration (%), (C) citric acid concentration (%), (D) soaking time (minutes), (E) soaking skin/water ratio, (F) extraction skin/water ratio (G) extraction temperature (°C), (H) extraction time (hours).Each variable is independent at 2 different levels (Table 1).The treatment combination of all independent variables can be seen in Table 2.The responses measured in the study were yield, gel strength, and viscosity.
Description: A=NaOH concentration, B=H2SO4 concentration, C=Citric acid concentration, D=soaking time, E=water/soaking skin ratio, F=water/extraction skin ratio, G=extraction temperature, and H=extraction time.

Research Procedure
The process of extracting gelatin from Spotted oceanic triggerfish skin follows research conducted by [5] with slight modifications.The raw materials used are waste goat skin that has been cleaned and then frozen, and at the time of use of goat skin in thawing (4 °C) overnight (±12 hours).The fish skin is then cut into pieces with a size of ± 4x4 cm, then soaked in NaOH solution with NaOH concentration, soaking duration and soaking water ratio according to treatment (Table 2), then washed with water 3 times.Furthermore, soaking in H2SO4 solution with H2SO4 concentration, soaking duration and soaking water ratio according to treatment and washed 3 times.Then soaking using citric acid with citric acid concentration, soaking duration and soaking water ratio according to treatment.After that, the material is washed 3 times.The next step is gelatin extraction with extraction duration, temperature and skin ratio according to treatment.The filtrate obtained from the extraction process is filtered and then dried in an oven at ±50°C for ± 3 days.

Analysis
Gelatin that has been dried is then ground so that dry gelatin is obtained in the form of fine grains (powder).After obtaining gelatin, the yield was calculated [6], and viscosity analysis was carried out using the RVA instrument and gel strength using a Texture Analyzer (TA-XT plus, Stable microsystem Ltd., UK).Response Surface Methodology (RSM) is used to determine the optimal extraction condition parameters to produce gelatin with the best characteristics.

Raw Material Analysis
The raw material used in this study is Spotted oceanic triggerfish skin which is waste from the fish slaughterhouse in Pasar Mahirah, Lampulo, Banda Aceh.In this study, an initial analysis of raw materials was carried out in the form of proximate analysis to determine water content, ash content, protein content and fat content.The results of the proximate analysis of Spotted oceanic triggerfish skin are presented in the following Table 3.Chemical component analysis is a method used to measure the content of water, ash, fat, protein and carbohydrates in foodstuffs.The chemical analysis of spotted oceanic triggerfish as a whole are 77% moisture content, 20.58% protein content, 0.59% fat content and 0.96% ash content [7].Based on the results of the analysis, the moisture content of fish skin in this study was 50.11%.Then the protein content of Spotted oceanic triggerfish skin is high, which is 23.16%.

Functional Quality Analysis
The yield, viscosity and gel strength data obtained from this study can be seen in Table 4 and the probability values of the three responses can be seen in Table 5.Based on the probability value of yield, viscosity and gel strength of gelatin (Table 5) it is known that there are several independent variables that have a real effect (Table 6) on yield, namely variables B (concentration of H2SO4), D (soaking duration), F (ratio of water / extraction skin) and G (extraction temperature).In viscosity, the influential variables are C (citric acid concentration), D (soaking duration), E (soaking water/skin ratio) and G (extraction temperature).While on gel strength, the influential variables are A (NaOH concentration) and G (extraction temperature).This is because these variables have a probability value of < 0.05.Based on Table 6, the influence of factors on response can be sorted sequentially.In yield, the order is G>F>B>D>A>H>C>E, in viscosity it is C>E>D>G>H>A>B>F and in gel strength it is G>A>E>H>B>F>C>D.This indicates that the G factor (extraction temperature) was chosen as the most important factor, and the 2nd and 3rd factors can be selected from A (NaOH concentration), B (H2SO4 concentration ), D (extraction time) and E (soaking water/skin ratio), and H (extraction duration).

Yield of Gelatin
The results of fish gelatin extraction from Spotted oceanic triggerfish waste using the [4] method with modifications, where 8 independent variables with 2 levels (-1 and 1) were used to get yields ranging from 2.7 -9.3%.From the gelatin yield data (Table 7), the average value of 5.50% can be calculated.Based on the results of the Design of Experiment analysis using Design Expert, results were obtained in the form of independent variables that affect gelatin yield, namely variables B (H2SO4 concentration), D (soaking duration), F (water/skin extraction ratio) and G (extraction temperature) (Figure 1).It can be seen in Table 6 that the higher the concentration of H2SO4 the higher the gelatin yield, which is shown by R.O 10, 14 and 15 which use levels 1 (0.3%) higher than levels -1 (0.15%) such as R.O 5, 7 and 9.The statistical fit of yield can be seen in Table 8, and the pareto diagram of the independent variables in yield can be seen in Figure 1.From the results of the study, it can be seen that the higher the acid concentration and the duration of soaking, the higher the yield produced .Based on Figure 1 above, the value of the effect that causes an increase in the value of the yield response is shown in orange and the value of the effect that causes a decrease in the value of the yield response is shown in blue.In Figure 1, the effect value of the extraction temperature (G) causes an increase in the yield response value (the highest value for positive effect), while the effect value of the water/soaking skin ratio (F) causes a decrease in the yield response value (the highest value for negative effect).This means that the higher the extraction temperature, the higher the gelatin yield will be produced, but if the higher the water/skin extraction ratio, the lower the amount of gelatin yield.

Viscosity
Gelatin viscosity analysis was carried out from goat-goat skin waste using the Rapid Visco Analyzer (RVA) instrument.The viscosity of gelatin obtained ranged from 6.21-7.57cP (Table 9).The results of the Design of Experiment analysis using Design Expert show variables that have a real effect on gelatin viscosity, namely citric acid concentration (C), soaking time (D), soaking water / skin ratio (E), extraction temperature (G) and extraction duration (H) (Figure 2).Based on Table 9, it can be observed that R.O 10 which uses level 1 soaking length (40 minutes) has a greater viscosity value than R.O 1 which uses level -1 soaking length (35 minutes).The length of soaking will affect the molecular weight of gelatin.Viscosity is related to the average molecular weight (BM) of gelatin and the distribution of molecules, while the molecular weight of gelatin is directly related to its amino acid chain length [8].The less molecular weight of gelatin, the faster the distribution of gelatin molecules in solution, thus giving a low viscosity value [9].The statistical fit of viscosity can be seen in Table 10, and a pareto diagram of the influence of independent variables on viscosity can be seen in Figure 2. According to [10], factors that affect viscosity include temperature, extraction time, acid concentration, and number of dissolved molecules.Based on the results of other research, the viscosity of mackerel gelatin is 5.51 cP [11], and 3.12 cP [12].Meanwhile, the results of this study using spotted oceanic triggerfish skin obtained viscosity ranging from 6.21-7.57cP.
The smaller the concentration of acid used, the viscosity value obtained will be smaller.This is thought to be because the low concentration of acid causes complete hydrolysis so that the amino acid chain formed is not long enough and the viscosity becomes low.The viscosity of the gelatin produced is highly dependent on the concentration of the dissolved gelatin, and the temperature used in gelatin extraction.The viscosity of gelatin will have an effect on the final product of a product.The higher the concentration of acid, the higher the viscosity [13].In Figure 2, the variables that have a positive influence on gelatin viscosity are variable C or citric acid concentration, D or soaking time, G or extraction temperature, H or extraction time.That is, the higher the value of the variable used, the higher the viscosity produced.Variable E or the ratio of water / soaking skin has a negative influence, namely the higher the ratio, the lower the viscosity value obtained.The use of acetic acid in the acid process has an influence on changes in collagen structure (triple helix) released into a single helix so that the viscosity produced changes [14].
The viscosity of gelatin from spotted oceanic triggerfish skin obtained in this study tends to meet the Indonesian National Standard (SNI) No. 3735 of 1995 because it ranges from 1.5-7.0cP.Different treatments will result in different viscosity values in each gelatin.The excessive drying process can cause the breaking of gelatin protein bonds, resulting in low viscosity quality and high moisture content will cause gelatin gel to be difficult to thicken [15].Gelatin with low viscosity will produce gels that are easily brittle, while gelatin with high viscosity will produce stronger gels [16].

Gel strength
The gel strength of gelatin from spotted oceanic triggerfish skin in this study was analyzed using a Texture Analyzer (TA-XT plus, Stable Microsystem Ltd., UK) using a modified method from FMC Marine Colloid.The results of the analysis obtained ranged from 80.33-97.67g / cm 2 .This value is still in the range of gelatin gel strength according to the Indonesian National Standard (SNI) No. 3735 of 1995, which is 50-300 Bloom.Based on research conducted by [17] red snapper skin gelatin can produce a gel strength value of 312.5 gbloom, while conducted a study on gelatin extraction from the head of curisi fish using acid treatment obtained a gel strength of 311.01 gbloom [12].Alfaro et al., (2014) conducted a study on the extraction of gelatin from African catfish and obtained a gel strength value of 234 gbloom [18].Likewise with research obtained from [19] where the strength of the gel obtained from large snapper gelatin amounted to 62.6 gbloom.The value of gel strength in this study ranged from 80.33-97.67g/cm2.The factors that can affect the high and low strength of the gel are pH, differences in concentration and extraction temperature.Fit viscosity statistics can be seen in Table 12, and a pareto diagram of the influence of independent variables on viscosity can be seen in Figure 3.The results of the Design of Experiment analysis showed that the independent variables that significantly affect the strength of the gelatin gel produced were variables A or NaOH and G concentrations or extraction temperature (Figure 3).The use of high concentrations of ingredients, both acids and bases, in the gelatin production process can cause the strength value of gels to increase or decrease, while the quality of gelatin produced from a production process is highly dependent on the extraction process carried out on collagen protein [20].shows that the higher the concentration of NaOH, the higher the strength value of the gelatin.The difference in gel strength is caused by the process of collagen hydrolysis due to differences in NaOH concentrations.The difference in hydrolysis method with NaOH results in differences in the size of collagen peptide chains.A NaOH concentration of 0.3% is able to convert collagen into gelatin effectively.This means that the size of the gelatin collagen peptide chain hydrolyzed with 0.3% NaOH is longer than with other treatments.The size of the length of the gelatin peptide chain is related to the molecular weight of the gelatin product.The longer the pepetida chain, the greater the molecular weight and the higher the value of the gel strength [21].Likewise, the higher the extraction temperature, the lower the gel strength value obtained.
The relationship between viscosity and gel strength is very strong, where the higher the viscosity, the higher the gel strength [16].The strength of the gel is one of the aspects needed to determine the quality of gelatin.The quality of gelatin can be assessed from various aspects, including gel strength and viscosity, the higher the value of gel strength and gelatin viscosity, the higher the quality of the gelatin [22].

Conclusions
The yield of gelatin from Spotted oceanic triggerfish skin ranges from 2.7 -9.3% The results of the Design of Experiment analysis using Design Expert obtained results in the form of independent variables that affect gelatin yield, namely variables B or H2SO4 concentration, D or soaking duration, F or water/skin extraction ratio and G or extraction temperature.The viscosity value obtained from the results of analysis using the Rapid Visco Analyzer (RVA) instrument ranges from 6.21 -7.57cP.In this case, the variables C (citric acid concentration), D (soaking time), E (soaking water/skin ratio), G (extraction temperature) and H (extraction time) have a significant effect on the gelatin viscosity of Spotted oceanic triggerfish skin.The strength of the gel obtained from the measurement results using a Texture Analyzer in this study ranged from 80.33 -97.67 g/cm2.In the results of the analysis, the results obtained in the form of variables that affect the strength of the gel produced are variables A (concentration of NaOH) and G (extraction temperature).

Figure 1 .
Figure 1.Pareto diagram on variables independent of gelatin yield

Figure 2 .
Figure 2. Pareto diagram on variables independent of gelatin viscosity

Table 1 .
Factors and levels used at the screening stage

Table 3 .
Analysis of the chemical composition of the skin of the spotted oceanic triggerfish

Table 4 .
Yield, gel strength and viscosity data

Table 5 .
Value probability Yield, viscosity and strength of gelatin gel

Table 6 .
The influence of factors on response

Table 9 .
Gelatin viscosity data

Table 10 .
Fit Statistics gelatin viscosity

Table 11 .
Gelatin gel strength data

Table 12 .
Fit Statistics gelatin gel strength