Isolation of fish skin and bone gelatin from tilapia (Oreochromis niloticus): Response surface approach

In this study, gelatin from fish collagen, as one of halal sources, was extracted from tilapia (Oreochromis niloticus) skin and bone, by using Response Surface Methodology to optimize gelatin extraction conditions. Concentrations of alkaline NaOH and acid HCl, in the pretreatment process, and temperatures in extraction process were chosen as independent variables, while dependent variables were yield, gel strength, and emulsion activity index (EAI). The result of investigation showed that lower NaOH pretreatment concentrations provided proper pH extraction conditions which combine with higher extraction temperatures resulted in high gelatin yield. However, gelatin emulsion activity index increased proportionally to the decreased in NaOH concentrations and extraction temperatures. No significant effect of the three independent variables on the gelatin gel strength. RSM optimization process resulted in optimum gelatin extraction process conditions using alkaline NaOH concentration of 0.77 N, acid HCl of 0.59 N, and extraction temperature of 66.80 °C. The optimal solution formula had optimization targets of 94.38%.


Introduction
Gelatin is a translucent, colorless, brittle (when dry), flavorless, edible, multifunctional natural macromolecule protein polymer which is extracted by thermal hydrolysis of collagen tissue found in bones, skin, and connective tissue of various animal and fish [1][2] [3]. Industries utilize gelatin extensively as a gelling agent, emulsifier, stabilizer, adhesives, viscosity agent and binder agent. Worldwide gelatin consumption is to reach 395.84 thousand metric tons by the year 2017. The vast majority of commercial gelatin is derived from porcine skin (46%), bovine hide (29.4%), bones (23.1%) and other sources (1.5%) [4]. Demand for alternative gelatin source is increasing due to religious reasons. Porcine gelatin cannot be used in kosher (Jewish) and halal (Muslims) foods, while Hindus do not consume bovine gelatin. The use of fish byproducts for alternative gelatin production is growing. Fish byproducts such as skin, bones, fins and scales account for ± 21% [5], even ± 50% for certain fish. Tilapia (Oreochromis niloticus) consumption in Indonesia is rapidly increasing and utilization of skin and bone are expected to reduce waste and increase the value of the waste. Fish gelatin properties influenced by many factors, such as species, water temperature fish habitats, and the 1 To whom any correspondence should be addressed.  gelatin extraction process, which may depend on pH, temperature, and time during pretreatment and extraction process [6]. The objectives of this research were to study the effects of pretreatments using alkaline and acid on gelatin extraction, to characterize tilapia gelatin, and to determine optimum conditions for gelatin extraction from tilapia fish skin and bone.

Method
In order to obtain the optimum conditions of gelatin extraction, a Response Surface Methodology (RSM) approach was used. RSM using design expert software (9.0.5 version) was used to obtain several optimum gelatin extraction conditions. Mathematical techniques combine with statistical techniques used in the RSM method to create and analyze Y response (e.g. yield, gel strength, etc.) which are influenced by independent variables or X factor. Three independent variables (X factors) used in this study, i.e. X1= alkaline (NaOH) concentration, and X2= acid (HCl) concentration in the pretreatment process, and X3= temperature of the extraction process which were tested at 5 different levels using 3 factors and 5 levels central composite rotatable design ( Table 1). Levels of independent variables were obtained after the central composite rotatable design was run ( Table 2). Response factors (=Y response) observed were yield [7], emulsion activity index [8], and gel strength [9]. The gelatin extraction process based on [10] method with adaptations. The raw materials (200 g) were soaked in a solution of NaOH (1: 5 w/v) with various concentrations (factor X1, N) for 1 hour. Then, the samples were washed with water (1: 5 w/v) three times, filtered and then squeezed. Subsequently, the samples were immersed in HCl (1: 5 w / v) with various concentrations (factor X 2, N) for 1 hours, then the samples were washed with water (1: 5 w/v) three times, filtered and then squeezed. After that, the samples were put into glass beakers, added with distilled water (1: 4 w/v) and covered with aluminum foil. Subsequently, the samples were extracted using a water bath with various extraction temperatures (factor X3, °C) for 3 hours. Next, the filtrate was obtained by filtering using cheesecloth and dried in a glass container at a temperature of 60°C for 72 hours.

Response Surface Model Building of Gelatin Extraction
The response surface models for every response investigated showed in Table 3. Based on the results, in the optimization of the gelatin extraction, the response variables optimized were yield, emulsion activity index, and gel strength.

Yield
The bone and skin tilapia gelatin yield ranged from 3.49% to 9.5%. Generally, the average of fish gelatin yield is 6-19% [11]. Response surface model of tilapia gelatin yield was linear ( Table 3). The RSM equation of the gelatin extraction optimization towards the yield response is: Yield = +5,81-1,01A+0,41B+1,49C, where A= the concentration of alkaline pretreatment, B= the concentration of acid pretreatment, and C= the extraction temperature. The yield response will increase proportionally with the decrease of alkaline pretreatment concentration, and the increase of acid pretreatment also extraction temperature which are indicated by negative and positive value, respectively. This presumably because acid produced a pH that was suitable for the extraction of gelatin. Acid and temperature increase also caused some cross-linkages easily to break with little damage to the polypeptide chain [12]. Therefore, a higher yield could be generated.  Figure 1 and 2 showed that yield increase at combination of lower alkaline concentration, and higher acid concentration and extraction temperature. Kittiphattanabawon et al., 2016 [13] was using only 0.1-3% HCl in the pretreatment process with 40-80 o C extraction temperature stated that gelatin extracted at higher temperature for longer time (>12 hours) had higher yield but poorer gel strength. However, Niu et al., 2013 [14] indicated that adjusting acids concentrations to appropriate levels resulting in optimal protein yield and gelatin physicochemical properties.

Gel Strength
The gel strength of commercial gelatin range between 50-300 g [9]. The skin and bone tilapia gelatin have gel strength between 2.83-297.67 g, which suitable for product such as wafers, dairy products, desserts, and marshmallow. Depend on fish habitats, gelatin of other fish such as cold water salmon has a gel strength of 195 g [15], while warmer water cat fish at 252 g [16]. Furthermore, da Silva et al., 2017 [17] was using NaOH, acetic acid and kumakuma (Brachyplatystoma filamentosum) fish skin from the Amazonian industries produced gelatin with high yield and gel strength, 19.7% and 244.3g. Response surface model of tilapia gelatin gel strength was mean (Table 3 and Figure 6).

Gelatin Extraction Optimization
The purpose of optimization was to minimize the effort required and to maximize the desired results. Through this approach, optimization of multi responses could be obtained using desirability functions. Table 4 shows the components which were optimized, targeted, limits, as well as the level of importance at the stage of formulating the optimization. The concentration of alkaline and acid, and extraction temperature will affect the quality of the resulting gelatin. Since yield, emulsion activity index (EAI), and gel strength responses have the highest level of importance (5=+++++), therefore only these responses that would be optimized.  There were 33 optimization solution formulas obtained from the optimization process which had the desirability values all over 0.91, except one with the lowest value of 0,89. Gelatin extraction process conditions recommended was using alkaline (NaOH) pretreatment concentration of 0.77 N, acid (HCl) pretreatment concentration of 0.59 N, and gelatin extraction temperature of 66.79°C. The optimal solution formula had optimization targets of 94.38% and was predicted to produce gelatin with yield of 9.4%, emulsion activity index of 26.97 m 2 /g, and gel strength of 120.96 g.

Conclusion
The decrease in alkaline pretreatment concentration combine with the increase of extraction temperature cause an increase in yield of the extracted tilapia gelatin. On the other hand, the decrease of alkaline concentration and extraction temperature resulted in high emulsion activity index (EAI). There is no significant effect of alkaline and acid pretreatment concentration, and gelatin extraction temperature on gel strength of the extracted tilapia gelatin. Gelatin yield were 3.49-9.5%, while EAI and gel strength were 8.71-40.47 m2/g and 2.83-297.67 g, respectively. Optimization using expert design program 9.0.5 via response surface methodology approach generates optimal formula for gelatin extraction with alkaline concentration (NaOH) of 0.77 N, acid (HCl) of 0.59 N and temperature of 66.80°C with desirability value of 94.38% and was predicted to produce gelatin with 9.40% of yield, gel strength of 127.37 g, and emulsion activity index of 26.97 m2/g.