Pangasius Fish Skin and Swim Bladder as Gelatin Sources for Hard Capsule Material

In this paper, we report the extraction and characterization of gelatin from the abundant industrial fishery waste of Pangasius skin and swim bladder and its application as the base material for hard capsule shells. The yield of gelatin ranged between 19 and 23%, content of moisture is 7.6–9.2%, ash is 1.1–1.7%, pH is 4.1–5.2, gel strength is 238–278 bloom, and viscosity is 65–74.7% mP. SDS-PAGE showed all gelatins have chains of α1, α2, and β-peptides. The skin, swim bladder, and mixed gelatins were successfully used in the production of hard capsule shells. The dimensions, weight, disintegration time, and water content properties of the hard capsules from these Pangasius wastes were akin to the standards of commercial capsules.


Introduction
Fish industries generate more than 50% of waste from the total weight of the processed fish [1]. Recently, to reduce the amount of waste generated, a lot of research studies have focused on turning these byproducts into a value-added product. Nam et al. [2] reported the conversion of catfish excesses into bioactive components such as fish protein hydrolysate (FPH), hydroxyapatite (HA), and lipid fraction. In addition, several studies have successfully reported the extraction of gelatin from the fish byproducts such as tuna skin [3,4], Pangasius skin [5], and Chinese giant salamander [6].
Gelatin is an animal-based protein derived mostly from the bones and skins of cows and pigs, collected from the slaughterhouse. It has been used in the food and pharmaceutical industries due to its ability to form a hydrogel in the pH range, devoid of ionic assistance or other additives [7]. Moreover, the physical form of the gel solution changed between the gel and solution reversibly according to temperature changes. Hence, gelatin has been employed as a base material for hard capsules. Interestingly, more than 15% of marketed drugs are delivered in gelatin-based hard capsules [8]. Hard gelatin capsules are preferred because they are easier to swallow than tablets, mask the bitter taste, extend the drug shelf life, and allow facile drug identification by using various colors and patterns on the capsule surface.
However, lately, there are concerns to use extracted gelatin from bovine skins and bones due to the possibility of conveying the bovine spongiform encephalopathy (BSE) diseases and preferably lifestyles such as vegetarian. Several countries have abandoned the use of the porcine-based capsule due to religious causes. e alternative for these mammalian-based gelatins is gelatin extracted from fish processing wastes including the skin, bone, swim bladder, and scale. Nevertheless, the commercial application of fish gelatin has been impeded due to the lower gelling temperature and gel strength in comparison to mammalianbased gelatin, particularly, gelatin from cold water fish.
Stimulatingly, several reports have shown that tropical fish have physicochemical characteristics parallel to bovine and porcine gelatins. e extraction of gelatin from Pangasius skin had a yield of 14.94% with an acidic method and had a yield of 14.30% with the base method [5]. e extraction of gelatin from the catfish swim bladder had a yield of 13.5% with an acidic and base process [9]. Interestingly, there are no published reports of the application of this fish waste gelatin as the base material for hard capsule shells. us, in this paper, we used the abundant and cheap tropical fish waste of Pangasius skin and swim bladder for gelatin extraction and then applied as a material for hard capsule shells.

Pangasius Waste Materials and Preparation.
e frozen Pangasius skin and swim bladder were obtained from the waste of the fish fillet industry located in West Java, Indonesia. e raw materials were cleaned from the meat, fat, and other impurities using a sharp knife and washed with running water. e cleaned product was cut into a square shape with a size of ±1 × 1 cm and stored at a low temperature of −18°C until used. e proximate analysis of this byproduct was conducted, including water, ash, fat, and protein content.

Gelatin Extraction.
Before gelatin is extracted, pretreatment is completed with an acid and base solution. Pangasius skin was obtained according to the previous report by immersion of 100 g of the sample in 0.05 N NaOH solution for 1 hour with a sample ratio of 1 : 4 at room temperature [3]. e sample was then washed with water followed by immersion using 0.2% H 2 SO 4 solution for 1 hour. Finally, the sample was soaked with 0.2% citric acid for 6 hours in the ratio of 1 : 4. e swim bladder sample was pretreated differently compared to the skin sample. 100 g sample was immersed in 0.05 N NaOH solution at room temperature with a sample ratio of 1 : 10 by weight for 1 hour. e sample was washed with distilled water to neutral pH followed by soaking using 0.2% citric acid solution for 1 hour while stirring with a sample ratio of 1 : 10 and then washed using distilled water until neutral. e pretreated samples of the skin, swim bladder, and mix of the skin and swim bladder (1 : 1 ratio) were extracted with deionized water at 65°C for 7 hours. e extracted samples were filtered, and then the filtrates were dried in an oven at 50°C for 24 hours to yield solid gelatin. e gelatin properties were characterized, including the ash content, pH, gel strength, viscosity, and protein profile analysis (SDS-PAGE).

Hard Capsule
Casting. 10 g gelatin powder is mixed with 10 mL distilled water at 85-90°C until all gelatin dissolved into homogenous solution. Following the reported method [10], the preheated pin bar is then dipped into the warm mixed solution and immediately removed when the film is formed and dried for approximately 30 minutes. Dried capsules were released from the pin bar, and then the properties of the capsules were analyzed including the weight and thickness, ash and moisture contents, and the time evaluation of capsule shell rupture.

Proximate Analysis of the Pangasius Skin and Swim
Bladder. Proximate analysis is conducted to determine the quality of the starting material that will be used for the gelatin extraction. Water, ash, fat, and protein contents of raw material affect the properties of the manufactured gelatin significantly [11]. e proximate result of the Pangasius skin and swim bladder is shown in Table 1. e proximate composition of the skin and swim bladder produced is quite different compared with the previous report given by Mahmoodani et al. [12] and Vijayan et al. [13] for the proximate analysis of the Pangasius skin and swim bladder, respectively. ese differences were due to different Pangasius species use. In the study of Mahmoodani et al. [12], Pangasius sutchi was used and originally from the waters of Penang, Malaysia, while the Pangasius species in the study of Vijayan et al. [13], namely, Pangasius hypophthalmus, was obtained from the waters of the western district of Godavari, Andhra Pradesh, India.
e Pangasius skin and swim bladder have ash content values of less than 0.5%. e value of the ash content less than 0.5% is a good-quality raw material for the manufacture of collagen and gelatin [9]. However, the amount of fat in the skin material is relatively high compared to that in the swim bladder, 12.3% and 0.25%, respectively. us, to improve the quality of the resulting gelatin, the optimization of the pretreatment process is required [14]. Because of the disparity in tissue composition where the Pangasius skin has a hypodermic layer or a subcutaneous layer in which it is composed of fat tissue, the fat content in Pangasius skin is higher than in the Pangasius swim bladder, where that layer is not stored as swimming bubbles. e protein content of the Pangasius skin and swim bladder is 39.8% and 27.6%, respectively, and it is in good agreement with the previous report. However, this value is relatively low compared to the protein content of the Pangasius bone which is 40.9%.

Physicochemical Properties of Extracted Gelatin.
e physicochemical properties of the Pangasius skin and swim bladder are shown in Table 2. Gelatin from the Pangasius skin and swim bladder was successfully extracted with the yield in the range of 19-23% (Table 2). e yield of gelatin can be influenced by several factors, including the type of material, the pretreatment process, and the extraction temperature [15]. e gelatin yield in this study was higher than in other previous reports of gelatin extraction from other fish resources. Gelatin yields 11.3% from tuna skin [15]. Gelatin yield of yellowfin tuna skin is 19.97% [3]. Gelatin extraction of the goldfish swim bladder had yielded 13.5% by pretreatment using NaOH, sulfuric acid, citric acid, and extraction temperatures of 45-50°C [9].
pH is an essential factor for the quality of gelatin. e results of pH values based on Table 2 show that swim bladder gelatin has the highest pH value of 5.16, whereas skin gelatin has the lowest pH value of 4.15. e pH value of gelatin is in accord with the standard of the Gelatin Manufacturer's Institute of America for edible gelatin which states that the pH value of gelatin is around 3.8-5.5 [16].
Gel strength is the most important physical property of gelatin. e strength of the gel illustrates the cohesion power between gelatin molecules and is proportional to the molecular weight. e results of gelatin gel strength values based on Table 2 show that mixed gelatin has the highest gel strength of 278 bloom, whereas skin gelatin has the lowest gel strength of 238 bloom. e results are in accord to the standard of the Gelatin Manufacturer's Institute of America for edible gelatin which states that the strength of gelatin gel is around 50-300 bloom [16]. e gel strength of this gelatin is comparable with gelatin from bovine [17] and other fish sources [18][19][20][21]. It is widely reported that gelatin from tropical fish species displays higher gel strength compared to gelatin from cold water fish varieties. e high bloom value of Pangasius waste gelatin found in the current investigation exhibits its suitability for the application as hard capsule shells. Beside gel strength, viscosity is also the essential property to assess the gelatin quality. e viscosity of extracted gelatin is shown in Table 2. Gelatin from the Pangasius swim bladder has the highest viscosity of 75 mP, while the skin and mixed gelatin showed the viscosity value of 61 and 71 mP, respectively. ese viscosity values were proportional to bovine and porcine gelatin and thus showed the potential of Pangasius waste gelatin for commercial purposes.

SDS-PAGE Profile of Gelatin from Pangasius Wastes.
Protein profile is one of the critical properties for the quality of extracted gelatin. e relative amount of the protein component, including α-and ß-protein fragments, has a significant effect on the physical properties of gelatin. In Figure 1, the SDS-PAGE analysis showed the protein profile of Pangasius waste gelatin.
e Pangasius skin, swim bladder, and mixed gelatin showed a similar protein pattern with three major bands around 128-230 kDa that were corresponding to α1-, α2-, and β-protein components akin to the previous reports. e emergence of α-and β-chains and other lower molecular weight protein fragments occurs during extraction due to the collagen degradation process. Gelatin of yellowfin tuna skin extracted at a temperature of 65°C had a molecular weight of β 250 kDa, α1 129.670 kDa, and α2 116.364 kDa [19]. e β-component shows high molecular weight (>200 kDa), while the α1-and α2-components show low molecular weight (>120 kDa) [22].

Hard Capsule Shells.
In the current study, gelatin from Pangasius waste was used as material for hard capsule shells. Figure 2 shows the image of capsules produced from the pangasius skin, swim bladder, and mixed gelatin. e transparency of the capsules was respectable and similar regardless of the gelatin origin, although the color of the capsules is slightly yellow. Physical properties of hard capsule shells from pangasius skin and swim bladder gelatin are shown in Table 3, and the properties were as good as the commercial mammalian-based gelatin capsules thus showing the feasibility of the use of gelatin from Pangasius wastes as the base material to produce the hard capsule shells.   International Journal of Biomaterials     Table 3. e produced capsule dimensions from three types of gelatin are different substantially (p < 0.05) due to the variance of dipping temperature and viscosity of the gelatin sources, while the influence on the filling of capsule base material also contributes to the difference in capsule thickness. e size of the capsules based on the dimensions of the capsules produced in this study is included in capsule size 0 [23]. Capsules have various variations and sizes. It is intended to adjust the capsule shell with medicinal ingredients to be inserted into the capsule, such as powder, paste, and liquid [7]. e values of capsule weight typically spread (p > 0.05) based on Kolmogorov-Smirnov analysis, which indicates the influence of the raw gelatin material on the weight of the capsules produced (p < 0.05). e water content of capsules is influenced by temperature and drying time of the dipping process, as well as the physical properties and the viscosity of gelatin [24]. e capsule disintegration time is normally spread (p > 0.05) based on Kolmogorov-Smirnov analysis ( Figure 3). Furthermore, the results of Duncan's test showed that the disintegration time of the skin gelatin capsule differs significantly with the swim bladder gelatin capsule and mixed gelatin capsule (Table 4).

Conclusions
e extraction of gelatin from the Pangasius skin, swim bladder, and mixture has been effectively performed with the yields in the range of 19 to 21%. e physicochemical properties of extracted gelatins are comparable to the mammalian-based gelatins with the values of gel strength being 239, 273, and 278 bloom for Pangasius skin, swim bladder, and mixture, respectively. e physical properties of hard capsule shells from these gelatins were comparable to the standards of commercial capsules.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.