Production and characteristics of sailfin catfish ( Pterygoplichthys pardalis) protein hydrolysate [version 1; peer review: awaiting peer review]

Background: The sailfin catfish (Pterygoplichthys pardalis) is a freshwater fish from the Loricariidae family, and is considered an invasive species in Indonesia. The fish is usually neglected and discarded. Its protein hydrolysate is the product of the breakdown of proteins into amino acids through the hydrolysis of acids, bases, or enzymes. Therefore, this study aims to determine the hydrolysate characteristics of sailfin catfish (Pterygoplichthys pardalis) proteins, produced with different pH and hydrolysis durations. Methods: The hydrolysis was carried out with variable pH (control, 5, 7, 9) and hydrolysis durations (12 and 24 hours) in three replicates. Results: The hydrolysis duration, pH, and interaction of both variables had significant effects (p<0.05) on the parameters of yield, antioxidant activity, degree of hydrolysis, protein levels, and ash content. Similarly, the moisture content, hydrolysis duration, and pH were significantly affected (p<0.05), while their interactions were not (p>0.05). The pH treatment had a significant effect on fat content (p<0.05), while the hydrolysis length and the interaction between the two had no significant effect (p>0.05). Based on this study, the best resulting production of fish protein hydrolysate (FPH) from sailfin catfish were a for a combination of pH 9 and a hydrolysis duration of 24 hours. The characteristics of FPH produced were as follows: a yield of 57.39%, antioxidant activity 63.99%, degree of hydrolysis 40.67%, water content 7.28%, ash content 7.63%, fat content 5.10%, protein content 34.51%, molecular weight 6.14 -118.17 KDa, total essential amino acids 49.30%, and nonessential amino acids 50.71%. Conclusions: Two factors affected FPH characteristics in sailfin catfish (Pterygoplichthys pardalis). The best processing conditions to obtain optimal characteristics for FPH were a yield of 57.39%, antioxidant levels of 63.99%, a DH of 40.67%, a moisture content of 7.28%, ash content of 7.63%, fat content of 5.10%, and protein content of 34.51%. Open Peer Review Reviewer Status AWAITING PEER REVIEW Any reports and responses or comments on the article can be found at the end of the article. Page 1 of 13 F1000Research 2021, 10:1089 Last updated: 27 OCT 2021


Introduction
The sailfin catfish (Pterygoplichthys pardalis) is a freshwater fish from the Loricariidae family and native to the Americas (Susanto, 2014). In Indonesia, sailfin catfish are present in various locations with high levels of pollution (Qoyyimah et al., 2016), and have a negative impact because they prey on other species. The fish can dominate the water and become a predator or competitor of domestic fish (Hadiaty, 2011). Sailfin catfish is considered as an under exploited fish (Guo et al., 2019). Despite being discarded as bycatch, the nutritional content of sailfin catfish is relatively high, especially the protein, fat, and carbohydrates contents (Panase et al., 2018). Hence, the use of proteins in less valuable fish species can increase the functional value of the product (Hsu, 2010). The production of fish protein hydrolysate (FPH) is considered the most important source of protein and bioactive peptides (Chalamaiah et al., 2012).
FPH is a product of the decomposition of fish proteins into smaller fragments of peptides that contain between two and 20 amino acids through hydrolysis by enzymes, acids, or bases (Latifah, 2013;Chalamaiah et al., 2012). FPH is produced mainly through protease activity controlled by additional enzymes or endogenous protease enzymes from raw materials (Swanepoel and Goosen, 2018).
Previous studies on the production of FPH using endogenous enzymes have been carried out on Alaska pollack (Je et al., 2005), ornate threadfin bream (Nalinanon et al., 2011), barramundi fish, silver warehou fish, salmon (Nurdiani et al., 2015), and parrotfish (Chlorurus sordidus) (Prihanto et al., 2019). During the production process, precise hydrolysis conditions such as temperature, pH, and hydrolysis duration need to be considered to achieve optimal results (Meldstad, 2015). Therefore, this study aims to determine the optimum conditions for the production of FPH from the sailfin catfish (Pterygoplichthys pardalis) protein and characterize its product.

Sample collection
About 5 kg of Sailfin catfish (Pterygoplichthys pardalis) were collected from the river in Blitar Regency, East Java, Indonesia. The sailfin catfish were washed with water to remove dirt and brought to the laboratory in a cool box at a temperature of 4°C.
Fish protein hydrolysate process A preliminary study was conducted to determine the best comparison of sample and Aqua Dest (w/v) for the production of FPH. At this stage, a whole sailfin catfish (Pterygoplichthys pardalis) was chopped and smoothed using a Philips food processor (model HR7627, 650 W), and the chopped fish (20 grams) was added to Aqua Dest distilled water with a ratio of sample to Aqua Dest as follows 1:0, 1:1, 1:2, and:3 (w/v). The hydrolysis process was carried out for 18 hours using a shaker at 150 rpm and a temperature of 30 AE 2 o C. Furthermore, the FPH was centrifuged at 3000 rpm for 30 minutes and the ratio with the highest yield was used for the subsequent investigation.
The main study was carried out to determine the characteristics of sailfin catfish FPH, and the investigated factors were variations in pH and duration of hydrolysis. The pH levels used were 5, 7, 9, and 6.4 (control) with 12 and 24 hours of hydrolysis, while in the sample comparison, 1:2 (w/v) Aqua Dest was used. Meanwhile, the hydrolysis process was carried out following the same procedure as the preliminary study, and the supernatant obtained was dried using a spray drying method (Prihanto et al., 2019).

Yield
The FPH yield was calculated from the percentage of the number of hydrolysate products obtained against the volume of initial raw materials as of the hydrolysis process. The yield was calculated using the following formula:

Antioxidant levels
The inhibitory effect of DPPH free radicals was determined according to the method by Donkor et al. (2012), with a slight adjustment on the wavelength used. The inhibitory effect of DPPH free radicals was determined according to the method by Donkor et al. (2012), with slight modifications on wavelength, where they originally used 515 nm. Briefly, a dissolved protein fraction (100 μL) was added to the 3900 μL solution of DPPH 0.075 mM in methanol. In a dark room, an aliquot was homogenized and incubated for 30 minutes at room temperature (28 AE 2°C). A UV-Vis spectrophotometer (Genesys 6, Thermofisher Scientific, USA) was used to measure the absorbance of the aliquot at a wavelength of 517 nm. The antioxidant activity was based on the equation below:

Degree of hydrolysis
The degree of dissolved protein hydrolysis in the production of fish protein hydrolysate was calculated according to the method by Hoyle and Merritt (1994). The dissolved protein fraction (2 mL) was added to 2 mL of 20% TCA and the solution was centrifuged at a speed of 5000 rpm for 10 minutes at room temperature. Furthermore, the supernatant was taken to determine the nitrogen content after adding a 20% TCA solvent using the Kjeldahl method (AOAC, 2005). The degree of hydrolysis (DH) was defined by the following equation:

Proximate analysis
The analysis of FPH proximal was carried out using standard methods from AOAC (2005). Protein levels were determined using the Kjeldahl method, while fat content was determined using the Soxhlet method. Furthermore, the water content analysis was conducted using the drying method, in an oven at a temperature of 105°C for 2 hours. The ash levels were also determined using the drying method in the furnace at 550 o C, for at least five hours until the stable weight was achieved.

Molecular weight (SDS-PAGE analysis)
An SDS-PAGE analysis was carried out based on the Laemli (1970) method. A sample of 15 μL was mixed with a 15 μL sample buffer and heated at 100°C for 15 minutes. The SDS-PAGE analysis used a 12% gel-separating concentrate and a 4% gel stacking, and the running process was started at a constant current of 20 mA 100 V for three hours. Meanwhile, staining was carried out by soaking the gel in a staining solution which contained one gram of Coomasie Brilliant Blue G-250 (Merck, Catalogue No. 1154440025), 450 mL of methanol, 100 mL of glacial acetic acid, and 450 mL of Aqua Dest. The molecular weight calculations were analyzed by comparing the bands appearing on the samples with the standard markers.

Amino acids analysis
The amino acid content was analyzed based on Boogers et al. (2008), using the ultra-performance liquid chromatography (UPLC) method. Moreover, the sample liquid (0.50 mL) was added to 2.0 mL of AABA's standard internal solution of 10 mM, diluted with HCl 0.1 N, and homogenized. The solution was filtered using a filter membrane of 0.22 μm, and 10 μL of the solution were taken and placed in a vial. The aliquot was added to 70 μL AccQ-fluor borate and vortexed.
Similarly, approximately 20 μL of the reagent fluor A was added and vortexed. The solution was allowed to stand for a minute and further incubated for 10 minutes at a temperature of 55°C, and the sample was later injected into the UPLC system.

Statistics
The experiment was optimized using the response surface method (RSM), which was presented in a contour plot using the Minitab 18 software tool (Minitab Pty Ltd., Sydney, NSW, Australia). Apart from optimization, all the data obtained were analyzed with the one-way variant analysis, followed by a post-hoc test. Furthermore, all data were analyzed in triplicate, and the results are presented as the mean AE SD.

Proximate content of raw material
The proximate content of sailfin catfish (Pterygoplichthys pardalis) is shown in Table 1. Based on the results, the sailfin catfish protein content was 30.42%, while the fat content was 5.24%.

Yield and antioxidant activity
After the centrifugation process, the sample formed five layers of different fractions. These layers included the oil, the light lipoprotein, the dissolved protein (liquid/soluble protein), the fine particles, and the rough particles at the bottom of the cuvette, as shown in Figure 1. The different sample and Aqua Dest ratios affected FPH production yields and antioxidants (P < 0.05). Meanwhile, the highest yield and antioxidants were obtained for a sample and Aqua Dest ratio of 1:2 (w/v) (Figure 2), with a soluble protein yield of 78.25 AE 1.75% and antioxidant levels of 46.70 AE 0.30%. These samples with an Aqua Dest ratio of 1:2 (w/v) were used to manufacture FPH in the advanced study and analyzed to determine the characteristics of FPH.

Yield
The hydrolysis duration and pH had a significant effect on the yield of FPH, which ranged from 20.29 AE 0.27% to 57.39 AE 0.17% (Table 2). The highest yield value (57.39 AE 0.17%) was obtained at pH 9 and after a 24-hour hydrolysis duration, while the lowest value was obtained in control treatment after a 12-hour hydrolysis duration, with a value of 20.29 AE 0.27%.

Antioxidant activity
The results showed that the antioxidant values of FPH ranged from 11.56 AE 1.59% to 63.99 AE 0.62% (Table 2). Furthermore, there was a significant difference (P<0.05) in antioxidant activity with variations in pH and duration of  hydrolysis. The highest antioxidant activity of 63.99 AE 0.62% was obtained at pH 9 with a 24-hour hydrolysis, while the lowest value (of 11.56 AE 1.59%) was obtained for the control treatment with a 12-hour hydrolysis.

Degree of hydrolysis (DH)
The duration of hydrolysis and pH significantly affected the DH (P < 0.05). Based on the results, the DH of the fish protein hydrolysis samples ranged from 22.08 AE 1.77% to 40.67 AE 1.19%. The highest DH (40.67 AE 1.19%) was obtained at pH 9 after a 24-hour hydrolysis duration, while the lowest value was obtained for pH 5 after 12 hours (22.08 AE 1.77%).

Proximate analysis
Based on the approximate FPH content as shown in Table 2, the hydrolysis duration, pH, and interaction between both factors gave significantly different protein content results (P < 0.05), which ranged from 8.51 AE 1.34% to 34.51 AE 1.56%. Furthermore, the highest protein content (34.51 AE 1.56%) was obtained at pH 9 for a 24-hour hydrolysis duration.
Different pH treatments gave significantly different results (P < 0.05), while the duration of hydrolysis and the interaction of both factors had no significant impact (P > 0.05) on fat levels. Meanwhile, fat content ranged from 4.37 AE 0.22% to 5.30 AE 0.13%. The fat content in the study decreased compared to the content of the raw materials of sailfin fish due to the separation of fat and lipoproteins during centrifugation, while some lipids still existed in the hydrolysate.
The results showed that the moisture content of the FPH ranged from 7.28 AE 1.06% to 13.84 AE 0.36%. Additionally, hydrolysis duration and pH variations gave significantly different results (P < 0.05), while the interaction of these  All treatments had significantly different ash levels (P < 0.05), with values ranging from 3.75 AE 0.33% to 7.63 AE 0.19%. In addition, there was a significant increase in ash levels with pH treatments of 5 and 9.

Molecular weight
The molecular weight characteristics of FPH from different treatments were visualized using the SDS-PAGE method ( Figure 3). Moreover, the 12-hour hydrolysis formed 11-protein bands with molecular weights ranging from 7.23-123.03 kDa, while the 24-hour hydrolysis formed a 13-protein band with a molecular weight range of 6.14-118.17 kDa.

Determining the optimum process
The response surface methodology (RSM) method is a common method for analyzing process optimization. Therefore, to achieve the best characteristics of FPH (in terms of yield, antioxidants, DH, moisture content, ash content, fat content, and protein content), two factors were optimized, namely pH and hydrolysis duration. The white area on the contour plot indicated the optimum condition for (Pterygoplichthys pardalis) hydrolysis process (Figure 4). It shows that the optimum pH for hydrolysis ranged from pH of 8.8 to 9 and hydrolysis duration for 22.8 -24 hours. Therefore, based on our experimental treatment, the best Sailfin catfish FPH processing was at pH 9 for 24-hour hydrolysis. The treatment that still yielded acceptable results based on FPH quality standard is shown from the formation of white areas on the contour plot ( Figure 4). The treatment received by the response ranges from a pH of 8.8 to 9 and a hydrolysis duration of 22.8 -24 hours. It results that the best FPH processing was for a pH 9 and a 24-hour hydrolysis.

Amino acid composition
The amino acid content analysis was carried out only on the FPH produced using the optimal treatments. The amino acids detected in the yielded FPH is shown in Table 3. The result showed that eight essential and seven non-essential amino acids were detected. The amino acids such as lysine, leucine, glutamic acid, aspartic acid, and glycine were dominant in the FPH samples from sailfin catfish.

Discussion
The protein and fat values obtained were higher than in a previous study by Munandar and Eurika (2016) which showed that the protein levels and fat content were 19.71% and 1.73%. According to Tunjungsari (2007), the chemical composition of fish meat varies depending on the species, age level, habitat, and feeding locations.
The yield obtained in this study was lower than the hydrolyzed protein yield of salmon waste protein of 65.17%, obtained without added enzymes, for a pH 2.5 and at room temperature, and for a hydrolysis duration of 18 hours (Nurdiani et al., 2015). According to Jamil et al. (2016), differences in yields from FPH are due to variations in species, parts of the fish used, types of enzymes used, and the applied hydrolysis conditions.
The antioxidant activity of FPH from sailfin catfish was previously processed without the addition of enzymes, and for pH 7, at a temperature of 55 o C, and a hydrolysis duration of six hours (Baehaki et al.,2015). Data on the antioxidant activity of snakehead (Channa striata) FPH were significantly higher compared to those of sailfin catfish. The FPH was processed using the addition of protease enzyme at pH 7 for 90 min at 55°C, which gave an antioxidant activity of 20.7% (Baehaki et al., 2020). The antioxidant peptides from fish proteins are considered safe and healthy due to their low molecular weight, easy absorption, low cost, and high activity (Sarmadi and Ismail, 2010).
The degree of hydrolysis measured in this study was lower than that of the yellowfin fish's (Limanda aspera) waste protein hydrolysis samples with the addition of mackerel digestive tract enzymes, at pH 10, a temperature of 50°C, and a 3-hour hydrolysis duration, by 67% (Jun et al., 2004); it was also lower than that in catfish (Pangasius pangasius) FPH obtained without the addition of enzymes, at pH 7, a 55°C temperature, and a 6-hour hydrolysis, by 63.21% (Baehaki et al., 2015). According to Hasnaliza et al. (2010), the increase in DH is due to an increase in dissolved peptides and amino acids in TCA, which was terminated during hydrolysis.
The protein levels obtained were similar to the hydrolysate protein of catfish (Pangasius pangasius) without enzymes, at pH 7, 55°C, and hydrolysis duration of 6 hours, with a value of 20.86% (Baehaki et al., 2015). The protein level in FPH was higher than that in raw material due to the dissolution of proteins during hydrolysis, and the separation of solids and non-protein substances during centrifugation (Chalamaiah et al., 2010). According to Bautista (1999), the hydrolysis duration affects the protein levels of FPH. The protein will increase by approximately 3% when the hydrolysis length is extended from 60 to 120 minutes.
In this study, the fat content produced was lower than the hydrolysate fat content of the salmon waste protein obtained without adding enzymes, at pH 2.5, and a hydrolysis duration of 18 hours, which was 18.90% (Nurdiani et al., 2015;Idowu et al., 2018). The process reduces the moisture of the product. This is due to the use of high-temperature spray drying methods for the processing of FPH, which removes most of the water content. According to Riansyah et al. (2013), the ability of the material to release water from its surface is more remarkable with increased drying air temperature used, and a longer drying process to produce a lower moisture content. This shows that the water content of the FPH produced was lower than that of the spray-dried FPH from Nile tilapia and Tambelo, which yielded water contents of 9.06 AE 0.09% and 10.16 AE 0.17%, respectively (Anwar and Rosmiati, 2013;Annisa et al., 2017). Mixing acidic and alkaline compounds in protein hydrolysate solutions leads to the formation of salt compounds, causing the ash levels to increase in protein hydrolysates (Wijayanti et al., 2015). In addition, the ash content in the hydrolysate was also influenced by the release of minerals during hydrolysis, especially from bones (Ido et al., 2018).
The longer hydrolysis time was more effective in breaking protein bonds into peptides and amino acids with low molecular weight. This hydrolysis process caused structural changes, where proteins slowly degraded into smaller peptide units (Rawdkuen et al., 2018). The low molecular weight of FPH also support its potential for application as a functional food product.
Optimal hydrolysis depends on the reaction conditions, such as the type of endogenous protease. Moreover, the raw material has specific optimum conditions for its endogenous enzymes. For example, the endogenous enzymes of the digestive tract of snapper and mackerel function best at pH 9 and temperatures of 45°C and 55°C, respectively (Singh and Benjakul, 2018). Meanwhile, parrotfish have showed better results when processed at pH 8-9 and after reaction times of 21.5-24 h (Prihanto et al., 2020).
Variations in the composition of the amino acids produced were related to differences in prevailing hydrolysis conditions such as enzyme type, pH, and hydrolysis temperature (Klompong et al., 2009) and the total essential amino acid contents were 49.30%. This result is higher than that of total essential amino acid in Nile tilapia (Oreochromis niloticus) and commercial hemp seed protein isolate (HPI), which were previously found to be 40.77% and 40.6%, respectively (Foh et al., 2011;International Quality Ingredients, 2019). Compared to other amino acids, glutamate showed the highest proportion with 12.35%. This result is in line with the FPH of Nile tilapia, which had the highest glutamic acid content of 21.03% (Foh et al., 2011). According to Chalamaiah et al. (2012), most reported FPH to have the highest content of amino acids of the types of aspartic acid and glutamate.

Conclusion
The FPH characteristics of the sailfin catfish (Pterygoplichthys pardalis) were affected by two factors, which were pH and hydrolysis duration. Based on the results, the best processing conditions to obtain optimal characteristics for FPH were a yield of 57.39%, antioxidant levels of 63.99%, a DH of 40.67%, a moisture content of 7.28%, ash content of 7.63%, fat content of 5.10%, and protein content of 34.51%. Furthermore, the treatments ranged from a pH of 8.8 -9 and a hydrolysis length of 22.8 -24 hours, which led to total essential and non-essential amino acid levels of 49.30% and 50.71%, respectively.