Protein Hydrolysate from Waste of Catfish Fillet Processing for Snakehead Fish Feed Formulation

The negative impact of fish processing industry waste needs to be minimized, by processing it into valuable products, one of which is fish feed. The objectives of this research were to determine the optimum dose of crude extract of B. cereus RGL.1.1 enzyme in hydrolyzing protein from the waste of catfish (Pangasius hypophthalmus) fillet processing and to evaluate the effectiveness of using protein hydrolysate in snakehead fish (Channa striata) feed. There were four doses of enzyme treatment for protein hydrolysis designed in a completely randomized design, namely, 0, 4, 6, and 8% (v/w) with three repetitions. Furthermore, to assess the quality of protein hydrolysate, an analysis of soluble protein level, hydrolysis degree, amino acid content, fatty acid content, and digestibility was carried out. The percentage of protein hydrolysate applied in the feed formula was 0, 15, 30, and 45% (v/w), which was designed in a completely randomized design with three replications. Absolute weight growth, specific growth rate, protein efficiency ratio, feed efficiency, and snakehead fish survival were measured to evaluate the effects of the feed formula. Results showed that the crude extract of B. cereus RGL.1.1 enzymes at a concentration of 6% (v/w) enhanced the availability of soluble proteins, amino acids, fatty acids, and feed digestibility. Protein hydrolysate application in snakehead fish feed formula up to 45% (v/w) can improve the growth performance (8.03%), protein efficiency ratio (25.66%), and feed efficiency (23.41%).


Introduction
Waste in the fsh processing industry can become a fundamental problem for our environment; it must be managed and treated seriously to reduce its negative impacts.Fish waste, if treated and processed properly, can not only reduce the environmental problem but also be benefcial to our lives.One of the benefcial products of fsh waste is fsh feed.Te fsh waste can be used as a source of raw materials for fsh feed because the waste consists of inedible parts of fsh such as ofal, skin, and bones that are still rich in protein and minerals and can be used as feed ingredients, especially after being processed into protein hydrolysates.Tis type of protein can replace fsh meals as a protein source [1].Tis hydrolysate is frequently used as a protein supplement and appetite stimulant [1,2] and has been shown to boost fsh growth and feed efciency [2][3][4].
Protein hydrolysate can be produced using protease enzymes.Enzymes can help reduce the risks of product deterioration and damage [5].Protease (E.C. 3.4.21) is an enzyme of the hydrolase group that helps in breaking down peptide bonds in protein molecules.Tis enzyme catalyzes the hydrolysis process involving the addition of water to a specifc substrate bond [6].Te protease enzyme is found in many organisms, including plants, animals, and microbes.Microorganisms are regarded to ofer advantages when utilized as enzyme producers since they are quick to generate, easy to grow, and whose environmental circumstances can be adjusted [7].One type of microorganism that can produce the protease enzyme is bacteria, one of which is Bacillus cereus [8][9][10].B. cereus RGL1.1, isolated from hot springs in the Rengganis crater, Bandung Regency [11], can be used to produce proteases that can hydrolyze fsh protein hydrolysates.However, the optimum dosage of protease from B. cereus RGL1.1 for catfsh protein hydrolysis is still unknown.As a result, more research is needed to determine the optimum dose of the enzyme to produce protein hydrolysates and to evaluate the impact of these hydrolysates on snakehead fsh growth and digestibility.
Te natural population of snakehead fsh has declined because of overfshing.Aquaculture is the best alternative to ensure snakehead fsh's long-term production.Nonetheless, there are problems with its implementation, particularly in the availability of feed.Since fsh meals and soybean meals must be imported from other countries, their prices are high in Indonesia.Furthermore, predatory fsh, such as snakehead fsh, require high protein levels in their feed to grow properly.Snakehead fsh grow best when fed with a 40% protein diet [12].For these reasons, it is necessary to fnd an alternative feedstuf of low price, one that is readily available and contains sufcient nutrients.Te best option so far is to use the waste of the catfsh fllet industry, which is prevalent in Indonesia.
Te objective of this experiment was to maximally use the waste of catfsh (Pangasius hypophthalmus) fllet processing industry to produce feed for snakehead fsh (Channa striata) by establishing the optimum dose of crude protease to produce protein hydrolysate, snakehead fsh feed formulation, and to observe the efects of protein hydrolysates on the feed digestibility and the growth rate of the fsh.

Materials and Methods
2.1.Materials.Fish waste was obtained from CV Kurnia Mitra Makmur Purwakarta, West Java, a catfsh (Pangasius hypophthalmus) fllet processing company.Te waste was taken in the frozen condition; it was practically fsh ofal including the intestine which was cleaned from fsh excrement before being frozen.

Determination of the Dosage of Fermentation Broth.
To hydrolyze the catfsh ofal, the experiment employed a fermentation broth of B. cereus RGL1.1.Te bacteria were cultivated in tryptic soy broth media under static conditions at a density of 10 8 -10 9 CFU/mL for 48 hours at 28 °C with intermittent shaking to obtain the fermentation broth.After centrifuging the fermentation broth at 10,000 rpm for 30 minutes, the supernatant was collected.Te procedure for producing the protein hydrolysate was based on [13].Before hydrolysis, fsh ofal is defrosted and chopped until crushed and then homogenized with aquadest in a ratio of 1 : 3. Te mixture is then adjusted to pH 8. Te catfsh ofal was hydrolyzed for 6 hours at 55 °C using the fermentation broth at concentrations of 0%, 4%, 6%, and 8% (v/w).Te hydrolysate is heated to 80 °C for 20 minutes to deactivate enzymes.Afterward, the sample is centrifuged for 20 minutes at 5000 x g to separate the supernatant from the sediment.Te protein hydrolysates in the form of supernatants are then analyzed further.In order to evaluate the quality of the protein hydrolysate, the Bradford method was employed to measure the soluble protein content.Tis method determines the total protein concentration present in a sample by measuring the binding of protein molecules to Coomassie dye under acidic conditions, which changes color from brown to blue.Additionally, the quality of the protein hydrolysate was further assessed by measuring the protein hydrolysis degree using the method suggested in [14].Tis method makes use of a nitrogen soluble index to determine the degree of hydrolysis by using trichloroacetic acid as a precipitating agent.
After producing the protein hydrolysate at the optimal dose, further analysis was conducted to determine its amino acid and fatty acid profles, as well as its digestibility.Te optimal dose is the one that produces the highest protein hydrolysates.To analyze the amino acid profle, highperformance liquid chromatography (HPLC) was used with a C-18 column, a fow velocity of 1 mL/min, and a pressure of 3000 psi.Te fuorescence detector with a wavelength of 350-450 nm was employed, and the device temperature was set to 27 °C.Te mobile phase consisted of 95% methanol and 1 M sodium acetate.For the fatty acid profle, gas chromatography (GC) was used.Te standard used for the analysis was SupelcoTM 37 Component FAME Mix.Nitrogen gas was used as the mobile phase, with a pressurized fow rate of 20 mL/min, and hydrogen gas served as the burner gas with a fow rate of 30 mL/min.Te analysis was performed using a 60-meter-long Quadrex Fused Silica Capillary Column 007 Cyanoprophyl Methyl Sil, with an inner diameter of 0.25 mm.Te temperature was maintained at 125 °C and was gradually increased by 5 °C per minute until it reached 225 °C.Te injector temperature was 220 °C, and the detector temperature was 240 °C.In order to test the digestibility of the protein hydrolysate, the method suggested in [15] was used with 0.5% Cr 2 O 3 as an indicator.For the tests, a feed mixture was prepared using 70% commercial feed and 30% fsh protein hydrolysate, and the optimal dosage was used as a reference.Commercial feed was used as a control.Te experiment was conducted in a 60 × 50 × 40 cm aquarium flled with 100 L of water and equipped with an aeration system.Te feeding experiment was carried out using twenty snakehead fsh weighing 10 g each, with a density of twenty individuals per aquarium.Satiated feeding was done three times a day.After fve days 2 Scientifca of feeding, feces were collected by siphoning and sieving them through a fne sieve.To avoid nutrient decomposition in the water, feces were collected an hour after feeding.Te feces were then dried in a 40 °C oven for 24 hours, wrapped in sealed plastic bags, and stored in a refrigerator at 5-6 °C.Te process of collecting fecal samples was conducted until enough samples were available for analysis.Both the feed and fecal samples were analyzed for their proximate and chromium (Cr 2 O 3 ) content.For the proximate analysis, crude protein was measured using the Auto Kjeldahl System (VELP Scientifca, Milano, Italy), crude lipid content was measured through the ether-extraction method using a Soxhlet extractor (Foss -ASN 3125-2005), moisture content was measured by drying the samples in an oven at 105 °C for 4 hours, and the ash content was measured using a furnace (550 ± 20 °C for 3 hours).In chromium analysis, approximately, 0.2 grams of ground samples were digested with the oxidation process, and the fnal solution was measured through simple photometry at 350 nm.

Te Efectiveness Test of the Use of Protein Hydrolysate in
Snakehead Fish Feed.A completely randomized design with four treatments and three replications was used to determine the efectiveness of using protein hydrolysate in snakehead fsh feed.Te four treatments of the percentage of protein hydrolysate applied in the feed formula were 0, 15, 30, and 45%.Te commercial feed used was "Prima Feed 1000" (PF1000) produced by CV Matahari Sakti.Te protein hydrolysate was mixed thoroughly with the mashed commercial feed before being transformed into pellets.Te proximate composition of the feed after the addition of protein hydrolysate is presented in Table 1.Snakehead fshes (C.striata) weighing 38.19 ± 0.14 g were acclimated for 14 days before being stocked in aquariums at a density of twenty individuals per aquarium.Fish are collected before and after treatment for proximate analysis.Te experiment lasted 40 days.Feeding treatment was administered three times a day.Te test parameters were absolute weight growth (AWG) [16], specifc growth rate (SGR) [17], feed efciency (FE) [15], protein efciency ratio (PER) [18], and survival rate (SR) [16].Te formula for each parameter is as follows: (i) Absolute weight gain (AWG) = Final weight (g) − Initial weight (g) (ii) Feed efciency (FE) = (Fish wet weight gain/feed intake) × 100 (iii) Specifc growth rate (SGR) = ((ln fnal weight − ln initial weight)/feeding period in days) × 100 (iv) Protein efciency ratio (PER) = Fish wet weight gain/protein intake (v) Survival rate (SR) = (Final number of the remaining fsh/initial number of fsh) × 100 2.4.Data Analysis.Te software program SPSS verse 22.00 was used to perform statistical tests.Data of the test parameters were collected and analyzed using analysis of variance (ANOVA); if the results were diferent signifcantly, the Duncan test at a 95% confdence interval was applied.

Results and Discussion
3.1.Determination of Enzyme Dosages for the Protein Hydrolysis Process.Te degree of hydrolysis was signifcantly diferent in each concentration of B. cereus RGL1.1 fermentation broth (P < 0.05) (Figure 1).Te highest degree of hydrolysis of catfsh ofal was observed with a fermentation broth concentration of 8%, followed by concentrations of 6% and 4%, respectively.Te control had the lowest degree of hydrolysis.Tis fnding was in line with the fndings of [19], where the authors used the enzyme papain to hydrolyze snakehead fsh fllet waste and discovered that the higher the concentration of papain, the greater the degree of hydrolysis produced.According to [20], the higher the enzyme concentration, the higher the level of dissolved nitrogen in protein hydrolysates due to peptide bond breakage.
Te substantial degree of hydrolysis in the 6% and 8% concentrations is further verifed by the soluble protein analysis (Figure 2).Te higher the concentration of the fermentation broth, the higher the level of soluble proteins.A rise in the concentration of soluble proteins implies that protein bonds are broken down into peptides or amino acids.Insoluble proteins are transformed into soluble nitrogen compounds during the hydrolysis process, which are subsequently broken into simpler compounds, such as peptides and amino acids, which are more easily absorbed by the body [19].Although a concentration of 8% results in the highest increase in hydrolysis and soluble protein, it is statistically insignifcantly diferent from a concentration of 6% (P > 0.05).As a result, protein hydrolysate at a dose of 6% was chosen for the next research step.
Except for lysine, practically, all the amino acids tested in catfsh ofal hydrolysate have increased (Table 2).Transamination occurs throughout the hydrolysis process, increasing the number of amino acids liberated [21].Te dominant amino acids in catfsh hydrolysate in this study were glutamic acid, lysine, aspartic acid, leucine, and isoleucine.Tis is slightly diferent from turbot fsh protein hydrolysate where the dominant amino acids are glycine, glutamic acid, aspartic acid, and alanine [22].
Te fatty acid composition of catfsh viscera hydrolyzed with B. cereus RGL1.1 is shown in Table 3. Apart from proteolysis, the B. cereus RGL1.1 crude enzyme extract produces lipolysis in catfsh ofal, resulting in alterations in its fatty acid composition.It is suspected that besides protease, the crude extract of enzymes extracted from the bacteria B. cereus RGL.1.1 contains lipase, which can break down fats into fatty acids.Tis is evident from the increase in the quantity of almost all types of fatty acids measured, such as myristate, stearic, oleic, linoleic, linolenic, EPA, and DHA.According to Subandiyono and Hastuti [23], freshwater fsh require essential fatty acids such as linoleic acid and/or linolenic acid for normal growth and development.Te results of this study suggest that catfsh waste protein hydrolysate can be a potential source of essential fatty acids that can be used in freshwater fsh feed.

Scientifca
Catfsh ofal protein hydrolysate digestibility difers signifcantly (P < 0.05) from that of the control (Table 4).Te use of a fermentation broth from B. cereus RGL.1.1 resulted in higher protein digestibility than the control in this research.Tis study's fndings support the claim by [24] that high protein digestibility in fsh protein hydrolysates is associated with increased solubility and the release of protein molecular structure bonds into smaller peptide units during hydrolysis.Te benefts of utilizing protein hydrolysate in digestibility, according to [25], are related to the composition of the resultant product, which comprises peptides with a low molecular weight that contribute to enhanced digestibility.
Fat digestibility rose signifcantly (P < 0.05) in this study when compared to controls.Fat digestibility increased from 80.10 ± 0.68% to 85.61 ± 1.45%.Te increase in fat digestibility found in this study is assumed to be related to lipase enzyme activity in B. cereus RGL1.1.B. cereus exhibits lipase activity, and it can degrade fats into simple molecules [26,27].

Te Efcacy of Using Catfsh Ofal Protein Hydrolysate in
Snakehead Fish Feed.Te efectiveness of applying protein hydrolysate from catfsh ofal for snakehead fsh feed has been studied and it was discovered that adding up to 45% of the protein hydrolysate tends to further enhance the fsh's growth.Tis is supported by the increase in snakehead fsh's absolute weight growth (AWG) and specifc growth rate (SGR) (Table 5).Te result of this study agrees with the fndings of the authors of [28], who found that protein hydrolysate could increase the growth of the Pseudosciaena crocea R fsh.Te authors of [29] found that using 30% fsh protein hydrolysate resulted in a higher tilapia growth performance.Although the growth trend of snakehead fsh fed with catfsh ofal protein hydrolysate in this study has increased, the DGR and AWG of control (dose of 0%) were not statistically diferent (P > 0.05)with dosages of 15% and 30%.However, the controls (0%) difered signifcantly (P > 0.05) with a dosage of 45%.Tis demonstrates that up to 45% dose of protein hydrolysate can increase snakehead fsh growth.According to reference [30], fsh protein hydrolysate has been shown to increase survival and growth rate, lower malformation levels, increase enzyme activity, modify nutrient transformation patterns in the digestive tract, increase nutrient absorption, and induce the formation of nonspecifc immune responses in larvae, fry, and adult fsh at certain levels.Te authors of [31] stated that a moderate inclusion of fsh protein hydrolysate in aquafeeds has the potential to improve growth, feed utilization, immune functions, and disease resistance of fsh.
Te feed efciency (FE) at doses of 15% and 30% did not difer signifcantly (P > 0.05) from that of the control (concentration of 0%), though it tended to have a higher trend than the control.Whereas, FE at 45% is higher and signifcantly diferent from FE at 0% (P < 0.05) (Table 5).Te FE of snakehead fsh-fed protein hydrolysate in this study was higher (37.42-46.18%)than the FE of snakehead fsh fed gold snail meal (12.74-21.47%).Te authors of [32] reported that the feed efciency of snakehead fsh decreased after being fed with golden snail meal.On the contrary, when snakehead fsh are fed with hydrolysate of catfsh ofal, feed efciency is increased.Fish protein hydrolysate is considered a suitable source of protein for human and animal nutrition due to its balanced amino acid composition and low molecular weight, allowing for a higher rate of absorption by the intestines [30].Tere is a suspicion that the low PER (protein efciency ratio) value observed in the 30% dose treatment is due to genetic variation among the fsh used in the experiment.Despite the fact that at the start of the experiment, all fsh were obtained from the same parent source and were of similar size (as per the complete randomized design), there is still a possibility of genetic variations that infuence the growth rate during the experiment.
Statistically, the protein efciency ratio (PER) values of snakehead fsh-fed hydrolysate protein at 15% and 45% doses difered signifcantly (P < 0.05) from those at a dose of 30% and control (without hydrolysate protein) (Table 5).It is suspected that the high value of protein efciency ratio observed at the doses of 15% and 45% is due to the presence of protein hydrolysate of catfsh in the feed.Tis hydrolysate has been converted into simple amino acids and peptide compounds, which are easily digestible by the snakehead fsh and therefore, can be utilized optimally for its growth.Te PER parameter is widely considered a good criterion for assessing the protein quality in aquatic feed.Te diference in PER values indicates a diference in feed protein quality [33].
When snakehead fsh consume protein hydrolysates, their protein content rises from 15.60% to around 16.28-17.24%and their fat content rises from 2.93% to around 3.51-4.00%.Meanwhile, the ash content has dropped from 5.13% to between 4.80 and 5.04% (Table 6).Te high protein level in snakehead fsh treated with hydrolyzed ofal catfsh compared to controls indicates that up to 45% of such material is sufcient to deposit the protein in the body (Table 6).Tis is consistent with the fndings of the authors of [34], who found that up to 50% fsh protein hydrolysate in carp juvenile feed is sufcient to induce muscle protein deposition.Te same thing can be seen in snakehead fsh body fat content, where the addition of catfsh ofal hydrolysate in snakehead fsh feed is signifcantly higher (P < 0.05) than in the control.According to several studies, fsh protein hydrolysate raises fat levels in fsh [35][36][37][38].
Te various analyses demonstrated that adding hydrolyzed catfsh ofal to the meal had no signifcant efect on snakehead fsh survival (P > 0.05) (Figure 3).Te survival rate of snakehead fsh was relatively high during the study, ranging from 93.33 to 96.67%.Tis implies that adding 45% catfsh ofal protein hydrolysate to snakehead fsh feed is still relatively safe and does not afect the fsh's survival.Tis  Scientifca research showed that the survival of snakehead fsh after being fed hydrolysate protein was in line with other studies such as [24,33].Fish protein hydrolysates, according to [30], can promote fsh survival and growth.Te authors of [25] reported the same thing, that fsh protein hydrolysate in diet can improve tilapia larvae' survival by stimulating the animal's immune system.Te authors of [39] also said that the presence of fsh hydrolysates may also improve the digestion, absorption, and utilization processes of animals and increase their immunity.

Conclusions
Te hydrolysis of catfsh fllet processing byproducts using crude extract enzyme B. cereus RGL.1.1 improves the availability of soluble proteins, amino acids, and fatty acids as well as the digestibility of this feedstuf.In the process of producing protein hydrolysates from catfsh fllet processing waste, a 6% enzyme dose can currently be used.Meanwhile, utilizing up to 45% catfsh ofal protein hydrolysate in feed can improve snakehead fsh growth performance, protein efciency ratio, and feed efciency.Data were expressed as mean ± SD.Diferent superscript letters in the same column show signifcantly diferent results (P < 0.05).

Figure 1 :Figure 2 :
Figure 1: Te degree of hydrolysis of the catfsh ofal, which was hydrolyzed using a fermentation broth of B. cereus RGL1.1.Data were expressed as mean ± SD.Diferent superscript letters in the bars have signifcantly diferent results (P < 0.05).

Figure 3 :
Figure3: Te survival rate (%) of snakehead fsh fed with feed containing protein hydrolysate from catfsh ofal (Data were expressed as mean ± SD).Te same superscript letters in the bars show no signifcantly diferent results (P > 0.05).

Table 1 :
Te proximate composition of snakehead fsh feed after the addition of diferent doses of protein hydrolysate.

Table 2 :
Amino acid composition of protein hydrolysate from catfsh fllet processing waste with and without hydrolysis.

Table 3 :
Fatty acid composition of protein hydrolysate from catfsh fllet processing waste with and without hydrolysis.

Table 4 :
Snakehead fsh (C.striata) digestibility after being fed feed that contains protein hydrolysate from catfsh fllet processing waste.Data were expressed as mean ± SD.Diferent superscript letters in the same column show signifcantly diferent results (P < 0.05).