Tra catfish (Pangasius hypophthalmus L.) accountsfor an essential part of Mekong Delta’s and Vietnamesefisheries’ yield. In 2019, their production area totaled6600 ha yielding 1.42 million tons, of which 60–70%were by-products [1–3]. The production expansionin order to meet the domestic and export demandhas caused a growing concern about the fisheries’by-product treatment, especially from Pangasiusprocessing. The Mekong Delta, where Pangasiusproduction and processing are more developed, needseconomical and environmentally friendly solutions forlarge amounts of by-products generated by the localmanufacturing facilities [4]. Recent years have seen aninterest in the utilization of Pangasius by-products tomanufacture value-added products, such as fish powder,fish skin, and viscera.Fish powder is the most popular by-product used asa primary protein supplement for animal feeds. Fish byproductsare a major source of lipids, native proteins,and hydrolysates, accounting for 10–20% of the total fishprotein [5, 6]. Presently, most fish powder manufacturersuse the traditional procedure with high pressure andtemperature, resulting in products with a low digestiveand absorptive index.20Hien B.T.T. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 19–26Enzymatic protein hydrolysis of fish by-productsis currently a promising approach to making productswith various applications and high nutritional values [5,7–10]. Proteases are among common enzymes usedin this process [6]. They hydrolyze protein in fish byproductsto smaller peptides that usually contain 2–20amino acids. Fish hydrolysate is a liquid of aminoacids – peptides produced by speeding up fish proteinhydrolysis using proteases under controlled conditions.When applied in animal feed products, fish hydrolysateimproves the digestion and absorption of proteins, aswell as feed intake, efficiency, and protein utilization[11–14]. Furthermore, protein hydrolysate enhancesthe attractiveness and palatability of the feed, thusincreasing its consumption [15].There have been several studies in Vietnam ondifferent aspects of enzymes for hydrolysis of catfishby-products. Nguyen Cong Ha et al. found that possiblesubstrate concentrations for an optimal enzyme tosubstrate ratio were 108.4 g/L for neutrase, 36.2 g/Lfor papain, and 135.8 g/L for bromelain [16]. NguyenThi Thuy et al. investigated Pangasius by-producthydrolysis using commercialized papain as a proteinsource for animal feeds [17]. Dang Minh Hien et al.also studied papain for application in Bacillus subtiliscultivation with satisfactory results [18]. Phan VietNam et al. hydrolyzed catfish by-product using acombination of enzymatic hydrolysis and thermaltreatment, having achieved over 30% hydrolysis, 80%nitrogen recovery, and a large amount of essentialamino acids [3]. The researchers showed the potentialof catfish by-product treatment with protease enzymesand possible application of hydrolysates in feedproduction. Therefore, an investigation of optimalhydrolysis conditions is essential for further commercialapplication.This study assessed optimal conditions for Pangasiushydrolysis, such as the type of enzyme, enzyme/substrate ratio, temperature, time, and the amount ofwater added by analyzing total nitrogen and aminoacids. As a result, we identified the initial conditions toproduce hydrolysates from Pangasius by-products forthe animal feed industry.STUDY OBJECTS AND METHODSStudy objects. This study featured the by-productsof industrially processed Pangasius c atfish ( Pangasiushypophthalmus L.) fillet.Materials. Pangasius by-products, includingheads, bones, and fins (Fig. 1), were collected from theprocessing factory of the Travel Investment and SeafoodDevelopment Corporation (Trisedco, Vietnam). Theywere minced into small pieces of 3–5 mm and storedat –20°C until use.The enzymes were obtained from ICFood Vietnam,including bromelain (active pH 5.5–7.0, 55–60°C,500 IU/g), papain (pH 4.5–8.5, 60–70°C, 500 IU/g),protease (pH 4.5–8.5, 50–60°C, 500 IU/g), andSEB-Neutral PL (pH 5.5–7.5, 35–60°C, 750 IU/g). Theyare commercial enzymes commonly used in animal feedproduction.Optimal hydrolysis conditions. The materials weredefrosted, mixed with 20% water, and heated to 55°Cbefore adding enzymes. Hydrolysis was performed ina pilot-scale hydrolysis equipment with a capacity of80 kg/batch. It was terminated by heating to 85–90°C in10 min.After hydrolysis reactions, the mixtures were filteredand analyzed for total nitrogen (Naa). The optimalhydrolysis conditions were obtained by optimizing asingle condition at a time in consecutive order. Theresulting condition in the previous experiments was usedas a constant condition in the later experiments. Theprotein recovery efficiency was evaluated using the ratioof nitrogen amino acid (Naa) to total nitrogen (Nts) in thehydrolysates.Evaluating the chemical composition of thematerials. We analyzed such parameters as crudeprotein, moisture content, lipid, ash, TVB-N, and totalaerobic microorganisms.Choosing the enzyme. We assessed four enzymes,namely protease, bromelain, papain, and SEB-NeutralPL. Hydrolysis reactions were performed with theenzyme/substrate (E/S) ratio of 0.3%, temperatureof 55°C, and 20% water added in 5 h. The hydrolyzedproducts were then analyzed for total protein and Naa tochoose the most effective enzyme. The chosen enzymewas used in the later experiments.Figure 1 Pangasius by-products used as study objects21Hien B.T.T. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 19–26Choosing the optimal enzyme/substrate ratio. Sevenenzyme/substrate ratios, namely 0.0, 0.1, 0.2, 0.3, 0.4,0.5, and 0.6%, were analyzed during hydrolysis. Thehydrolysis reactions were performed at a temperatureof 55°C, with 20% of water added in 5 h. The chosenenzyme/substrate ratio was then used in the laterexperiments.Choosing the optimal temperature. Fivetemperatures (45, 50, 55, 60, and 65°C) were assessed.The hydrolysis was performed with the optimalenzyme and enzyme/substrate ratio from the previousexperiments and with 20% water added in 5 h.Choosing the optimal amount of added water. Waterin amounts of 0, 5, 10, 15, 20, 25, and 30% was usedfor hydrolysis under the optimal conditions chosen fromthe previous experiments in 5 h. The products were thenanalyzed to choose the optimal ratio of water requiredfor hydrolysis.Choosing the optimal hydrolysis time. Hydrolyseswith the optimal conditions from the previousexperiments were performed in five different periods,from 3 to 7 h. The products were then analyzed tochoose the optimal reaction time.Optimizing the hydrolysis procedure. A Box-Behnken design was used to investigate the effect ofthree factors: A – hydrolysis time, B – enzyme/substrateratio, and C – temperature on the Naa/Nts ratio [19].An optimal scenario was obtained, and a verificationexperiment with the optimum conditions was performedin the lab.Analysis methods. The following Vietnamesestandards (abbreviated TCVN) were used to analyzethe chemical quality parameters in this study: TCVN3705:1990 for crude protein; TCVN 3708:1990 for aminoacid nitrogen; TCVN 3700:1990 for water amount;TCVN 9215:2012 for vaporized acid-base; TCVN3706:1990 for ammonia nitrogen; and TCVN 5165:1990/TCVN 4884:2005 for total aerobic microorganisms.Data analysis. Each experiment was done intriplicate, each time with three samples, and the resultswere averaged. The data were processed and charted inMS Excel 2007 and model analysis was performed inDesign Expert (version 10).RESULTS AND DISCUSSIONMaterials’ quality. The quality of the by-productsused for hydrolysate production was evaluated throughchemical and microbiological parameters, as shown inTable 1.The materials’ crude protein and moisture contentswere 12.76 and 59.27%, respectively, similar to otherresearch in Vietnam [20]. TVB-N, a freshness qualityindicator, was 13.45 mg/100 g, which was much betterthan the best quality limit of 25 mg/100 g. Although thetotal microorganism count was 1.13×105, a large numberrelating to long storage before use, the TVB-N resultindicated that the materials were still fresh and did notcontain any spoilage compounds that could adverselyaffect the quality of hydrolysates [21].Optimal hydrolysis conditions. Choosing thehydrolysis enzyme. The Naa/Nts ratios obtained from thehydrolysis with four enzymes under study are shownin Fig. 2.Under the same hydrolysis conditions, SEB-neutralPL resulted in the highest Naa amount (36.12 ± 0.31%),equivalent to 1.4 times from bromelain hydrolysis and1.3 times from protease or papain. The SEB-neutral PLenzyme was chosen to be used in the later experimentsin this study.Choosing the enzyme/substrate ratio. The effectof the enzyme/substrate ratios on the Naa/Nts ratio ispresented in Fig. 3.Table 1 Chemical and microbiological parametersof the materialsParameters ResultsCrude protein, % 12.76 ± 0.48Moisture, % 59.27 ± 0.33Lipids, % 21.19 ± 0.32Ash, % 7.02 ± 0.24TVB-N, mg/100 g 13.45 ± 0.37pH 6.41 ± 0.17Nitrogen from amino acid, % 0.07 ± 0.0028Total aerobic microorganism, CFU/g 1.13 ± 0.18×105Figure 2 Proteases effect on Naa/Nts ratio Figure 3 Enzyme/substrate ratio vs. Naa/Nts ratio10152025303540Protease Bromelin Papain SEB-NeutralPLNaa/Nts ratio, %Enzyme protease1015202530354045500 0,1 0,2 0,3 0,4 0,5 0,6Naa/Nts ratio, %E/S ratio, %20253035404550Naa/Nts ratio, %3035404550Naa/Nts ratio, %10152025303540Protease Bromelin Papain SEB-NeutralPLNaa/Nts ratio, %Enzyme protease1015202530354045500 0,1 0,2 0,3 0,4 0,5 0,6Naa/Nts ratio, %E/S ratio, %253035404550Naa/Nts ratio, %3035404550Naa/Nts ratio, %22Hien B.T.T. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 19–26The lowest Naa/Nts ratio demonstrated the reactionwithout enzyme (14.96 ± 0.38%), and the highest wasfrom 0.6% enzyme added (45.22 ± 0.32%), though theratios of 0.4 and 0.5% were not much different from0.6%. The enzyme/substrate range of 0.3–0.5% waschosen for further analysis, and the enzyme/substrateratio of 0.4% was chosen for the later experiments.Choosing the hydrolysis temperature. Figure 4 showsthe effect of hydrolysis temperatures on Naa/Nts ratios.The results showed that the higher the temperature,the better the hydrolysis performance till it reachedoptimum at 55°C. The effective ratio went down as thetemperature increased beyond that. Thus, 55°C waschosen for use in later experiments, and the range of 55–60°C was used in the optimal analysis.Choosing the amount of added water. The amountof added water influences the enzyme’s dispersaland contact with substrates. As we can see in Fig. 5,hydrolysis performance increased steeply when thewater amount went up from 0 to 10%. Then it sloweddown considerably with water increasing from 10onward to 30%, suggesting an equivalent efficiency.To economically use other substances in the hydrolysisreaction, we chose 10% added water to use in laterexperiments.Choosing the hydrolysis time. The samples werehydrolyzed in 3, 4, 5, 6, and 7 h under the conditionsfrom the previous experiments (0.4% SEB-neutralPL, 55°C, and 10% added water). The hydrolysisperformance increased sharply from 34.44 ± 0.34% (3 h)to 43.90 ± 0.26% (5 h), then slowing down and reachinghighest at 44.65 ± 0.30% (7 h). The difference betweenthe samples hydrolyzed in 6 and 7 h was not statisticallysignificant (P > 0.05). Although nitrogen solubilityFigure 4 Hydrolysis temperature vs. Naa/Nts ratioFigure 5 Effect of water amount on Naa/Nts ratioFigure 6 Effect of hydrolysis time on Naa/Nts ratioTable 2 ANOVA resultsFactors Sum of square Degrees of freedom Mean square F ratio P-value (P < 0.05)Model 390.41 9 43.38 155.85 < 0.0001 significantA 116.13 1 116.13 417.24 < 0.0001B 24.12 1 24.12 86.65 0.0002C 70.27 1 70.27 252.47 < 0.0001AB 4.82 1 4.82 17.31 0.0088AC 5.45 1 5.45 19.59 0.0069BC 4.67 1 4.67 16.76 0.0094A² 58.95 1 58.95 211.81 < 0.0001B² 112.44 1 112.44 403.98 < 0.0001C² 12.68 1 12.68 45.57 0.0011Residual 1.39 5 0.2783Lack of fit 1.18 3 0.3934 3.72 0.2190 insignificantStandard deviation 0.2115 2 0.1057R2: 0.9964 Expected R²: 0.9506A – Enzyme/substrate ratio, B – Temperature, and C – Hydrolysis duration101520Protease Bromelin Papain SEB-NeutralPLNaa/Enzyme protease101520250 0,1 0,2 0,3 0,4 0,5 0,6Naa/E/S ratio, %152025303540455045 50 55 60 65Naa/Nts ratio, %Temperature, oC2530354045500 5 10 15 20 25 30Naa/Nts ratio, %Water added, %3032343638404244463 4 5 6 7Naa/Nts ratio, %Time, hour1015Protease Bromelin Papain SEB-NeutralPLEnzyme protease1015200 0,1 0,2 0,3 0,4 0,5 0,6E/S ratio, %152025303540455045 50 55 60 65Naa/Nts ratio, %Temperature, oC2530354045500 5 10 15 20 25 30Naa/Nts ratio, %Water added, %3032343638404244463 4 5 6 7Naa/Nts ratio, %Time, hour101520253035Protease Bromelin Papain SEB-NeutralPLNaa/Nts ratio, %Enzyme protease10152025303540450 Naa/Nts ratio, %152025303540455045 50 55 60 65Naa/Nts ratio, %Temperature, oC2530354045500 Naa/Nts ratio, %3032343638404244463 4 5 6 7Naa/Nts ratio, %Time, hour23Hien B.T.T. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 19–26Figure 7 Factors relative influences on the dependent variable in the statistical model: (a) Influences of E/S ratio and duration;(b) Influences of temperature and E/S ratio; (c) Influences temperature and durationNaa/Nts, %C: Duration, hA: E/S ratio, %6.05.55.04.54.00.30 0.35 0.40 0.45 0.5034363840424446338Design-Expert® SoftwareFactor Coding: ActualNaa/Nts, %Design points above predicted valueDesign points below predicted value29.56 45.47X1 = А: Е/S ratioX2 = C: DurationActual FactorB: Temperature = 55C: Dur ation, h5045403530256.05.55.0 4.54.0 0.35 0.40 0.45 0.500.30Design·Expert® SoftwareFactor Coding: ActualNaa/Nts, %Design points above predicted valueDesign points below predicted value29.56 45.47X1 = А: Е/S ratioX2 = B: TemperatureActual FactorC: Duration = 5A: E/S ratio, %0.30 0.35 0.40 0.45 0.50605856545250B: Temperature, °C3540353Naa/Nts, %B: Temperature, °C50454035302560Naa/Nts, %5652 50 0.300.400.450.500.3554586.05.55.04.54.0B: Temperature, °C50 52 54 56 58Design·Expert® SoftwareFactor Coding: ActualNaa/Nts, %Design points above predicted valueDesign points below predicted value29.56 45.47X1 = B: TemperatureX2 = C: DurationActual FactorА: Е/S ratio = 0.4B: Temperature, °C5045403530256.0C: Duration, h5.04.0 50 5254 56 58 604.55.5C: Duration, h604535 403Naa/Nts, %abcA: E/S ratio, %A: E/S ratio, %Naa/NtsN , % aa/Nts, %24Hien B.T.T. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 19–26increases with time in enzyme hydrolysis, after acertain period, the formation of hydrolysis productsand the reduction of peptide bonds during hydrolysisinhibit enzyme activities and decrease hydrolysisrate [22, 23]. Therefore, the effective range of durationwas determined to be from 4 to 6 h.Optimizing Pangasius by-product hydrolysis withSeb-Neutral PL enzyme. We studied the influence ofdifferent variables on the outcome of hydrolysis byperforming Box-Behnken Designs in Design-Expert. Inparticular, we used the following factors: A – enzyme/substrate ratio (0.3–0.4–0.5%); B – Temperature(50–55–60°C); and C – Hydrolysis duration (4–5–6 h).The ANOVA results are presented in Table 2.The model was significant with the F ratio of 155.85(P < 0 .000) a nd t he l ack o f fi t o f 3 .72 ( P = 0 .2190).The variables AB, AC, BC, A2, B2, and C2 all hadP < 0.05 and therefore were significant and includedin the regression formula. The model to determine therelationship between the dependent variable Naa/Nts andenzyme/substrate ratio, temperature, and duration, aswell as their interactions, was presented in the followingformula:y = a + b1·A + b2·B + b3·C + b12·AB + b13·AC ++ b23·BC + b12·A2 + b22·B2 + b32·C2Naa/Nts ratio = 43.79 + 3.81·A + 1.74·B + 2.96·C ++ 1.10·AB + 1.17·AC – 1.08·BC – 4.00·A2 –– 5.52·B2 – 1.85·C2 (1)In formula (1), b1, b2, and b3 were positive, showingthat the hydrolysis performance (Naa/Nts) was directlyproportional to the analyzed factors: enzyme/substrateratio, temperature, and duration. ǀb1ǀ<ǀb3ǀ<ǀb2ǀ suggestedthat the enzyme/substrate ratio had a more substantialinfluence on the performance of the hydrolysis reactionthan the other factors. The coefficients b12, b22, and b32were negative, suggesting that the graphs were parabolicfaces with concave surfaces facing downwards andhaving extreme points. The coefficients b12 and b13were positive, showing a positive interaction betweentemperature and time, with the enzyme/substrate ratioincreasing hydrolysis performance. At the same time,b23 was negative, indicating that the interaction betweentemperature and time was inversely proportional tothe Naa/Nts ratio. This could be explained by the natureof enzyme reactions, where high temperature andprolonged duration might cause unstable enzymes andreduce enzyme hydrolysis activities. The effects of thesefactors on the Naa/Nts ratio were graphically presentedin Fig. 7.Based on the Box–Behnken design model, theoptimum hydrolysis conditions (enzyme/substrateratio = 0 .48%, t emperature = 5 6.52°C, a nd d uration =5.77 h) was chosen to conduct a laboratory experiment.The results of the laboratory experiment in comparisonwith the predicted values are shown in Table 3.There were no significant differences betweenthe predicted and experimental values. Therefore, weproposed the following optimum hydrolysis conditionsfor Pangasius by-products: 10% added water, 0.48%SEB-Neutral PL enzyme, 57°C, and 6 h.Product quality. The results of the hydrolysatequality evaluation are presented in Table 4.The resulting Pangasius hydrolysate was a highqualitysource of nutrition with 12.41 g/L nitrogen fromamino acids, accounting for 46.08% of total nitrogen(26.94 g/L). Its other parameters fully complied withthe current national standard QCVN 01-190:2020/BNNPTNT, making it suitable for use as a supplementto animal feed to improve its amino acid content andaroma.CONCLUSIONWe investigated the initial hydrolysis conditions toproduce hydrolysates from Pangasius hypophthalmus L.by-products. The regression model prediction andlaboratory verification determined the followingoptimum hydrolysis conditions: 10% water amount,0.48% SEB-Neutral PL enzyme, 57°C temperature,and 6 h hydrolysis time. The hydrolysates yielded fromthe proposed hydrolysis procedure satisfied the qualityrequirements for animal feed supplements and compliedwith the national standard QCVN 01-190: 2020/BNNPTNT. Thus, our study results were commerciallyapplicable for feed production and provided a solutionfor by-product treatment in the fisheries.CONTRIBUTIONB.T.T. Hien designed the study concept. P.T. Diemdeveloped the methodology. L.A. Tung, T.T. Huong,Table 3 Predicted vs. experimental resultsValue Enzyme/substrateratio, %Temperature,°CDuration,hNaa/Ntsratio, %Predicted 0.48 56.52 5.77 46.25Experimental 0.48 57 6 46.08 ± 0.31Table 4 Quality parameters of the resulting hydrolysateParameters ResultsTotal nitrogen, g/L 26.94 ± 0.16Nitrogen from amino acid, g/l 12.41 ± 0.08Naa/Nts ratio, % 46.08 ± 0.31TVB-N, mg/100 g 42.73 ± 0.63Lipid, % 2.6 ± 0.02Escherichia coli Not detected in 1 gSalmonella Not detected in 25 gSensoryColor: deep brownSmell: aroma characteristic of fish protein hydrolysates,no off-aroma.25Hien B.T.T. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 19–26REFERENCES1. VASEP General information of Shark catfish aquaculture [Internet]. [cited 2021 Jun 10]. Accessed from: https://vasep.com.vn/san-pham-xuat-khau/ca-tra/tong-quan-nganh-ca-tra.2. Nguyen TNH, Huynh TD. Research on increasing the extraction yield of gelatin from Pangasius hypophthalmus byultrasonic. 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(In Vietnamese).and N.H. Hoang performed the validation experiment.N.H. Hoang and N.K. Bat conducted formal analysis.N.V. Nghia and B.T.T. Hien drafted, reviewed, andedited the manuscript. All the authors were involved inthe investigation, as well as read and agreed to the finalversion of the manuscript.CONFLICT OF INTERESTThe authors declare that there is no conflict ofinterest.