2.1 Tilapia enzyme extracts
A fish farm from Bolívar (Buenos Aires, Argentina) provided a viscera batch from O. niloticus juveniles. The stomachs and intestines of 24 fish weighing 21.1 ± 3.02 g were removed from the visceral mass and placed on ice. For each organ, pools of 4 individuals were separately homogenized in ice-cold water (1:1 w/v) at pH 2 for stomach pools or pH 8 for intestine pools that were adjusted with 0.1 N HCl and 0.1 N NaOH, respectively. These preparations were centrifuged (10000 g for 30 min at 4°C) and the resulting supernatants were stored at -20°C as stomach enzyme extract (SE, n = 6) and intestine enzyme extract (IE, n = 6).
First, SE acid proteolytic activity was evaluated according to Anson (1938) using as a substrate solution 0.5% (w/v) bovine hemoglobin (Sigma H 2625) in 200 mM glycine-HCl buffer. The reaction was incubated for 30 min at 25°C. The absorbance was measured at 280 nm using a microplate spectrophotometer with Gen5™ Software (Epoch BioTek). Total acid protease activity was expressed as activity unit per mL of enzyme extract (U/mL), where U = Abs 280 × mL total / 0.051 × min (0.051 represents the molar extinction coefficient of Tyrosine).
Secondly, the methodology carried out by García-Carreño (1992) and adapted by González-Zamorano et al. (2013) was employed to determine the alkaline protease activity in IE. For this, 0.5% (w/v) azocasein (Sigma A 2765) in 50 mM Tris-HCl buffer at pH 8 was used as the substrate. The incubation was performed for 30 min at 25°C. The readings were performed in the microplate spectrophotometer setting the wavelength at 366 nm. All assays were run in triplicate. Total protease activity was expressed as activity unit per mL of enzyme extract (U/mL). A unit of enzyme activity (U) was defined as a change in absorbance per minute.
Finally, to estimate specific activities, SE and IE soluble protein concentrations were determined according to Bradford (1976) where bovine serum albumin (Sigma A9647, St. Louis, MO) was used as the standard. Specific activity was expressed as the activity unit per mg of soluble protein (U/mg protein).
2.2 Protease inhibition assay
The inhibitory effect of PM on tilapia alkaline proteases was evaluated following an adaptation of El-Sayed et al. (2000) methodology. For this, pea Pisum sativum waste was provided by a regional producer (Buenos Aires, Argentina). The legume seeds were grinded to obtain a meal (PM0). One fraction of this PM was next extruded at 150°C for 15 sec with the aim of inactivating the inhibition factors (PM150). Commercial FM was obtained from Coomarpes Ltda. (Mar del Plata, Argentina) and used as a control treatment without inhibitors in the following protocol.
Prior to the inhibition assay, all meals were exposed to an in vitro simulation of tilapia stomach digestion. For this, 100 mg/mL of PM0, PM150 and FM were separately homogenized with 5 µL of SE and 955 µL of 200 mMGly-HCl buffer at pH 2. These mixtures were incubated for 60 min at 25°C. Then, they were centrifuged for 10 min at 2000 g. Subsequently, for the alkaline protease inhibition assay, 5 µL of each supernatant was mixed with 250 µL of 50 mM Tris-HCl buffer at pH 8. Immediately, 5 µL of IE was added to the reaction tubes that were next incubated for 60 min at 25°C under continuous agitation. Finally, alkaline protease activity was determined as detailed in section 2.1. To determine the inhibition rate, the alkaline proteolytic activity of IE in absence of meals was set as 100%. All assays were run in triplicate.
2.3 In vitro digestibility of the protein sources
The in vitro protein digestion of PM0, PM150, and FM was determined using a pH-stat method based on Yasumaru and Lemos (2014) methodology. Tilapia digestion includes an acid phase in the stomach, followed by an alkaline one carried out at the intestine level; thus, determining the hydrolysis degree in both conditions becomes essential. The assessments were carried out using a pH-meter (Orion Star A211) and an automatic titrator (TIM 856). SE (1:3 w/v) and IE (1:1 w/v) extracts were utilized for these assays.
A proper volume of distilled water adjusted to pH 2 with 0.1 N HCl was added to each protein substrate (PM0, PM150, or FM), obtaining a final solution of 80 mg of protein per mL. The proper amount of protein was estimated based on the proximal composition of each ingredient. Then, 130 Μl SE were added to every solution, and then, incubated at pH 2 and 25 ± 0.2°C in a magnetic stirrer. After the acid digestion, the resulting solutions were adjusted to pH 8 by the addition of 0.1 N NaOH, to simulate the alkaline protein digestion. Once the desired pH was reached, each solution was incubated with the IE (10 µL of enzyme per mL of solution). The solutions were constantly mixed in a magnetic stirrer and maintained at pH 8 and 25 ± 0.2°C. The pH was stabilized by the addition of the titrant (0.1 N HCl or 0.1 N NaOH). These assays were carried out in triplicate.
The hydrolysis degree of protein (AcHD) exposed to stomach acid conditions was calculated based on the formula proposed by Diermayr and Dehne (1990):\(AcHD \left(\%\right)=\left[(V \times N)/E\right]\times \left(1/P\right)\times {F}_{pH}\times 100\). Where V is the volume of acid consumed in the hydrolysis reaction (mL); N represents the normality of such acid; E is the mass of substrate protein (g); P denotes the number of peptide bonds cleaved (mol/g protein), for proteins which amino acid composition is not determined, P is generally suggested as 8.0; and FpH is 1.08 (correction factor for pH 2.0 at 25°C). On the other hand, the alkaline hydrolysis (AkHD) estimated at intestinal conditions was calculated according to Adler Nissen (1986):\(AkHD\left(\%\right)=B\times Nb\times 1/a\times 1/MP\times 1/{H}_{tot}\times 100\). In which B is the volume of alkali consumed (mL); Nb represents the normality of the alkali; α is the average degree of dissociation of the α-NH groups (1/α = 1.50 for pH 8.0 at 25°C); MP denotes the mass of substrate protein (g); and Htot is a total number of peptide bonds in the protein substrate [7.6–9.2meqv/g protein, according to the source of protein]. Finally, a total protein digestibility (TPD) was estimated for each meal (FM, PM0, and PM150) by the sum of the acid (AcHD) and alkaline (AkHD) hydrolysis degree.
2.4 Preparation of shrimp hydrolysate
SH was elaborated through enzyme autolysis using Pleoticus muelleri processing waste, composed of cephalothoraces, based on the method described by Leal-Goncaves et al. (2010). The raw material was grounded in distilled water (1:1 v/v) and next submitted to digestion in a jacketed stirred reactor connected to a thermostated bath at 45°C for 10 min. A sample was taken to determine alkaline protease activity as previously described, and afterward, enzyme deactivation was performed by raising the temperature (100°C, 10 min). The solid and liquid fractions were separated by centrifugation at 10000 g for 15 min. The obtained supernatant, defined as SH, was employed to estimate the hydrolysis degree (HD) and its antioxidant properties in the following section.
2.5 Shrimp hydrolysate characterization
The HD of SH was estimated according to the methodology proposed by Baek and Cadwallader (1995). For this, 500 µL of HD were mixed with 1000 µL of 0.3M TCA, incubated at room temperature for 20 min and next filtered. Twenty-five microliters of the resultant filtrate were mixed with 225 µL of distilled water, 1250 µL of 0.5N NaOH, and 250 µL of 1.0 N Folin and Ciocalteu’s phenol reagent (Sigma F9252). The resulting solution was incubated at 30ºC for 15 min and then centrifuged at 2000 g for 10 min. Supernatant absorbance was measured at 578 nm. A sample of SH was taken from the reactor prior to hydrolysis reaction (initial time), and used as blank for HD. The assay was run in triplicate. HD was defined as follows:\(HD\left(\%\right)=({Abs}_{t}-{Abs}_{t0})⁄{Abs}_{max} \times 100\). Where: Abst represents the absorbance after ten minutes of hydrolysis, Abst0 is the absorbance at zero time and Absmax is the maximum amount of 0.3 M TCA soluble peptides as tyrosine determined after the hydrolysis of 0.1 g shrimp substrate with 4 ml 6 N HCl at 110°C for 24 hr.
The DPPH radical scavenging activity by DH was evaluated by the method of Shimada et al. 1992. An SH sample of 1500 µL was added to 1500 µL of DPPH in 95% ethanol. The mixture was homogenized, and incubated in the absence of light for 30 min. After standing, its absorbance was measured at 517 nm. The sequestration capacity of the DPPH free radical, defined as scavenging effect (SE), was calculated as follows: SE%= (1− (Abssample / Abscontrol)) x 100. Where Abscontrol is the absorbance without sample and Abssample is the absorbance with sample.
2.6 Proximate and amino acid analyses
PM150 and SH proximate compositions were determined at the “Laboratorio de análisis industriales” of the “Universidad Tecnológica Nacional” (UTN-FRMdP, Mar del Plata, Argentina) and at the “Instituto Nacional de Tecnología Industrial” (INTI, Mar del Plata, Argentina), as recommended by the Association of Official Analytical Chemists (AOAC 2013). Amino acid profiles were conducted at the “Laboratorio Fares Taie” (Mar del Plata, Argentina).
2.7 Experimental diets
Two isonitrogenous (33% crude protein) and isocaloric (378 kcal/100 g) diets, referred as Control diet and PM150 + SH diet, were formulated to feed Oreochromis niloticus juveniles. Feed formulae were designed according to the nutritional requirements defined for this species by FAO (2022) and NRC (2011). The control fed ingredients were selected based on a formulation routinely used by the “Laboratorio de Acuicultura” (UTN-FRMdP) for Nile tilapia production. PM150 + SH diet was formulated by linear programming using Excel Solver from Windows 2010, in which SBM was totally replaced by 10% of SH and 25% of PM150. According to Leal-Goncaves et al. (2010), liquid SH was mixed with PM150, and the dough was dried at 65°C for 24 h. Table 1 shows the porcentual formulation and proximate composition of the formulated feeds.
Table 1
Diet formulation and proximate composition of the formulated feeds
| Formulated feeds |
Ingredients (%) | Control | PM150 + SH |
Pea meal | - | 25 |
Shrimp hydrolysate | - | 10 |
Soybean meal | 16.05 | - |
Fish meal | 20 | 20 |
Wheat gluten | 13.07 | 13.07 |
Cornmeal | 6.93 | 6.93 |
Vegetable oil | 3 | 3 |
Cornstarch | 7 | 7 |
Vitamins and minerals premix | 1 | 1 |
Wheat bran | 32.95 | 14 |
Proximal composition (% of dry matter basis) |
Moisture | 6 | 8.7 |
Ash | 7.1 | 6.5 |
Lipids | 8.5 | 5.6 |
Protein | 32.8 | 34.5 |
Carbohydrates | 45.6 | 44.7 |
Gross energy (Kcal/100g) | 390.1 | 367.2 |
Table 1
2.8 Fish and experimental conditions
All experimental work involving fish was revised and approved by the Institutional Animal Welfare & Ethical Review Committee at “Universidad Nacional de Mar del Plata” (UNMdP, CICUAL 6-2041/19RD 378). Sex-reversed Nile tilapia (n = 56, mean weight = 7.28 ± 0.186 g) were obtained from the “Laboratorio de Acuicultura” (UTN-FRMdP). Groups of seven fish were stocked in each of eight 70-L plastic tanks (60 × 35 × 25 cm, width × length × height) equipped with continuous aeration and a biologic filter. After 7 days of acclimatization, the PM150 + SH and control diet were randomly assigned to four tanks.
Fish were fed the experimental diets for 40 days at a feeding rate of 5% of aquaria biomass in three equal rations. Daily feed was adjusted on a weekly basis by batch weighing in a water container following a 24 h deprivation period. Although no significant feed scrap was seen, the aquaria were siphoned once daily and submitted to a 50% water replacement due to feces accumulation and keep water quality. The water temperature was maintained at 28 ± 1ºC and pH 7.5 ± 0.15; additionally, the photoperiod used was a 10 h light/14 h dark cycle. To monitor growth, fish were weighed and measured before and after the onset of the feeding trial. In the end, fish were sacrificed and their livers and gastrointestinal (both counted as viscera) were excised and weighed.
2.9 Growth performance
Growth performance and organosomatic indexes were assessed by weight gain (WG), specific growth rate (SGR), specific total large rate (SLTR), condition factor (K), hepatosomatic index (HSI) and viscero-somatic index (VSI). Calculations were carried out using the following formulae: WG = 100 × [Final body weight – Initial body weight]/Initial body weight; SGR = 100 × [ln(Final body weight) – ln(Initial body weight)]/Days of feeding trial; STLR = 100 × [ln(Final total large) – ln(Initial total large)]/Days of feeding trial; K = 100 × [Final body weight (g)/Total length (cm)3]; HSI = 100 × [Liver weight (g)/Body weight (g)]; VSI = 100 × [Visceral weight (g)/Body weight (g)].
2.10 Statistical analysis
Data sets were presented as mean ± standard error (SE). Statistical analysis was carried out using NCSS10 software for Windows. Data were checked for normality and variance homogeneity using Shapiro-Wilks and Levene tests. When assumptions were met, data were analyzed using one-way analysis of variance (ANOVA) followed by a Tukey’s multicomparison test to determine significant differences. If data violated these conditions, a Kruskal-Wallis test was used. Differences between treatments were then determined using a Mann-Whitney U test. Just protease inhibition assay data were analyzed differently through a Student's t-test. Differences are reported as statistically significant when P˂0.05.