Antioxidant and antiproliferative activity of enzymatic hydrolysates from red tilapia (Oreochromis spp.) viscera.

The antioxidant and antiproliferative activity of red tilapia (Oreochromis spp.) viscera hydrolysates (RTVH) was evaluated. For that, the hydrolysates was applied to three cancer cell lines (HepG2, Huh7 and SW480) and the control (CCD-18Co). Finally, the line on which the hydrolysate had the greatest effect (SW480) and the control (CCD-18Co) were subjected to the ApoTox-Glo Triplex Assay to determine apoptosis, toxicity, and cell viability. The result showed that hydrolysate had a dose-dependent cytotoxic effect selective on the three cancer cell lines, compared to the control cells. There is a relationship between the antioxidant capacity of RTVHs and their antiproliferative capacity on cancer cells evaluated, which achieved cell viability by action of RTVH of 34.68 and 41.58 and 25.41 %, to HepG2, Huh7 and SW480, respectively. The action of RTVH on cancer cell line SW480 is not due to the induction of apoptosis but to the rupture of the cell membrane.

Optimization of fractionation with membranes of antioxidant enzymatic hydrolysate of Californian red worm (Eisenia fetida) protein.

Problem: Earthworm is a valuable source of biologically and pharmacologically active compounds, with applications in the treatment of various types of diseases; however, the main application they have been given is in the production of organic fertilizer. One of the alternatives for obtaining bioactive compounds is by means of enzymatic hydrolysis. Aim: This study proposes the optimization of the fractionation of the antioxidant enzymatic hydrolysate from Californian red worm (Eisenia fetida) protein. Methodology: For this purpose, the worms were separated and hydrolyzed using the enzyme Alcalase 2.4L for 4000s. The obtained hydrolysate was fractionated by means of a crossflow tangential ultrafiltration system, with a 3 kDa molecular weight cut-off ceramic membrane. A response surface design of the composite central factorial type was implemented to evaluate the effect of pH, transmembrane pressure, and flow factors on the response variables transmission, volume reduction factor (VRF) and permeate flow resistance. The transmissions focused on the antioxidant peptides, measured by three conventional methods such as TEAC, FRAP, ORAC, also known as TTEAC, TFRAP and TORAC, respectively. The evaluated resistances were the total resistance (Rtotal), fouling resistance (Rfouling), and gel resistance (Rgel). Result: The results showed that the three factors evaluated affect all the response variables either in their linear or quadratic terms or by some interaction. For each response variable, a mathematical model was obtained, with statistical significance and a non-significant lack of adjustment. The models obtained were used for a multi-objective optimization process in which transfers were maximized, and resistances were minimized. The efficiency of the optimum ultrafiltration process was 25 %. Conclusion: The neutral-alkaline pH is ideal for the ultrafiltration process of bioactive peptides, as it is where the highest transmissions of peptides with antioxidative capacity are found. Under optimal conditions, the 3 kDa membrane permeate was found to exhibit higher antioxidant capacity than the retentate and feed. Based on this, the fraction of less than 3 kDa emerges as a potential multifunctional ingredient, thanks to its antioxidant properties.

Kinetic-conceptual model of the hydrolysis of bovine plasma proteins - ALCALASE® 2.9L: The role of inhibition by product.

In the present work, the inhibitory effect of the peptide fractions, obtained through enzymatic hydrolysis of bovine plasma was evaluated, on the enzyme used in the reaction (Alcalase 2.4 L). In this sense, Ultra-filtered peptide fractions of different molecular sizes (A: Fraction>10; B: Fraction 10-3 kDa; and C: Fraction <3 kDa), were used to verify the impact on the total hydrolysis rate. The Fractions between 3 and 10 kDa were refined to fit a conceptual kinetic model which considers inhibition by product and substrate. Additionally, the inactivation of the enzyme through the reaction time was evaluated and its effects incorporated into the model. It was shown that some peptides released in the successive stages of the reaction can in turn inhibit the activity of the hydrolyzing enzyme. The model evaluated suggests a time-varying expression of inhibition parameters as a function of the initial substrate concentration in the reaction. This is based on the kinetic changes of the product profiles for each reaction time in the evaluated operating conditions (S0 variable). A greater inhibitory effect due to the products is evidenced when the reaction occurs with a higher load of the initial substrate (S0 = 20 g/L).

Quality of red tilapia viscera oil (Oreochromis sp.) as a function of extraction methods.

This study aimed to propose a simple and efficient heating-freezing method for oil recovery from red tilapia (Oreochromis sp.) viscera, suitable for industrial application and that does not affect its composition. Three methodologies for oil extraction were studied: a) direct heating (69 °C and 29 min) of samples followed by separation of the oil by decantation, b) direct heating with subsequent freezing and c) solvent extraction assisted by ultrasound. For the oil obtained by each methodology, the following factors were determined: peroxide and iodine values, oxidative stability index, yield percentages and fatty acid profile and, to evaluate the changes thereof, a thermal analysis by differential scanning calorimetry was performed. An oil extracted by centrifugation from fresh viscera was used as control. Results showed yields of 92,126%, 60,99% and 55,36% for the oil obtained by heating and freezing, heating and decanting and solvent extraction, respectively, the other evaluated parameters were similar among each other. The content of PUFA was not affected by heating when compared to the control oil, although a decrease was observed in the solvent extracted oil. This behavior was corroborated with the thermal analysis, which showed that the higher PUFA content, the lower the melting temperatures of the oils and the energy required for phase change. A principal component analysis allowed determining that while there are no differences in the abundance of fatty acids C20:1, 14:0, 18:0, 16:1 and C16:0, there are differences for fatty acids C18:1 and C18:2 depending on the method of extraction used in the oil obtention. The results of this study show that the heating-freezing extraction method is a good alternative for acquiring value-added products and facilitates their implementation in rural areas. Furthermore, allows obtaining a product with high content of polyunsaturated fatty acids (at least a third of the total content).

Scaling up the Enzymatic Hydrolysis of Bovine Plasma Protein to Produce an Antioxidant from a Biological Source.

BACKGROUND: In modern society, there is a tendency to consume products with natural origins and minimum chemical additives. This has encouraged the replacement of synthetic antioxidants for the ones obtained from natural sources, such as the antioxidants acquired from enzymatic protein hydrolysates. OBJECTIVE: In this study, the process of enzymatic hydrolysis of proteins from bovine plasma, which produces hydrolysates with an Antioxidant Capacity (AC), was scaled up from 1 to 5 L. METHODS: An experimental design was developed in 1 L to evaluate the effect of the Substrate concentration (So) on the time needed to reach a Degree of Hydrolysis (DH) of 20% as well as the AC. RESULTS: The best conditions in the 1 L reactor controlled by a Titrando 842 were transferred to 5L in a BioFlo310 reactor. These conditions were achieved at a ratio of 80g/L of the substrate and 0.89 AU of Alcalase 2.4L/g of the substrate in order to obtain a level of 16.36 ± 0.21min of the 20% of DH and antioxidant capacity of 58.98 ± 1.80%. CONCLUSION: The results showed that DH depends significantly on So, while the antioxidant capacity only depends on the DH. Additionally, the dimensional analysis using Re as a scaling criterion allowed us to obtain the same results in the model (1 L) and the prototype (5 L).

Optimization of the Red Tilapia (Oreochromis spp.) Viscera Hydrolysis for Obtaining Iron-Binding Peptides and Evaluation of In Vitro Iron Bioavailability.

Iron deficiencies continue to cause significant health problems in vulnerable populations. A good strategy to combat mineral deficiency includes fortification with iron-binding peptides. This research aims to determine the optimal conditions to hydrolyze red tilapia viscera (RTV) using Alcalase 2.4 L and recovery of iron-binding protein hydrolysate. The result showed that under the optimal hydrolysis condition including pH 10, 60 °C, E/S ratio of 0.306 U/g protein, and substrate concentration of 8 g protein/L, the obtained hydrolysate with 42.5% degree of hydrolysis (RTVH-B), displayed the maximal iron-binding capacity of 67.1 ± 1.9%. Peptide fractionation was performed using ultrafiltration and the <1 kDa fraction (FRTVH-V) expressed the highest iron-binding capacity of 95.8 ± 1.5%. Iron content of RTVH-B and its fraction was assessed, whereas iron uptake was measured indirectly as ferritin synthesis in a Caco-2 cell model and the result showed that bioavailability of bound minerals from protein complexes was significantly higher (p < 0.05) than iron salt in its free form, increased 4.7 times for the Fe2+-RTVH-B complex. This research suggests a potential application of RTVH-B as dietary supplements to improve iron absorption.

Effects of Enzymatic Hydrolysis Conditions on the Antioxidant Activity of Red Tilapia (Oreochromis spp.) Viscera Hydrolysates.

BACKGROUND: Fish is an essential source of nutrients for human nutrition due to the composition of proteins, vitamins, and minerals, among other nutrients. Enzymatic hydrolysis represents an alternative for the use of by-products of the aquaculture industry. OBJECTIVE: We propose to evaluate the effect of stirring speed, temperature, and initial protein concentration on the degree of hydrolysis of proteins and antioxidant activity of red tilapia (Oreochromis spp.) viscera hydrolysates. METHODS: The effect of stirring speed, temperature, and initial protein concentration on the degree of hydrolysis of proteins and antioxidant activity was evaluated using an experimental design that was adjusted to a polynomial equation. The hydrolysate was fractioned to determine the antioxidant activity of the fractions, and functional properties were also measured. RESULTS: Stirring speed and protein concentration presented a statistically significant effect (p <0.05) on all the response variables. However, the temperature did not present a statistically significant effect on the degree of hydrolysis. DISCUSSION: The best conditions of hydrolysis were stirring speed of 51.44 rpm, a temperature of 59.15°C, and the protein concentration of 10 g L-1. The solubility of the hydrolysate protein was high at different pH, and the hydrolysate fraction with the highest antioxidant activity has a molecular weight <1 kDa. CONCLUSION: The degree of hydrolysis and the biological activity of red tilapia viscera hydrolysates (Oreochromis spp.) are affected by temperature, substrate concentration, and stirring speed. The optimal conditions of hydrolysis allowed to obtain a hydrolysate with antioxidant activity are due to the peptides with low molecular weight.

In-vitro antioxidant capacity and cytoprotective/cytotoxic effects upon Caco-2 cells of red tilapia (Oreochromis spp.) viscera hydrolysates.

The antioxidant capacity of red tilapia viscera hydrolysates (RTVH) with different degrees of hydrolysis (DH) as well as their ultrafiltration membrane fractions, were analyzed using different chemical assays. Their protective effects against oxidative stress were evaluated using H2O2-stressed human intestinal differentiated Caco-2. The highest antioxidant capacity was obtained with a DH of 42.5% (RTVH-A) and its <1 kDa fraction (FRTVH-V). RTVH-A and FRTVH-V did not show cytotoxic effects at a concentration of ≤0.5 mg/mL,prevented the decrease in cell viability, and suppressed intracellular reactive oxygen species (ROS) accumulation induced by H2O2. However, pretreatment with RTVH-A after adding H2O2, showed a greater decrease in glutathione levels. Moreover, FRTVH-V allowed for a recovery close to that of control levels of cell proportions in the G1 and G2/M cell cycle phases; and a decrease in the cell proportion in late apoptosis. These results suggest that RTVH-A and FRTVH-V can be beneficial ingredients with antioxidant properties and can have protective effects against ROS-mediated intestinal injuries.