Orientador: Prof. Dr. Ricardo Pereira Ribeiro

Data da Defesa: 01/07/2022



INTRODUCTION AND OBJECTIVES - Fish consumption is recommended for its nutritional diversity. Despite this, the fish is highly perishable, hindering processing and resulting in greater losses of raw material. Fish protein hydrolysates (FPHs) can be an alternative to reduce waste and assign value to fish by-products, featuring various bioactive activities and making essential amino acids more available in peptides. These hydrolysates are being researched in several applications as anti-inflammatory, anticancer, antimicrobial, antioxidant and enzymatic inhibitors. These bioactivities can be diversified according to hydrolysis conditions, as well as fish species and their part used as substrate. These conditions combined with adequate peptide fractionation and isolation can also potentiate some specific bioactivity. Due to the great potential of FPHs, recent work has sought to optimize their acquisition through new technologies for fish treatments, such as high-pressure processing, microwave, ultrasound and thermal treatments to achieve a better protein cleavage with lower losses. The objectives of this work were: Chapter 1 - To carry out a literature review on the important aspects in the production of FPHs and their peptides, the most explored bioactive properties and applied methods to obtain larger bioactivities and to ensure viable applications; Chapter 2 - Selecting the best enzymatic hydrolysis conditions in the production of FPHs obtained from Nile Tilapia residues to relate it to its bioactive ability to inhibit the enzyme acetylcholinesterase (AChE) as well as evaluate changes in the protein structure responsible for this property; Chapter 3 - Investigate whether the substrate pretreatments, by thermal heating by sterilization and homogenization by Ultra-turrax, could affect the antioxidant and functional properties of FPHs after enzymatic hydrolysis and, analyze the technological properties of a real food system, salad dressing, when incorporated with FPHs.
METHODS - For the review article, a survey was conducted to evaluate the publications related to FPH (production, bioactive activities, encapsulation methods, technologies applied to substrates) published from 1940 to 2022, in which only peer-reviewed articles published in journals from January 2015 to 2022 were included. For the experimental article described in Chapter 2, the byproducts of Nile Tilapia were first homogenized: viscera, carcass, skin and bones and evaluated their centesimal composition. Then the by-products were solubilized in distilled water (1:2, p:v), adjusted pH (7,2-8,8), added the alcalase enzyme (0,53-1,9, [E:S] % v/p), adjusted to temperature (42-58; °C) and the solution kept under agitation by 120 min, according to the experimental planning (DCCR, with complete planning 23, 6 axial trials and 3 repetitions at the central point, totaling 17 trials), in which, the response variables were the hydrolysis yield, hydrolysis degree (DH) and AchE inhibition (20, 30, 50 mg.mL-1; %). To evaluate the relationship between the FPHs characteristics when obtained under different experimental conditions, Principal Component Analysis (PCA) was performed. The selected FPHs were characterized by Fourier transform spectroscopy (FTIR) and reverse phase HPLC amino acid profile analysis. The AChE activity and inhibition kinetics assay was performed in three FPH concentrations (20, 30 and 50 mg.mL-1) using electric eel (Electrophorus electricus) as a source of enzyme (expressed in enzymatic activity compared to control). Finally, a molecular Docking assay was performed to evaluate a possible interaction site of the FPHs responsible for enzyme inhibition of AChE. For the experimental article described in Chapter 3, were homogenized Nile Tilapia muscles submitted to three pretreatments: Control (C, without treatment before hydrolysis), Sterilization (EST, thermal treatment in autoclave at 120 ºC for 15 min) and Ulta-turrax (UT, ultra-turrax homogenization at 20,000 rpm for 15 min). After pretreatment the substrates were submitted to enzymatic hydrolysis. For this, the samples were solubilized in distilled water (1:2 w/v), the pH was adjusted to 7.5 with NaOH 1 M or 0.1 M HCl, added alcalase enzyme (0.8 % v/p [E:S]) and, the temperature adjusted to 55 ºC. Hydrolysis was maintained by agitation for 120 min. FPHs were characterized by degree of hydrolysis (DH), yield, free amino acids, antioxidant ability (FRAP, DPPH and ABTS analyses), acetylconilesterase inhibition activity (AChE), solubility, emulsifying ability, Fourier transform spectroscopy (FTIR), cytotoxicity and genotoxicity. FPHs were applied in salad dressing to evaluate its action as an emulsifier and antioxidant for 0, 15 and 30 days. The analyses evaluated in the sauce were rheological behavior, texture analysis, color and oxidative stability.
RESULTS, DISCUSSIONS AND CONCLUSIONS - From the review of the literature, carried out in the review article (Chapter 1), it can be verified that the bioactive properties most found in FPHs were those with antioxidant, antimicrobial, anticancer and antihypertensive properties. These bioactivities are dependent on the conditions of hydrolysis, fish species and, fractionation and isolation of specific peptides. New technologies for treating by-products can reduce process losses and achieve better results by cleaving proteins. On the other hand, encapsulation and film application techniques can increase bioactivity, bioavailability, and control release when applied to food, resulting in improved health. In Chapter 2, hydrolysis adjusted to 55 °C, pH 7.5 and enzyme concentration of 0.8% (Enzyme:Substrate) was selected by Principal Component Analysis (PCA) because it has greater inhibition potential than other experiments. Molecular characteristics have shown that higher temperatures possibly result in wider amide A bands. The results of enzyme kinetic inhibition of acetylcholinesterase (AChE) demonstrated mixed type inhibition behavior of FPH. The amino acid profile was similar for all the FPHs evaluated, however, the FPH with higher Ache inhibition showed a higher amount of total and essential amino acids. In addition, according to the Docking Molecular analysis, it has been observed that arginine is the most likely amino acid to bind to Ache, demonstrating that basic amino acids can be a key factor for this bioactivity. In Chapter 3, an extensive characterization of the functional and bioactive properties of FPHs was performed, comprising antioxidant activity (DPPH, ABTS and FRAP), acetylcholinesterase inhibition and emulsifying properties. The degree of hydrolysis (DH) obtained was 37.9, 37.66 and 40.55 % for the Control (C), EST and UT samples, respectively. Treatment with UT resulted in a sample with less radical removal ability. Ache inhibition was evaluated at three concentrations (15, 45 and 60 mg.mL-1) demonstrating to be a potential property of FPH. The cytotoxic assays in Allium cepa L. showed that toxicity to FPHs is not expected. As proof of concept, the FPHs were used as an emulsifying/antioxidant agent to prepare a salad dressing. The emulsifying activity index (EAI) and the emulsifying stability index (ESI) of FPH indicated better emulsifying capacity and stability in basic pH, probably due to the hydrophobic character of proteins. The FPH provided an increase in protein content, pseudoplastic behavior, characteristic color and texture. In addition, the FPHs helped in the oxidative stability of salad dressing, demonstrating potential application in emulsified foods, acting in the elimination of radicals generated in lipid oxidation. Finally, in general, we can conclude that FPHs have AChE inhibition properties, and this potential may be related to the binding to basic amino acids, mainly arginine. We can also verify the antioxidant and emulsifying potential of FPHs and its ability to assist in the oxidative stability of emulsified foods such as salad dressing.
Key-words: Fish protein hydrolysates (FPHs); Ache inhibition; pretreatment; antioxidant property; emulsifying property.


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