Os resultados obtidos nesta Tese estão apresentados em forma de Artigo Científico submetido em periódico de impacto na área de Ciência e Tecnologia de Alimentos e Patente.
Artigo: CHELATING AND ANTIOXIDANT CAPACITY OF PEPTIDES FROM
CHICKEN COMBS AND WATTLES.
(Submetido ao Periódico Journal of Functional Food. Fator de impacto: 3,973. Qualis: A1).
Patente: PROCESSO DE OBTENÇÃO DE PEPTÍDEOS BIOATIVOS DERIVADOS
DE CRISTAS E BARBELAS DE FRANGO.
Artigo: CHELATING AND ANTIOXIDANT CAPACITY OF PEPTIDES FROM
CHICKEN COMBS AND WATTLES
CHELATING AND ANTIOXIDANT CAPACITY OF PEPTIDES FROM CHICKEN COMBS AND WATTLES
Taliana Kênia Alencar Bezerraa*, Ana Maria Barbosa Lima Sousaa, Lorena
Lucena de Medeirosa, Marcelo Antônio Morganob, Maria Teresa Bertoldo
Pachecob, Marta Suely Madrugaa
a Post-Graduate program in Food Science and Technology, Department of Food Engineering, Technology Centre, Federal University of Paraiba, 58051-900, Joao Pessoa, Paraiba, Brazil.
b Institute of Food Technology (ITAL), Applied Chemistry and Nutrition Center, 13073-001 Campinas, São Paulo, Brazil.
* Author to whom correspondence should be addressed; E-mail:
taliana.kenia@hotmail.com; Tel.: +55-83-3216-7576; Fax: +55-83-3216-7269.
Abstract
The objectives of this study were to establish the best conditions for obtaining hydrolysates that contain bioactive peptides with mineral chelating and antioxidant properties. The raw material was characterized, and both the comb and wattle showed high protein concentrations (61 a 83% b.s.) and high proportions of collagen (47 a 51% b.s.); consequently they are good potential sources of protein hydrolysates. Five proteolytic enzymes were tested on extracts of comb and wattle mixtures, and Alcalase
presented the best hydrolytic performance (DH 19%); showing that in an enzyme to substrate (E:S) ratio of 5% and 240 minutes of hydrolysis. Hydrolysates with the > and < degrees of hydrolysis were evaluated, and the hydrolysate of the comb and wattle mixture obtained using the optimal process showed excellent Fe2+ chelating (>94%) and antioxidant capacities. This optimized hydrolysate reduced Fe3+ (>95 mg/100g equivalente trolox) and inhibited ABTS● (>46%) and DPPH● (>41%) radicals.
Keywords: protein hydrolysate, free radicals, chicken by-products, enzymatic process,
bioactive compounds.
1 Introduction
The increased production of broiler chickens has resulted in an increase in the quantity of by-products generated during the slaughter process, as only the carcass has commercial value. The full exploitation of these by-products with innovative processing and industrialization technologies is of great economic importance since it adds value to the entire production chain (Lafarga & Hayes, 2014; Martínez-alvarez, Chamorro, & Brenes, 2015).
By-products derived from the slaughter of chickens, including the head, skin, feathers, comb, wattle, bone, meat scraps, blood, fatty tissues, feet and internal organs, may account for 37% of the total live weight of the animal (Toldrá, Aristoy, Mora, & Reig, 2012). The comb and wattle are not exploited to their full potential. Though these by-products are an excellent source of collagenous protein (61% dry basis), they have not been properly explored and exploited (Rosa, Hoelzel, Viera, Barreto, & Beirão, 2008).
Currently, the food and pharmaceutical industries express great interest in the research and application of various transformation methods to exploit the by-products of animal slaughter. The processes used to extract and/or obtain a final product with a specific technological or biological potential differ according to the chemical composition of the by-products (Mora & Toldrá, 2012).
One of the transformation methods applied to the by-products of animal slaughter involves the protease-mediated hydrolysis of proteins. This process results in the release of peptides with different molecular weights and specific amino acid sequences. These features give peptides diverse bioactive and technological potential after their release from the protein chain (Mora, Reig, & Toldrá, 2014).
Peptides obtained from protein hydrolysates, especially those acquired from collagen hydrolysate, have received special attention from researchers due to their inherent bioactivities. Previous studies have reported that these products, which result from the breakdown of collagen from various sources, may have mineral chelating, antioxidant, antihypertensive, antithrombotic, antiulcerative, healing and osteoprotective properties as well as properties that improve the absorption/bioavailability of minerals (Choi, Sabikhi, Hassan, & Anand, 2012; Gómez-Guillén, Giménez, López-caballero, & Montero, 2011; Lee et al., 2013).
Bioactive peptides with antioxidant activities are widely researched due to their actions in combating diseases related to oxidative stress, such as cancer, diabetes, atherosclerosis, neurodegenerative diseases, inflammatory diseases and cell aging (Lee et al., 2013).
Oxidation occurs when some molecules lose electrons via electron transfer to another molecule, a process that generates free radicals. Antioxidant peptides have the
ability to stop chain reactions, such as lipid peroxidation, by donating a pair of electrons to free radicals (Duan et al., 2014).
Several studies have been carried out to obtain and isolate antioxidant peptides from different protein matrices, such as duck skin, ray skin, fish skin, viscera and others (Chi et al., 2015; Lassoued et al., 2015; Lee et al., 2013; Memarpoor-yazdi, Asoodeh, & Chamani, 2012); however, no research was reported on the use of chicken by-products to obtain protein hydrolysates.
In view of these arguments, the objective of this research was to develop a study of enzymatic hydrolysis applied to the extraction of bioactive peptides, with iron chelating capacity and antioxidant activities, from the mixture of ridges and ridges (1: 1 w / w) chicken byproducts.
2 Material and Methods
2.1 Materials
The chicken combs and wattles were acquired from a slaughterhouse located in the state of Paraíba (Brazil). The enzymes Alcalase (Bacillus licheniformis) and Flavourzyme (Aspergillus oryzae) were supplied by Novozymes Latino Americana Ltda. (Paraná, Brazil), and the enzymes Protamex (Bacillus licheniformis and Bacillus
amyloliquefaciens), Brauzyn (Carica papaya) and Colagenase (Clostridium histolyticum) were obtained from Prozyn Biosolutions (São Paulo, Brazil).
2.2 Acquisition of the protein hydrolysate from a 1:1 mixture of chicken combs and wattles
2.2.1 Selection of enzymes for protein hydrolysis of the 1:1 mixture of chicken combs and wattles
Initially, the hydrolytic activities of the enzymes [Alcalase (Bacillus
licheniformis), Flavourzyme (Aspergillus oryzae), Protamex (Bacillus licheniformis and Bacillus amyloliquefaciens), Brauzyn (Carica papaya) and Colagenase (Clostridium histolyticum)] were evaluated. The optimal pH and temperature of each enzyme were
indicated by the manufacturers.
The chicken combs and wattles were ground and homogenized at the ratio of 1:1 (w/w). This mixture was transferred to a beaker in a pre-heated, temperature-controlled water bath. Ultra-pure water was added to the mixture at a ratio of 1:2 (w/v), with constant stirring. During the entire hydrolysis process, the temperature and pH were controlled according to the optimal performance values of each added enzyme. The enzyme to substrate ratio (5%) and the total hydrolysis time (240 minutes) were determined according to the maximum levels to be used in the Full Factorial Experimental Plan, which was performed after the enzyme selection.
During hydrolysis, the pH was controlled by the addition of 0.5 mol / L NaOH. After 240 minutes of hydrolysis, the enzyme was inactivated at 90°C for 15 minutes. The hydrolysate was centrifuged (12,000 x g for 30 minutes), and the supernatant was filtered and lyophilized to obtain a hydrolysate powder. The enzyme with the highest hydrolytic activity was determined via the 22 Full Factorial Plan to obtain the best hydrolysis conditions.
2.2.2 Optimization of protease-mediated hydrolysis of extracts from the chicken comb + wattle mixture (1:1)
The conditions of protease-mediated hydrolysis of the chicken combs and wattles were optimized via a 22 Full Factorial Plan, which consisted of 4 factorial points and 3 central points for 7 experiments in total (Table 2). The independent variables investigated were X1 or E:S (enzyme/substrate ratio) and X2 or T (hydrolysis time in minutes). The response function was measured as the percentage of the degree of hydrolysis (% DH).
The following model was used: where Y is the response function predicted by the model, 0 is the average coefficient (or the constant), 1 and 2 are linear coefficients, and 3 is the interaction coefficient. In this model, E:S (enzyme to substrate ratio) and T (hydrolysis time) are the independent variables, and the dependent variable is the degree of hydrolysis. After adjustment to the experimental data, the model (Equation 1) was verified by Analysis of Variance (ANOVA) and the coefficient of determination (R2). The surface response and desirability function were constructed using STATISTICA 5.0 (Statsoft Inc. Corporativo Tulsa, OK, USA) (Statsoft, 2004).
For validation of the model, a new test at the optimum point was performed in triplicate, and the results were compared via t-tests (p ≤ 0.05) with the response function estimated by the model.
The hydrolysates with the highest and lowest degree of hydrolysis were selected and analyzed in terms of their electrophoretic profiles, hydrophobicity, total and free amino acids, mineral iron chelating capacities, and antioxidant capacities. This process
of hydrolysis of extracts from the chicken comb + wattle mixture (1:1) has been patented and has number BR 10 2016 027430 3.
2.3 Analytical Procedures
2.3.1 Chemical characterization of the chicken comb, wattle, and comb + wattle mixture (1:1 w/w)
The chicken combs, wattles, and the comb + wattle mixture (1:1 w/w) were characterized for their moisture, ash, protein and collagen contents according to procedures no. 39.1.03, 39.1.09, 39.1.15 and 990.26, respectively, in the AOAC method (2010). Lipid measurements were obtained using the method of Folch, Lees and Sloane Stanley (1947).
2.3.2 Measure of the degree of hydrolysis (DH)
The degree of hydrolysis of the proteolytic enzymes in the mixed extract of chicken combs and wattles (1:1 w/w) was determined using the equation described by Adler-Nissen (1986):
(Equation 2), where DH (%) is the degree of hydrolysis; B is the NaOH consumption (0.5 moL L-1) in mL; Nb is the normality of NaOH (0.5 moL L-1); 1/α is the average degree of dissociation of the group α-NH2; PM is the protein mass in g; and htot is the total number of peptide bonds in the protein substrate (using a value of 7.6 for meats).
2.3.3 Hydrophobicity profile of the comb + wattle mixture (1:1 w/w) and protein hydrolysates
The chicken comb and wattle mixture (1:1 w/w) and the hydrolysate proteins that resulted in the highest and lowest degrees of hydrolysis of the mixture were diluted in ultra-pure water and homogenized in an Ultra-Turrax tube disperser (Ika, Staufen, Germany) for 10 minutes at 18,000 rpm. The solution was then placed in an ultrasonic bath for 10 minutes (Unique, 1400, São Paulo, Brazil) and centrifuged at 2060 x g for 10 minutes. The samples were filtered using filter paper and transferred to a syringe containing a filter with pores measuring 0.45 μm in diameter.
The hydrophobicity profile was separated using a Nova-Pak C18 column (4.6 m x β50 mm, 4 μm particle size, cartridge, Waters, Ireland) connected in a high efficiency liquid chromatograph (Varian, Waters 2690, California, USA). The injection volume of the soluble extract (0.2 g / mL) was β0 μL. The mobile phase consisted of eluent A (1% trifluoroacetic acid in ultra-pure water) and eluent B (1% trifluoroacetic acid in acetonitrile). A linear gradient of eluents A and B was applied for 60 minutes at a flow rate of 1 mL/minute. Detection was performed at 218 nm.
2.3.4 Electrophoresis of chicken combs, wattles, the comb + wattle mixture (1:1 w/w), and the protein hydrolysates
The profiles of the protein and peptide fractions were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate the higher molecular weight bands (225 kDa to 38 kDa) (Laemmli, 1970) and by Tricine-SDS- PAGE to separate the lower molecular weight bands (38 kDa to 3.5 kDa) (Schagger &
Joagow, 1987). The samples were diluted in reducing buffer (0.5 mol / L Tris-HCl, pH 6.8, 10% SDS, 10% glycerol, 5% -mercaptoethanol and 0.1% Coomassie Blue G250), homogenized using a vortexer and sonicated for 10 minutes. Soon after, they were heated to 90 °C for 5 minutes in a dry bath (Loccus, São Paulo, Brazil).
For SDS-PAGE, a stacking gel was prepared at a concentration of 3.5% polyacrylamide in 0.5 mol / L Tris-HCl buffer, pH 6.8 and 1% SDS. The separation gel was set up using a gradient of 7.5 to 17.5% polyacrylamide in 3 mol / L Tris-HCl buffer, pH 8.8 and 1% SDS. The run was performed under constant amperage (25 mA). At the end of the run, the gel was removed from the plate, fixed in 12.5% TCA for one hour, and stained with Coomassie brilliant blue R250. Removal of the excess dye was performed with the aid of a discoloration solution of methanol, acetic acid and water (1:3.5:8 v/v/v). The molecular weights of the protein fractions were compared using molecular weight markers (GE Healthcare Life Sciences, Piscataway, NJ, USA).
For Tricine-SDS-PAGE, polyacrylamide gels of different concentrations were used: separation gel (16% T and 3% C), spacer gel (10% T and 3% C) and stacking gel (4% T and 3% C). After the runs, the gel was fixed for 1 h in methanol, acetic acid and water (5:1:4 v/v/v), stained with Coomassie brilliant Blue G250 (0.025% Coomassie Blue in 10% acetic acid) for 48 hours and discolored in 10% acetic acid. The molecular weights of the low-weight fractions were compared using molecular weight markers (GE Healthcare Life Sciences, New Jersey, USA).
2.3.5 Profile of total and free amino acids of the chicken comb + wattle mixture (1:1 w/w) and the protein hydrolysates
The total and free amino acids were hydrolyzed, extracted and derivatized in a pre-column with phenylisothiocyanate (PITC) according to the methods proposed by White, Hart and Fry (1986) and Hagen, Frost and Augustin (1989), respectively. Separation of the phenylthiocarbamyl amino acid derivatives (PTC-aa) was performed on a C18 reverse-phase (PICO-TAG, 3.9 x 150 mm) high efficiency liquid chromatograph (Varian, Waters 2690, California, USA). The mobile phases employed consisted of an acetate buffer of pH 6.4 and a solution of 40% acetonitrile. Sample injection was performed manually (β0 μL), and detection occurred at 254 nm. Separation of the amino acids was performed at a constant flow rate of 1 mL / minute at 35 °C. The chromatographic run time was 45 minutes. Quantification was performed using calibration curves of the separated and identified amino acids. The results were expressed as g of amino acid per 100 g of sample.
2.3.6 Minerals profile of the chicken comb + wattle mixture (1:1 w/w) and the protein hydrolysates by ICP OES
The samples were previously treated using the dry digestion method (AOAC, 2012) by the incineration in a muffle furnace at 450 °C until formation of ash-free black dots. The ashes obtained were solubilized in 5% hydrochloric acid solution (v / v) and filtered on quantitative filter paper. Determination and quantification of the mineral profile (Fe, Ca, P, Zn, K, Mg, Mn, Na) was performed using an inductively coupled plasma optical emission spectrometry (ICP OES) (Agilent Technologies, 5100 VDV,
Tokyo, Japan) equipped with a source of radiofrequency generator of 40 MHz (1200 W), a sequential optical detector, a peristaltic pump (12 rpm), a spray chamber and nebulizer sea spray (flow rate 0.70 L/min). For the system, liquid argon (12 L / min) with a minimum purity of 99.996% (Air Liquide, Brazil) was used as plasma gas, and the radial view of ICP OES. The quantification was obtained by calibration curve for each mineral detected, in ranges of 0.001 to 61 mg / 100 mL.
2.3.7 Fe2+ chelating capacity of the chicken comb + wattle mixture (1:1 w/w) and the protein hydrolysates
The Fe2+ chelating capacity was evaluated based on interruption of the formation of the F2+ - Ferrozine complex at 562 nm in a UV-VIS spectrophotometer (Quimis, Q798U, São Paulo, Brazil) according to the method described by Stookey (1970). The hydrolysate soluble extracts were homogenized with FeCl2 at 2 mmol / L and subsequently read in a spectrophotometer. After the first absorbance reading, a Ferrozine solution of 5 mmol / L was added, and the samples was shaken vigorously for 1 minute. For formation of the complex, the sample was left to rest for 10 minutes, and a new absorbance reading was then performed.
The percent inhibition of the ion complex formation (F2+-Ferrozine) was evaluated by a decrease in the color intensity, as greater inhibition indicates greater competition among peptides to form stable complexes with Fe2+ (thereby immobilizing Fe2+). EDTA was used as the chelating standard. The result was expressed as the percent inhibition of the ion complex.
2.3.8 Ferric reducing antioxidant power (FRAP) of the chicken comb + wattle mixture (1:1 w/w) and the protein hydrolysates
The ferric reducing capacity was evaluated using the Ferric Reducing Antioxidant Power (FRAP) method described by Benzie and Strain (1999). The FRAP reagent was prepared by mixing acetate buffer (300 mmol / L - pH 3.6) with a solution of 10 mmol / L TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mmol / L HCl and 20 mmol / L FeCl3at 10:1:1 (v:v:v). A 100 μL aliquot was added to γ.4 mL of the FRAP reagent.
After homogenization, the triplicate assays were maintained at 37 °C for 30 minutes. According to the antioxidant potential, the ability of the hydrolysates to reduce iron (Fe3+) to the ferrous form (Fe2+) was verified at 593 nm in a UV-VIS spectrophotometer (Quimis, Q798U, São Paulo, Brazil); the device was zeroed with a blank containing solvent and the FRAP reagent. Based on the calibration curve prepared with different concentrations of Trolox (1000 μmol / L), the results were expressed as the equivalent of mg of Trolox / 100 g of sample according to the antioxidant potential of the hydrolysates.
2.3.9 Sequestering activity for the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH●) of the chicken comb + wattle mixture (1:1 w/w) and protein hydrolysates
The ability of the protein hydrolysates to sequester the DPPH● radical was determined according to the method described by Brand-Williams, Cuvelier and Berset (1995). A γ50 μL aliquot was added to 3.15 mL of the DPPH● (%) radical in methanol. After homogenization, the zero time reading of the blank assay, containing the free radical and solvent, was performed, and all assays were performed in triplicate and
maintained at 25 °C for 30 minutes. Then, the radical elimination activity according to the antioxidant capacity of the hydrolysates was verified at 517 nm in a UV-VIS spectrophotometer (Quimis, Q798U, São Paulo, Brazil) in triplicate; the equipment was zeroed with methanol. The antioxidant potential of the samples were expressed as the percent inhibition of the DPPH● radical.
2.3.10 Sequestering activity for the 2,2-azino-bis (3-ethylbeothiazoline)-6-sulphonic acid radical (ABTS●+) of the chicken comb + wattle mixture (1:1 w/w) and the protein hydrolysates
The ability to sequester the ABTS●+ radical was determined according to the method proposed by Re et al. (1999). A 50 μL aliquot was added to 950 μL of the ABTS●+ radical (%) in methanol. After homogenization, the zero-time reading was obtained using a blank solution containing only the free radical and solvent; all assays were performed in triplicate and maintained at 25°C for 6 minutes. Then, the radical elimination activity was verified according to the antioxidant capacity of the hydrolysates at 734 nm in a UV-VIS spectrophotometer (Quimis, Q798U, São Paulo, Brazil); the equipment was zeroed with ethanol. The antioxidant potential of the samples were expressed as the percentage of inhibition of the ABTS●+ radical.
2.4 Statistical Analysis
The data obtained while optimizing the hydrolysis of the chicken comb and wattle mixture (1:1 w/w) were evaluated by analysis of variance (ANOVA) using the Statistica software, version 5.0 (Statsoft, 2004). From these results, the effects of the
studied variables were estimated, and the coefficients of the model for the experimental response were determined using a significance level of 5% (p ≤ 0.05).
The results (hydrophobicity profile, total and free amino acids, iron mineral chelating capacity and antioxidant activities) of the protein hydrolysates obtained from the chicken comb and wattle mixture (1:1 w/w) with the highest and lowest degrees of hydrolysis were analyzed by analysis of variance (ANOVA) using the Statistical Analysis System software, version 11.0 (SAS, 2014). Significance was assigned at 1% and 5%.
3 Results and discussion
3.1 Chemical characterization of the chicken combs, wattles, and comb + wattle mixture (1:1 w/w)
The chicken combs and wattles (Table 1) exhibited elevated concentrations of protein, particularly collagen. The wattle stood out due its total protein content, whereas the comb contained a high collagen content. In the comb, the high proportion of collagen matched the anatomical structure; according to Rosa et al. (2008), the combs are composed of dense connective tissues, cartilage, mucosa and epithelial tissue (Rosa et al., 2008).
The proportion of protein found in the comb, wattle and, consequently, the mixture (61 a 83% b.s.) was superior to that found by Abedinia, Ariffin, Huda and Nafchi (2017) in duck foot (50%). These results indicate that as by-products of chicken slaughter, the comb and wattle have excellent potential for use as a mixture to obtain protein hydrolysates.
The wattle had higher mineral residue contents than the comb. This high mineral level will allow for the enrichment of protein hydrolysates obtained from a mixture of chicken combs and wattles.
3.2 Acquisition of the protein hydrolysate
3.2.1 Selection of enzymes for hydrolysis of extracts of the chicken comb + wattle mixture (1:1 w/w)
For hydrolysis of the extract obtained from the chicken comb and wattle mixture (1:1 w/w), proteolytic enzymes, or proteases, with different specificities were applied to the proteins to be hydrolyzed. These enzymes exhibit distinct specificities that effectively act on different regions of protein chains, allowing for the release of peptides with different amino acid sequences (Lario et al., 2015).
As shown in Figure 1, after the actions of five enzymes on the mixture of combs and wattles (1:1 w/w) were evaluated, the enzyme of microbial origin, Alcalase (Bacillus licheniformis), exhibited a higher degree of hydrolysis compared with the other enzymes tested (Colagenase, Papain, Flavourzyme and Protamex). The hydrolysate processed with Alcalase showed a degree of hydrolysis of 19% under the assay conditions. This value was higher than the 15% for Alcalase-mediated hydrolysis of bovine collagen reported by Zhang, Tian, Zhang and Wang (2013) and the 11% for
Bacillus subtilis protease-mediated hydrolysis of hydrolysates derived from ray skin
reported by Lassoed et al. (2015).