Poultry cruor hydrolysate is a new potential source of hemoglobin: obtaining of active peptides

doi:10.21203/rs.3.rs-2949263/v1

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Poultry cruor hydrolysate is a new potential source of hemoglobin: obtaining of active peptides

https://doi.org/10.21203/rs.3.rs-2949263/v1

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published 22 Jan, 2024

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The hydrolysates of animal proteins from Agri-Resources have been the subject of numerous studies for their potential which makes it possible to produce molecules with high added value thanks to their richness in bioactive peptides obtained after enzymatic hydrolysis. The poultry cruor represents an important co-product from slaughterhouses. The aim of this study is to characterize this new source of peptides to be valorized as feed additives. In this work, the conditions of peptides production were fist studied, and revealed that hydrolysis and discoloration are both optimal at pH 3, and that the optimal initial substrate concentration is at 9% (w/v). The potential of poultry cruor to contain bioactive peptides was then studied in silico by comparing poultry with bovine hemoglobin derived peptides using bioinformatic tools. The blast results showed the presence of high similarities between poultry hemoglobin and bovine hemoglobin sequences with identities of 71.13% and 64.34% for α and β chains respectively. The prediction of cleavage sites of poultry hemoglobin was also carried out using peptidecutter software and compared to bovine hemoglobin peptides. The results the presence of similar peptides of poultry cruor hydrolysates comparing to bovine hemoglobin hydrolysates with generation of many new peptides. Mass spectrometry analysis was carried out to determine of bioactive peptides in poultry cruor hydrolysate based on those defined in previous studies. Results revealed the presence of 28 bioactive peptides with mainly opioid and antibacterial peptides. The antibacterial activity was then inspected in vitro against 6 different strains. Results revealed bacterial growth inhibition with interesting MIC values (10 mg/mL against M.luteus E.coli and S.aureus,1.25 mg/mL against K.rhizophilia and 20 mg/mL against S.entirica and L.innocua). The antioxidant activity was also evaluated using different tests. The β-carotene bleaching inhibition activity revealed a RAA of 79.23 ± 1.4%. The DPPH^•+ trapping assay an antiradical activity of poultry cruor hydrolysate of 829.35 ± 21.12 µmol/mL and 708.85 ± 0.66 µmol/mL at 40 mg/mL and 20 mg/mL of hydrolysate respectively which is greater than BHT at 0.1 mg/mL and neokyotorphin. The ABTS radical scavenging method revealed inhibition percentages are higher than 90% for hydrolysate concentration above 10 mg/mL which are higher than those obtained with BHT at 0.5 mg/mL. and Finally, the Total antioxidant capacity (TAC) assay showed that the studied hydrolysate have a TAC comprised between that of BHT at 0.3 mg/mL and 0.1 mg/mL. Consequently, these important biological activities found in poultry cruor hydrolysate make it a new interesting alternative natural additive in food industry.

Hemoglobin

hydrolysate

poultry

cruor

antibacterial

antioxidant

Poultry cruor is an important co-product of slaughterhouses. The objective of our study is to valorize this co-product as a new potential source of molecules with high added value thanks to their richness in bioactive peptides obtained after enzymatic hydrolysis. This work focuses on the identification and characterization of these new peptides and the demonstration of their antioxidant and antimicrobial activities to investigate their potential to be used as additives in the food industry.

This work focused on the study of poultry cruor peptides to exploit their bioactive potential. For this, the main stages of this study focused on:

Optimization of the hydrolysis process to obtain purified peptides
The search for the possible presence of active peptides by bioinformatics analysis and mass spectrometry
Research of the antibacterial activities of purified peptides
The study of their antioxidant activities

Today, the agri-food industry is gradually turning to the valorisation of its co-products. These are rich in bioactive compounds. Thus, many studies were focused on animal proteins hydrolysates from agricultural resources. Their potential would make it possible to generate products with high added value thanks to their richness in bioactive peptides obtained by enzymatic hydrolysis. Among these co-products we cite those from slaughterhouses and especially animal blood. These co-products are usually transformed into blood meal and used as low-cost fertilizer, animal feed or discharged as effluent [1, 2]. Blood is made up of 40% cruor which contains 90% hemoglobin. This protein widely described as a rich source of bioactive peptides obtained by enzymatic hydrolysis [3–5]. The biological activities characterized to the date from hemoglobin are very varied such as antioxidants [6], opioid [7, 8], antihypertensive [3], immunomodulatory, secretagogue, antimicrobial [9, 10] and antithrombic. Industrial interest in these peptides is strongly growing especially for application in animal and human food fields. More broadly, preparations of active peptides or enriched fractions can be sought as alternatives to synthetic preservatives which may have toxic effects and widely used today both in the food, pharmaceutical and cosmetic industries [11, 12]. In this context, poultry blood from slaughterhouses may represents a rich source of active peptides and a source of high-quality protein, due to its high isoleucine and lysine content [13]. Because of its functional properties and excellent nutritional qualities, poultry blood has been considered co-product which can potentially be used as a raw material for the food industry and animal feed [14]. Moreover, it is an inevitable by-product of the meat industry and its valuation is largely underexploited today.

Some studies have recently focused on poultry blood protein hydrolysates as an inexpensive source of protein and iron. Hence the interest of studying chicken blood as an important alternative as a food additive [15]. Previous studies have already investigated the ability of the east chicken blood corpuscle to produce antioxidant hydrolysates and peptides. this showed that these peptides possess good antioxidant properties which makes them a promising source for formulating functional foods [16].

The aim of this study is to define the optimal conditions to produce bioactive peptides from poultry hemoglobin or cruor by enzymatic hydrolysis, then to characterize its peptide’s population and to look up their eventual biological activities.

2.1 Peptides production from poultry cruor

Poultry hemoglobin or cruor hydrolysis was carried out using 200 mL reactors. The hemoglobin concentration was given by the Drabkin test [17]. The hydrolysis was performed in an acidic medium using H3PO4 (3M) and porcin pepsin (3200 – 4000 units.mg-1 protein) with an enzyme/ protein ratio = 1/50. Pepsin was prepared in 25 mL of sodium acetate-acetic acid buffer (0.1 M, pH 3.5), The pH decreasing was done gradually, passing through pH stages up to 3. The introduction of pepsin was occurred early at pH =5. The hydrolysis was carried out of 24 hours at 23°C. The hydrolysis is stopped by increasing the pH to 8.5 with NaOH (5M) for enzyme denaturation. The obtained products are called ‘whole hydrolysates or coloured hydrolysates. These experiences were carried out in triplicate.

2.2 Peptide’s purification

For further uses the peptides were purified. For this, the whole hydrolysates were centrifuged at 4000 x g for 15 minutes at room temperature to extract nucleic acid in the pellet. Then, Haem fraction was extracted by lowering pH supernatant using phosphoric acid HP₃O₄ (2 M) using a pH range [initial pH - 5 - 4.5 - 4 - 3.5 - 3] for hydrolysate discoloration [5, 18] The samples were then centrifuged at 10,000 x g for 3 min in order to separate the haem contained in the pellet and the discoloured peptides in the supernatant. Samples are then stored at 4°C for further analysis.

2.3 Determination of the degree of hemoglobin hydrolysis

The degree of hydrolysis represents the percentage of the number of cleaved peptide bonds out of the total number of peptide bonds in poultry hemoglobin. The cleavage of the peptide bond is determined by the orthophthaldialdehyde (OPA) method described by church et al. [19].

2.3 Analysis of peptide hydrolysate by UPLC

Peptides profile analysis was carried out on an Ultra Performance Liquid Chromatography UPLC Waters H-Class Bio System using a C18 Uptisphere CS Evolution (85 Å x 2,6 µm). All the samples were diluted to 1% (W/V) and then filtered at 0.2 μm.The volume of injection was 10 µm. The hydrolysate was eluted using two solvents: Eluent A corresponds to a milliQ water with 0.1% (V/V) of trifluoroacetic acid (TFA), Eluent B is a solution of water, acetonitrile and TFA in the proportions (40:60:0.1 V/V/V). The flow rate was 0.6 mL/minute and the solvent gradient is: 95% of A over 3 minutes, (95% - 87%) A over 3 minutes, (87% - 65%) A over 19 minutes than (65% - 5%) A over 4 minutes. Chromatographic analyses were completed with Millenum software using an UV absorbance scan between 200 nm and 390 nm [20, 21].

2.4 Identification of peptides by LC MS / MS

LC MS/MS analyses were performed using a UFLC-XR (Shimadzu) coupled to a QTRAP^(R) 5500 MS/MS triple quadrupole associated with a linear spectrometer ion trap mass (AB Sciex, Foster City, CA, USA) and a Turbo VTM as ion source. Assays were carried out using a 5500 V IonSpray in positive mode of ionization. The nebulizer gas is air and nitrogen broadcast with a flow rate of 30 psi. The Turbo VTM source was used with air as an auxiliary gas at 550°C and 50 psi. All analyses were performed using a C18 column. The samples were filtered through PVDF filters (0.20 μm), and then 5µL of each sample were injected. The mobile phase is composed of solvent A: ultrapure water/formic acid (1000:1, V/V) and solvent B: acetonitrile/ultrapure water/formic acid (600:400:1; V/V/V). The elution program is 95% (V/V) of the solvent A during 5 minutes, from 5% to 65% (V/V) of the solvent B for 23 minutes, then it goes from 65% to 95% of solvent B for 4 minutes to remove the inorganic salts. The flow rate was maintained at 0.6 mL/min. All the data have been acquired using BioAnalyst^TM 1.5.1 (ABSciex, Darmstald, Germany) and Peaks^(R) 7 (Waterloo, Canada).

2.4 Comparison of peptide sequences by bioinformatic approach

For peptides sequences comparison, the software BLAST^®((Basic local Alignment Search Tool) was used. It’s available online at https://blast.ncbi.nlm.nih.gov/Blast.cgi. The algorithm used by blast is the Smith-Waterman algorithm, which allows a quick alignment and a good sensitivity.

2.4 Determination of antibacterial activity

The antimicrobial tests of poultry hemoglobine or cruor hydrolysates were performed according to the method of Adje et al. [18]. Pre-cultures were carried out by inoculation of 50 µL of bacteria into 5 ml of Müller-Hinton medium (MH). Strains were incubated for 18-24 hours at 37°C. Then, absorbance at 600 nm was determined using MH medium as control in order to realize a serial dilution using Tryptone Sel (TS) or phosphate buffer, depending on the type of bacteria, to obtain a final Colony Forming Unit of 10⁶CFU/mL. Agar plates were inoculated using the flooding method. For that, 5 µL of hydrolysates were deposited by spotting on the agar. Plates were incubated at 37°C for 24 hours.

2.5 Minimal inhibitory concentration (MIC)

The minimal inhibitory concentration (MIC) was determined by bacterial growth inhibition in 96-well microplate [22]. For that, bacterial foaming colony unit was adjusted to 10⁶ CFU/mL. Then, 50 µL were disturbed in the microplate’s wells containing 50 µL of hydrolysates and 50 µL of MH broth. MIC corresponds to lower concentration of hydrolysate that inhibit completely the strain growth after incubation at 37°C during 24 h.

2.6 Antioxidant assay using the β-carotene bleaching method

β-carotene bleaching was tested as described by Koleva et al. [23]. A stock solution containing β-carotene (4 mg), linoleic acid (100 μL) Tween 40 (800 μL) was prepared. Then mixture was then dissolved into 4 mL of chloroform which was completely evaporated under vacuum in a Rotavapor at 40°C. Then, 400 mL of distilled water was added. The obtained solution was vigorously stirred in order to obtain a β-carotene / linoleic acid emulsion. 500 μL of aqueous hydrolysate solution (4 mg/mL) or butylated hydroxytoluene (BHT) at 0.5 mg/ml were added to 5 mL of the emulsion. The absorbance of the tested samples was measured at 470 nm before and after heat for 2 hours at 50°C. The BHT was used as a positive control. The tests were carried out in triplicate. The relative antioxidant activity of the extracts (AAR) was calculated as follows:

Where t₀ refer to the absorbances of the test sample and control measured before heat, and t_2h to the absorbances of the test sample and control measured after 2 hours of heating.

2.8 Trapping of DPPH^• radicals

The ability of hydrolysates to capture the DPPH^•+ (2,2-diphenyl-1-picrylhydrazyl) radical was determined according to Molyneux method, (Molyneux., 2004). For this, an ethanolic stock solution of DPPH^•+ (0.02% W/W) was prepared between 12 to 16 hours before use. The tested hydrolysates were prepared at 40 mg/mL^-1 and then diluted in water (4% V/V). Then, 500 μL of the tested sample was mixed with 250 μL of ethanol (99.5%), then 375 μL of the DPPH solution are added. The obtained solution was mixed by vortex. Mixture’s absorbance at 517 nm was measured after 1 hour of incubation in obscurity at 30°C, against pure ethanol which is used as control. The radical scavenging activity (RSA) is given by the following formula:

Absorbance control is the absorbance of pure ethanol and Absorbance blank is the absorbance of the mixture without DPPH.

2.9 ABTS^● Radical Scavenging Assay

ABTS^● radical cation discoloration assay was used to analyze the antioxidant activity of poultry cruor hydrolysates [25]. ABTS (2,20 -azinobis (3-ethylbenzothiazoline-6-sulphonic acid)) radical cation (ABTS^•) was produced by reacting 7 mM of ABTS solution with 2.45 mM of potassium persulphate. The mixture was incubated at room temperature in the dark for 12–16 h, then was then diluted with phosphate buffer (50 mM, pH 7.4) to reach an absorbance at 734 nm lower than 1. The hydrolysate was previously prepared different concentrations: 2.5, 5, 10, 20 and 40 mg/mL in the DMSO. The mixture was incubated at 30°C for 10 minutes, and absorbance was then read at 734 nm. Antioxidant activity was calculated using the following formula:

Where the initial absorbance refers to the absorbance of the buffered ABTS solution and the final absorbance refers to the absorbance of ABTS solution mixed with the tested samples.

2.10 Total Antioxidant Capacity (TAC)

The total antioxidant capacity (TAC) of poultry cruor hydrolysates was determined according to the method described by Prieto et al., [26]. Hydrolysates were dissolved at 40 mg/mL in distilled water. Then 300 μL was mixed with 3 mL of a reagent solution containing sulfuric acid (0.6 M), sodium phosphate (28 mM) and ammonium molybdate (4 mM). The mixture was incubated for 90 minutes at 95°C. After cooling, the solutions absorbance was measured 695 nm against the blank containing distilled water incubated under the same conditions. A Trolox solution at different concentrations, ranging from 50 to 3000 μmol/L, was used for calibration.

The antioxidant activity is expressed as an equivalent concentration of Trolox (μmol/L) using the equation given by the calibration line:

Where A₆₉₅is the absorbance at 695 nm, C_troloxis the equivalent concentration in Trolox (μmol/L), and a and b are the origin and slope of the calibration line respectively

3.1 Production of peptides from poultry cruor or hemoglobin

The aim of this study is to define the optimal conditions of poultry cruor or hemoglobin hydrolysis and hydrolysate purification. Cruor or hemoglobin hydrolysis was carried out at pH 3 and 23°C during 24 hours as used in similar studies with bovine cruor [6, 27]. The obtained whole hydrolysates were analysed using UPLC. Cruor or hemoglobin chromatograms are almost similar. Chromatograms showed that in current conditions, hemoglobin was totally denatured with a complete disappearance of α and β chains with a hydrolysis degree (DH) of 11.8% (Figure 1). These profiles showed that final peptides given by hemoglobin subunit hydrolysis are generally hydrophilic with small molecular weight. Haem was however still present which means that purification steps should be realized. The purification of peptides consisted initially of removing the nucleic acids. The experiments showed that for a pH range from 8 to 5 a better separation between the nucleic phase and the hydrolysate was obtained at pH 8 where the nucleic acids are recovered in the pellet while retaining the haem and peptides in the supernatant as shown in figure 1. Then, in a second time peptides and haem fractions were separated by discoloration. The discoloration of the hydrolysates was carried out by precipitation of haem through a gradual lowering of the pH ranging from 5 to 3. The discoloration is observed in UPLC at a wavelength of 392 nm [28]. The chromatograms show the presence of haem residues at pH between 5 and 3.5 and its complete disappearance pH 3 which corresponds to maximum discoloration (100%). The precipitation of the haem by lowering the pH can be explained by bonds breaking between the haem and the peptides of the α and β chains since it stabilizes its hydrophobic bonds by non-polar residues. In fact, lowering the pH causes a progressive breaking of the haem - peptide bonds. This dissociation is all the easier when the peptides have low molecular weight [29, 30].

3.2 Optimization of initial hemoglobin concentration

A study of the increase in the initial hemoglobin concentration was then carried out. This aims to reduce the processing steps of the final product and therefore increase the yield of active peptides produced as well as the reduction in the cost of the process, which would influence the price of the final product. Indeed, the concentration of the substrate is a parameter that influences the hydrolysis of hemoglobin [31] .To optimize the initial concentration of 1% 2% 5% and 8% and 9% (w/v) of hemoglobin were tested. The experiments carried out have shown that beyond an initial substrate concentration of 5% a significant increase in the viscosity of the medium was detected during the increase in pH. This step is necessary for stopping the hydrolysis of cruor. In fact, the increase of viscositycauses an important problem of homogeneity in the reaction medium. This is probably due to the richness of the cruor in nucleic acids as it is a characteristic in the blood of poultry [32]. This also causes a problem of separation between the different the nucleic fraction and the peptide fraction. A closer study on the parameters that could possibly influence the viscosity of the medium when the hydrolysis has been stopped. For this, the effect of pH was investigated by adding an additional step after stopping the hydrolysis by reducing the pH of the reaction medium with H3PO4 (1M). Thus, a descending range of pH was carried out ranging from 8.5 to 5.5 to determine the optimal pH of separation between the peptide fraction and the nucleic fraction for each concentration of hemoglobin. The optimal pHs of the final hydrolysates were chosen in such a way as to allow the separation between the peptide fraction and the nucleic fraction, as well as to prevent the passage of haem into the nucleic fraction in order to have a maximum yield of haem during discoloration. Thus, the optimum pHs for separation of the peptide/nucleic acids fractions are 8 for [HB]= 2%, 6.5 for [HB]= 5%, 6 for [HB]= 8% and 5.5 for [HB]= 9% (w/v). The peptide profiles of the hydrolysates sampled at pH 8 and those sampled at their defined optimum pH were compared by RP-HPLC analysis. These profiles show a good conservation of the peptide population in terms of quality and quantity, as well as of the haem. This proves that the decrease of pH does not impact the yield of peptides and haem. Then, the discoloration was carried out by applying a range of pH from 5 to 3. The results show that a progressive discoloration of the peptides with the best discolorations obtained at pH 3 for all the tested concentrations (Figure 2).

3.3 Peptide population Prediction in silico

This study was performed to compare the poultry hemoglobin to bovine hemoglobin by blast Gallus gallus and Bos taurus databases. The results show that the α and β chains of bovine or poultry hemoglobin are highly similar in pairs (Figure 3a). The both of α chains of are composed of 142 amino acids, with no Gaps during sequence alignment. The score obtained is 210 which indicates that the two compared sequences are very similar. The Expect value (e-value) is 3.10^-76, which is so close to zero. The α chains of Gallus gallus and Bos taurus have many similarities in their amino acid sequences: 101 out of 142 are strictly aligned, the value of the identity obtained is 71.13%. The same study was carried out in order to align the amino acid sequences of the β chains of hemoglobin Gallus gallus and Bos taurus. The β chain of Gallus gallus contains 147 amino acids against 145 for the β chain of Bos taurus. The score obtained (201), the expect value (8.10^-73) and the value of gaps (0%). The identity between these two β chains is 64.34% (Figure 3b). Several changes between the two compared sequences were detected. For hemorphins V the sequences are quite close, so their effect can probably be preserved. For the RNA-RYH segment, the determinant is affected by a change from R to K, the activity may be preserved. However, some bioactive peptide sequences have been found such as YPWTQR (Nyberg et al., 1996), YPWTQ and YPWT [33] which are opioid peptides as well as YR which is opioid and antihypertensive [34]. We also find there the hematopoietic peptide KLHVDPE [35].

3.3 Bioinformatic approach to predict peptides derived from the pepsic hydrolysis of poultry cruor

The sequences of α and β chains of hemoglobin Gallus gallus was submitted to the PeptideCutter software. The predicted cleavage sites of the α chains by peptic hydrolysis at pH higher than 2 are shown in Figure 4. For the α chain there are cleavage 36 sites (Figure 4a). The cleavage sites predicted by PeptideCutter, for the peptic hydrolysis, of the β chains at pH higher than 2, are presented in Figure 3b. There were 44 potential cleavages have been identified (Figure 4b). The predictions show an encouraging hydrolysis result. In comparing with the Bos taurus repository, similar peptides to known peptides are identified. However, fewer peptides seem to be generated in poultry hemoglobin due to amino acid changes between the two sequences. The main limit of this approach is the fact of imposing the study of the main blood protein, hemoglobin. This approach therefore does not take into account other proteins that can be digested during enzymatic hydrolysis of cruor where interesting new bioactive peptides could be generated.

3.4 Mass Spectrometry Analysis of poultry cruor hydrolysate and identification of the active peptides

The LC-MS/MS analysis were carried out on poultry cruor hydrolysate. The datas were acquired using the Mass Lynx software. The selection criteria are reduced by trying to process only the most abundant peptides. For this, the sequences which correspond to more than 5 MS/MS spectra are selected. The profile displays the presence of totally 121 peptides with masses (m/z) included between 503.3 Da and 2221.25 Da for the α chain. For the β chain, 79 peptides were detected with masses included from 507.31 to 2761.46 (Figure 5). To go further, the mass analysis was coupled to an ESI source (ions electron spray) data to have more voluminous MS/MS analysis data. The data was acquired using Peaks^® software. The sequences of α and β chains of bovine hemoglobin were taken as model proteins for the detection of peptides by Peaks^®software to obtain a list of all the detected peptides. Compared to the identified peptides derived from bovine hemoglobin, 28 active peptides were detected in the poultry cruor hydrolysate (Table 1). Totally, 8 opioid peptides have been detected. Opioid peptides can be potential pharmacological substitutes for pain treatments activity as they are described as simulating the action of opium. These opioid peptides belong to the family of hemorphins or LVVh-7 which are derived from the β chain [33]. The attachment of these peptides to a specific opiate receptor enables them to perform their opioid activity. The poultry cruor hydrolysates contain also an interesting number of antibacterial peptides. There is totally 8 detected antibacterial peptides in the α chain and 6 in the β chain. The α1-23 peptide, was the first peptide antimicrobial isolated by Froidevaux et al. [22] following the peptic hydrolysis of bovine hemoglobin which have shown activity against Micrococcus luteus A270. Then, 30 peptides with antimicrobial activity were found after HPLC fractionation of bovine hemoglobin hydrolysates: 24 are derived from the α chain of hemoglobin and 6 are derivatives of the β chain [5]. They demonstrate antibacterial activity against Micrococcus luteus, Listeria innocua, Escherichia coli and Salmonella enteritidis strains. New antibacterial peptides have been afterward identified from peptic hydrolysis of bovine hemoglobin by Adje et al., [36]. Furthermore, Other peptides with interesting activities were detected in poultry cruor hydrolysates such as hematopoietic peptides, which consists on peptides helping to form arm red blood cells in the bone marrow and bone. This activity has been observed in peptides derived from chemical hydrolysis [35, 37]. Other activities were also found in poultry cruor generated peptides which are mostly observed in bovine hemoglobin such as, bradykinin potentiators [38], antihypertensives [36] and coronary-constrictors [39].

3.5 The antibacterial activities of the poultry cruor or hemoglobin hydrolysates

The antibacterial activity of poultry cruor hydrolysates was tested against 6 strains mainly responsible in food alteration: Micrococcus luteus ATCC 9341, Listeria innocua CIP 80.11T, Staphylococcus aureus DSM 7526, Salmonella entirica CIP 70.52, Escherichia coli ATCC 25922 and Kocuria rhizophilia CIP 53.45. The results in agar plates showed that at a concentration of 20 mg/mL the hydrolysate was active against all the tested strains (Table 2a). The highest sensitivity was observed with Staphylococcus aureus DSM 7526. The hydrolysates were then assessed for their MIC values. The MIC is 20 mg/mL against Salmonella entirica CIP 70.52 and Listeria innocua CIP 80.11.T, 10 mg/mLagainst Staphylococcus aureus DSM 7526 and Micrococcus luteus ATCC 9341 and considerably lower against Kocuria rhizophilia CIP 53.45 (1.25 mg/mL) (Table 2b). The hydrolysates have an inhibitory effect on the bacterial growth of these strains. This effect is highly similar to that observed in bovine or porcine hemoglobin hydrolysates against as reported in previous study [40] Salmonella enterica strains is known to be resistant to at least 9 antimicrobials[41]. The addition of these hydrolysates would be a good alternative to chemical food preservative to prevent contaminations. In poultry feeding, these are promising candidates for the development of a supplement to reduce the use of antibiotics [42].

3.6 β-carotene bleaching inhibition activity

The antioxidant capacity is determined by measuring the inhibition of volatile organic compounds and conjugated diene hydroperoxides from linoleic acid’s oxidation [43]. This method thus corresponds to the measurement of the ability for a compound to reduce at least the oxidation of linoleic acid and carotene β in emulsified aqueous system. In this study, the lipid peroxidation inhibition of poultry cruor hydrolysate was determined by evaluating their capacity to inhibit oxidation of linoleic acid in an emulsified medium. Poultry cruor hydrolysate was tested at 40 mg/mL and 20 mg/mL and compared to neokyotorphin (0.5 mg/mL), which is a synthetic antioxidant peptide derived from hemoglobin, and a standard antioxidant, BHT (0.5 mg/mL) (Figure 6a). The obtained results are expressed in relative antioxidant activity (RAA) and shows that all the tested samples have the ability to inhibit the oxidation of β-carotene. The closer the RAA value is to 100%, the nearer is the antioxidant activity of the tested product to that of the BHT. The RAA of neokyotorphin is 93.74 ± 1.8%, which means that this peptide therefore has a lipid protection function close to thatobserved in the presence of BHT. The RAA of the hydrolysate at 40 mg/ml (79.23 ± 1.4%) is lower than that of BHT and neokyotorphin but which remains considerably high given the molecular mixture contained in cruor compared to BHT and neokyotorphin which are pure. The decrease in the concentration of hydrolysates to 20 mg/ml shows a slight decrease in RAA (71.1 ± 1.2%) which means that Relative antioxidant activity increased with increasing protein hydrolysates concentrations. For comparison, the RAA antioxidant activity of the phenolic compounds of olive oil extracted in the traditional way is 67.40 ± 1.02% compared to BHT at 0.5 mg/mL [44], which is lower than the RAA of poultry cruor hydrolysate at 20 mg/mL.

3.7 Trapping of DPPH^• radicals

The ability of hydrolysates to capture the DPPH^● radical as described by Molyneux [24] by spectrophotometry method. The DPPH^●+ radical is stable at room temperature and has a characteristic blue color. Antioxidants present in the sample reduce it by capturing the Hydrogen ion from it causing discoloration of the solution and releasing an antioxidant radical. The results were then standardized using Trolox Equivalent Antioxidant Capacity (TEAC). Poultry cruor hydrolysate was tested at a concentration of 40 mg/mL and 20 mg/ml. BHT was used as a positive control at 0.5 mg/mL and 0.1 mg/mL and neokyotorphin at 2.5 mg/mL as reference antioxidant peptide that showed an interesting activity against meat oxidation [6]. The results show that the hydrolysates have a considerable DPPH^● free radical scavenging at the two tested concentration and that it’s slightly higher at 40 mg/mL than at 20 mg/mL (829.35 ± 21.12 µmol/mL and 708.85 ± 0.66 µmol/mL respectively) (Figure 6b). The DPPH^● free radical scavenging capacity of poultry cruor hydrolysate at its 2 tested concentrations is higher to that of BHT at 0.1 mg/ml (436.32 ± 12.02 µmol/mL) and pure neokyotorphin (272.72 ± 13.65 µmol/mL) but still remains lower than that of BHT at 0.5 mg/mL (2181.6 ± 60.13 µmol/mL).

3. 8 Measurement of antiradical activity by the ABTS radical scavenging method

The antiradical activity by the ABTS^●+assay is based on the ability of molecules to quench the ABTS radical cation. This test has been performed using different concentrations of poultry cruor hydrolysate ranging from 0.5 mg/mL to 0.125 mg/mL and compared to BHT at 0.5 mg/mL and the neokyotorphin at 2.5 mg/mL. The results showed that the tested hydrolysates have an excellent ability to inhibit ABTS^●+ radicals, specifically for concentrations above 10 mg/mL where the inhibition percentages are above 90% (Figure 6c). These values are higher than those obtained for the reference antioxidant BHT (66.5 mg/mL). The IC₅₀ which corresponds to the concentration above which 50% of ABTS^●+ radicals are inhibited was determined. This value corresponds to 2.6 ± 0.04 mg/mL of poultry cruor hydrolysates. This high antioxidant activity of poultry cruor hydrolysate comes probably from the synergy of the peptide blend. Similarly, this capacity to inhibit ABTS^●+ radicals have been observed in bovine hemoglobin hydrolysates [45]

Total antioxidant capacity (TAC)

The antioxidant capacity of poultry cruor hydrolysates has been determined by a method based on an oxidation-reduction reaction which consists of the reduction of molybdenum (VI), present in an acid medium in the form of molybdate ions, to molybdenum (V) [26]. In this experiment, the hydrolysates were tested at a concentration of 40 mg/mL and compared to different concentrations of BHT ranging from 0.1 to 0.5 mg/mL. The results were calculated in Trolox equivalent and showed that at this concentration the hydrolysates have a total antioxidant capacity comprised between that of BHT at 0.3 mg/mL and 0.1 mg/mL with a TAEC of 1956 ± 85 µmol/L (Figure 6d). Poultry cruor hydrolysates have demonstrated important antioxidant properties. Many properties of the peptides could influence the antioxidant such as their hydrophobicity, their molecular size, and the polarity of their amino acid residue [46]. Antioxidant activity can be due to the total denaturation of hemoglobin which promotes the «Zipper» mechanism and favors smaller peptides generation having important radical scavenging activities

This study aimed to characterize the poultry cruor or hemoglobin hydrolysate which is a new potential source of hemoglobin. The initial hemoglobin concentration optimisation revealed that the best yield of peptides was obtained at 9% hemoglobin. Bioinformatic alignment was used to compare the poultry cruor hydrolysate sequences in silico to bovine hemoglobin hydrolysate which is well-known as bioactive peptides source. Results showed the presence of some similarities between the derived peptides from the two products with prediction of new peptides generation for the poultry cruor hydrolysate. Mass spectrometry analysis showed the presence of bioactive peptides derived from poultry cruor hydrolysate. The antibacterial activity was verified for the studied hydrolysate and showed an inhibitory effect on the bacterial growth against strain responsible in food alteration with interesting MIC values. The antioxidant activity was also evaluated with different methods. The results revealed that these hydrolysates have good antioxidant properties compared to the reference chemical antioxidant: BHT with good ability to trap DPPH^•+ free radicals and to inhibit ABTS^• radicals. To conclude the studied product is a promising new natural source of bioactive peptides which make it a new alternative to be valorised as food additive.

Acknowledgements

The authors are grateful to the Advanced High Throughput Technologies Platform for Biorefineries Catalysts Design ‘REALCAT’ for allowing the implementation of MALDI-ToF mass spectrometry analysis.

Funding :

This work was supported by Charles Viollette institute. The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing interests :

The authors have no relevant financial interests to disclose.

Autors contribution :

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Oumaima Zouari and Rémi Przybylski. Mass spectrometry analysis were performed by Barbara Deracinois and Christophe Flahaut. This study was supervised by Naima Nedjar and Rémi Przybylski. The first draft of the manuscript was written by Oumaima Zouari and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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