Proteolysis coupled with membrane separation for the isolation of bioactive peptides from defatted smooth hound byproduct proteins

doi:10.21203/rs.3.rs-2971192/v1

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Research Article

Proteolysis coupled with membrane separation for the isolation of bioactive peptides from defatted smooth hound byproduct proteins

https://doi.org/10.21203/rs.3.rs-2971192/v1

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Journal Publication

published 30 Oct, 2023

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The defatted viscera from smooth hound were the raw material used to produce four protein hydrolysates using three different microbial proteases (Neutrase, Esperase and Purafect) and the endogenous enzymes, named VPH-N, VPH-E, VPH-P and VPH-EE, respectively. Hydrolysates showed different degrees of hydrolysis (DH) depending on the enzyme used, where the VPH-P had the highest DH (30%). The amino acids (AA) characterization showed that Gly was the prominent AA (≥22%), followed by Ala, Glx, Lys, Asx, and Pro. In addition, the assessment of the antioxidant, ACE-inhibitory and antimicrobial activities revealed the high potentiality of the Purafect-hydrolysate. Therefore, it was selected to be fractionated by ultra-filtration according to their molecular weight (MW). Data showed that FIV (MW˂5 kDa) exhibited the best antioxidant activities. The differences detected in the bio-activities may be attributed, not only to the peptides size, but also to their sequences and hydrophobic AA contents. The antibacterial activity showed that the best inhibition values were recorded with FIV, particularly against S. typhi. Thus, the present results demonstrated the high potential of FIV from the VPH-P to be used a promising natural source of bioactive peptides in functional food formulations.

Defatting

Viscera

Protein hydrolysis

Membrane fractionation

Antioxidant activity and antibacterial activity

The FAO’s global discard estimated a total capture of one million seabirds, 8.5 million sea turtles, 225 000 sea snakes, 650 000 marine mammals and 10 million sharks, amounting to a total of around 20 million individuals, which are discarded annually in global fisheries [1]. The annual discards from global marine capture fisheries between 2010 and 2014 was 9.1 million tones, which represent 10.8% (10.1% − 11.5%) of the annual average catch of 2010–2014 [2]. These by-products may include muscle, skin, fins, frames, head, scales, roe and viscera. These large amounts are mainly discarded without any attempt of valorization.

Among the most consumed fish sharks, the smooth hound species are worldwide produced. In Tunisia, Mustelus mustelus is the most consumed hound shark, where its marketing and filleting processes generate a huge quantity of visceral mass that needs a scientific exploitation route, through the production of bioactive and functional protein hydrolysates [3]. In fact, fish viscera, due to their high protein content, represent a good protein matrix for fish protein hydrolysates production with interesting biological activities, such as Nile tilapia (Oreochromis niloticus) [4], rainbow trout [5], yellowfin tuna (Thunnus albacares) [6] and abalone (Haliotis discus hannai) [7].

In the recent years, among the investigated bioactive peptides are those owned with antioxidant, antibacterial and Angiotensin converting enzyme (ACE)-inhibitory potentials. Recent studies on ACE-inhibitory activities in fish hydrolysates are complemented with the analysis of antioxidant activity, since hypertension and oxidative stress have become the most serious health problems affecting society [8, 9]. These bioactive peptides are released upon exogenous and/or endogenous enzyme hydrolysis of fish proteins under controlled conditions [10]. However, their bioactivity is strongly linked to different parameters such as the source of protein, degree of hydrolysis (DH), type of the enzyme used, amino acid composition, peptide sequence and size [11]. Thus, several studies have used the membrane separation technique for the fractionation of peptides according to their molecular weights (MW) [12, 13]. Compared to other peptides’ separation methods, such as the gel chromatography separation, membrane technology offers several advantages such as higher productivity, high throughput of product, ease of high-scale production and easy equipment cleaning [14].

Of particular, fish byproducts, especially liver and viscera are known by their high fat contents. Thus, several methods have been used to remove lipids from these fish tissues. For instance, in the case of viscera, heat treatment followed by centrifugation is a very conventional practice, because it has the dual purpose of inactivating enzymes and removing fat [15, 16]. On the other hand, solvents have demonstrated a great defatting efficiency using a mixture of minced raw material and alcohols, with constant stirring for a fixed period of time [17]. Advantageously, this technique reduced the characteristic odor of fish and bitter flavors, However, it may affect the hydrolysis efficiency and the properties of the final protein hydrolysates [3, 18].

Thus, in the present study, the effect of defatting and enzymatic hydrolysis coupled to ultra-filtration process, on the amino acid composition and nutritional value of the peptide fractions recovered from smooth hound viscera protein hydrolysates, was evaluated. In addition, their biological activities, in terms of antioxidant and antimicrobial activities were assessed.

2.1. Raw material preparation and defatting

The raw material used in this study was the viscera of the smooth hound (M. mustelus) fish. Viscera mass were obtained following the processing of fresh fish fillets available in the local fish market, Sfax, Tunisia. The biological material was directly brought to the research laboratory in iced conditions. Upon arrival, they were immediately well rinsed with cold tap water to remove contaminants, then, stored in specific bags at -20°C until use.

Before hydrolysis reaction, viscera were defatted using different methods: either chemical solvents (acetone or isopropyl alcohol) or trichloroacetic acid precipitation. The defatted viscera were then physic-chemically characterized to select the best method allowing the highest lipid removing yield. The best defatting method is described as the following: the material was cut into small pieces and homogenized in a Moulinex® blender in the presence of a mix of isopropanol/distilled water (w/v/v) to remove the lipids. The mix was then kept under continuous stirring for 60 min, at room temperature, and then centrifuged at 9500 g at 4°C, for 30 min. The supernatant containing isopropanol enriched in lipids was removed, while the pellet obtained after filtration presents the defatted viscera, which will be used as a protein matrix for the enzymatic hydrolysis.

2.2. Enzymes

Neutrase®, Esperase® and Purafect® were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Crude enzymes extract from viscera were prepared as described in Abdelhedi et al. [19] and referred to the endogenous viscera enzymes.

2.3. Protein hydrolysates preparation

Defatted viscera material was first cooked in distilled water at 95°C for 15 min, with a solid/solvent ratio of 1:1 (w/v) to inactivate endogenous enzymes. The mixture was homogenized and the pH was adjusted to the values recommended by the manufacturer for each enzyme: Purafect (10.0, 50°C), Esperase (9.0, 50°C) and Neutrase (7.0, 50°C). For the autolysate, the crude endogenous enzymes (EE) were used at pH 8.0 and 50°C. The solutions were allowed to equilibrate for about 30 min before the reaction was initiated. Then, the enzymes were added at a ratio of 6/1 unit of enzyme/mg of protein. During the reaction, the pH of the mixture was kept constant at the initial value by continuous addition of NaOH solution (4 N). Hydrolysates obtained by treatment with the endogenous enzymes (EE), Neutrase (N), Esperase (E) and Purafect (P), were noted as VPH-EE, VPH-N, VPH-E and VPH-P, respectively. During the reaction, the degree of hydrolysis (DH %) was calculated simultaneously with the NaOH addition, as described by Adler-Nissen [20]. The undigested viscera proteins (UVPs), treated at the same conditions and with no enzyme addition, was used as control.

Finally, the hydrolysis reaction was stopped by heating the different solutions at 95°C for 20 min and the protein hydrolysates were then centrifuged at 9500 g for 20 min at 4°C. The supernatants were freeze-dried using a freeze-dryer (Bioblock Scientific Christ ALPHA 1–2, IllKrich-Cedex, France) and the resulted powders, referring to the smooth hound viscera protein hydrolysates (VPHs) were stored at -20°C for further use.

2.4. Physic-chemical analysis

The moisture, ash, lipids and protein contents in the fresh raw material and the freeze-dried hydrolysates were determined according to the AOAC methods [21]. A factor of 6.25 was used to convert the nitrogen value to protein.

2.5. UF fractionation

VPHs was fractionated by ultra-filtration membrane separation, in a tangential flow filtration mode, using Millipore UF systems, a Labscale™ TFF System (USA) with membranes having molecular weight cut-offs (MWCO) of 50, 10 and 5 kDa. Manometers before and after UF membranes were used to measure the inlet and the outlet pressures in order to control the trans-membrane pressure, which was maintained at 1 bar. Besides the pressure, the permeate flux was followed and calculated during UF separation. The recovered fractions were noted FI (MW˃50 kDa), FII (10˂MW˂50 kDa), FIII (5˂MW˂10 kDa) and FIV (MW˂5 kDa) and their total protein content was quantified using the Lowry method [22]. The collected fractions were freeze-dried to be characterized.

2.6. Amino acids composition

Samples diluted at 3 mg/ml were mixed with Norleucine (v/v), the internal standard, in vacuum-sealed glass tubes and subjected to dry under nitrogen-vacuum cycles. The protein samples (50 µl) were then hydrolyzed at 120°C for 24 h, using 300 µl of 6 M HCl containing 1% (v/v) phenol. Samples were then derivatizated with phenyl isothiocyanate (PITC) and dissolved in 300 µl of 5 mM sodium phosphate buffer (pH 7.4) containing 5% (v/v) of acetonitrile (ACN). The PITC-derivates were quantified by reverse phase-High Performance Liquid Chromatography (RP-HPLC) with Agilent liquid chromatography system (Agilent Technologies, Palo Alto, CA, USA) equipped a PicoTag® column (300 mm × 3.9 mm, Waters, Ireland). The eluents used were (A) sodium acetate (0.07 M, pH 6.55 and containing 2.5% ACN) and (B) ACN: water: methanol (45:40:15). The flow rate was fixed at 1 ml/min and the temperature was set at 52°C. The amino acids (AA) were detected at 254 nm and the results were expressed as AA residue/100 AAs in the sample. All analyses were performed in duplicate.

2.7. Free amino acids content

In the free amino acids (FAA) content, samples were firstly deproteinized with ACN and then derivatizated as previously described for the amino acids composition determination. Results were expressed as mg of amino acid per g of sample.

2.8. RP-HPLC analysis

The RP-HPLC analysis of all samples was performed using liquid chromatography system (Agilent Technologies, Palo Alto, CA, USA) equipped with a diode array detector and a C18 column (250 mm × 4.6 mm, Waters, Milford, MA, USA) at 30°C. Elution was performed using solvent (A) trifluoroacetic acid (TFA) in bidistilled water (0.1%, v/v) and solvent (B) ACN: bidistilled water (60:40, v/v) containing 0.085% TFA (v/v). Both mobile phases A and B were filtered through a 0.45 µm filter and degassed prior to any analytical run. The elution started with 100% solvent A for 2 min, followed by a linear gradient from 0 to 25% of solvent B during 30 min and reached 100% after 15 min. The flow rate used was fixed at 1 ml/min and the separation was monitored at a wavelength of 214 nm.

2.9. Antioxidant activities assessment

2.9.1. Reducing power assay

The ability of samples to reduce iron Fe³⁺ was determined according to the method of Yildirim et al. [23]. The sample solution (0.5 ml), prepared at different concentrations, was mixed with 1.25 ml of 0.2 M potassium phosphate buffer (pH 6.6) and 1.25 ml of 1% (w/v) potassium ferricyanide solution. The reaction mixtures were then incubated at 50°C for 20 min, followed by the addition of 0.5 ml of 10% trichloroacetic acid (TCA). After 5 min, the reaction mixtures were centrifuged at 2700 g for 10 min. Finally, 1.25 ml of the supernatant from each sample mixture was mixed with 1.25 ml of 0.2 M potassium phosphate buffer (pH 6.6) and 0.25 ml of 0.1% ferric chloride. After a 10-min reaction time, the absorbance of each tube solution was measured at 700 nm. The control was conducted in the same manner, except that phosphate buffer was used instead of sample. Butylated hydroxyanisole (BHA) was used as positive control. Analyses were done in triplicate.

2.9.2. Iron chelating activity

The iron (Fe²⁺) chelating effect was estimated by the method of Decker and Welch [24]. A volume of 100 µl of each sample solution was mixed with 50 µl of 2 mM FeCl₂ and 450 µl of distilled water. The mixtures were incubated at room temperature for 5 min. Then, 200 µl of 5 mM ferrozine solution (3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate) were added to initiate the reaction. The mixtures were thereafter shaken and left to stand for 10 min at room temperature. Control tube was prepared with the same manner with substituting the hydrolysate by water. EDTA (ethylene-diamine-tetra acetic acid) was used as positive control. The absorbance of the solutions was measured at 562 nm, and the inhibition percentage of ferrozine–Fe²⁺ complex formation was calculated as follows:

Metal chelating activity (%) = [(AC + AB − AS) / AC] × 100

where AC, AB and AS represent the absorbance of the control, the blank and the sample reaction tubes, respectively.

2.9.3. β-carotene bleaching protection

The prevention of β-carotene from bleaching was determined according to the method of Koleva et al. [25] and Vázquez et al. [26]. The β-carotene/linoleic acid emulsion was freshly prepared by dissolving 0.5 mg of β-carotene with 25 µl of linoleic acid and 200 µl of Tween-40 in 1 ml of chloroform. Then, the chloroform was completely evaporated under vacuum in a rotatory evaporator at 50°C and 100 ml of distilled water were added and the solution was vigorously stirred. The resulting β-carotene/linoleic acid emulsion was ready to be immediately used. A volume of 2 ml of the emulsion was transferred into test tubes containing 0.5 ml of each sample prepared at different concentrations. Control tube was prepared in the same conditions by adding 0.5 ml of distilled water to the emulsion. The absorbance of every test tube was measured at 470 nm before and after incubation for 1–2 h at 50°C. BHA was used as a positive control. Analyses were carried out in triplicate. The antioxidant activity was evaluated in terms of β-carotene bleaching using the following equation:

Antioxidant activity (%) = [1- (A₀ - A_t) / (A₀’- A_t’)] × 100

where A₀ and A_t are the absorbances of the test sample measured before and after incubation, respectively; and A’₀ and A’_t are the absorbances of the control measured before and after incubation, respectively.

2.10. Antibacterial activity

The antibacterial activity was evaluated using the agar diffusion method [27] against three Gram-negative bacteria: Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13883) and Salmonella typhi (ATCC 19430) and two Gram-positive bacteria: Staphylococcus aureus (ATCC 25923) and Micrococcus luteus (ATCC 4698). Briefly, culture suspension of each bacteria strain (CFU = 10⁶) was spread on a Luria-Bertani (LB) agar. Then, about 60 µl of sample solution (20 mg/ml) were loaded into wells (6 mm in diameter) punched in the agar layer. Thereafter, the Petri dishes were kept for 1 h at 4°C, and then incubated for 24 h at 37°C to initiate bacteria growth. The antibacterial activity was evaluated by measuring the clear growth inhibition zone diameter (mm) around the wells. Gentamicin discs of 30 µg (BD BBL™ Sensi-Disc™, US) were assayed under the same conditions and used as control.

2.11. Statistical analysis

Data are expressed as mean ± SD (Standard Deviation) from at least three independent experiences and statistically analyzed using SPSS ver. 17.0 (SPSS Inc., Chicago, USA). A one-way analysis of variance (ANOVA) and Duncun test were then applied to compare means at a confidence interval of 95%.

3.1. Preparation of protein hydrolysates from defatted smooth hound viscera

In the present study, the raw material used was the viscera mass of smooth hound (M. mustelus) fish. Their proximate composition showed high protein content (76.68 ± 0.75%), with ash and lipid contents of 2.02 ± 0.09 and 22.0 ± 1.66%, respectively (Table 1). The high fat level found in viscera led to proceed by a defatting step before hydrolysis, in order to remove the lipid fraction from viscera mass. As lipids are essential compounds in the formation of the biological structure of viscera, their elimination may influence the quality of the proteins, and therefore the resulting hydrolysates. In this work, the extraction of lipids was performed using 3 methods using conventionally polar/non-polar solvents (isopropyl alcohol and acetone) or by TCA precipitation for protein recovery. Results showed that 38% of fats were solubilized by acetone, with a mass yield of 16.77%, while the TCA precipitation resulted in a mass yield of 35%. However, the use of isopropanol eliminates 78.5% of the total lipid content, with 71% of protein recovery and a yield of around 66%. Thus, the isopropanol-defatting method was adopted and the resulting viscera exhibited protein and lipid contents of 93.94 ± 0.47% and 7.26 ± 0.16, respectively (Table 1).

Table 1

Physic-chemical characterization of fresh and defatted smooth hound viscera and their freeze-dried enzymatic protein hydrolysates
		Protein (%)	Fat (%)	Ash (%)
Fresh viscera	-	76.68 ± 0.75^d	22.01 ± 1.66^a	2.02 ± 0.09^c
Defatted fresh viscera	-	83.94 ± 0.47^b	7.27 ± 0.16^b	7.5 ± 0.07^b
UVPs	-	81.41 ± 0.98^c	3.26 ± 0.28^c	7.53 ± 1.07^b
VPH-EE	14.15	87.09 ± 2.68^a	2.13 ± 0.05^c	8.10 ± 0.54^a
VPH-N	6.85	83.40 ± 0.42^b	2.28 ± 0.60^c	7.44 ± 0.60^b
VPH-E	20.71	87.78 ± 0.89^a	2.91 ± 0.39^c	9.70 ± 0.01^a
VPH-P	30.1	87.08 ± 1.20^a	2.65 ± 0.21^c	8.94 ± 1.06^a
DH: Degree of hydrolysis (%); UVPs: Undigested viscera proteins; VPH-EE: Viscera protein hydrolysate prepared after endogenous enzymatic treatment; VPH-N: Viscera protein hydrolysate prepared with Neutrase; VPH-E: Viscera protein hydrolysate prepared with Esperase; VPH-P: Viscera protein hydrolysate prepared with Purafect.
Results are expressed in % based on dry weight matter. a,b,c,d different letters in the same column indicate significant difference at p ≤ 0.05

Thereafter, defatted viscera were enzymatically treated using three different microbial enzymes and the crude endogenous proteases extracted from the same raw material. All the enzymes were used at the same level (6 U/mg of protein) and the reaction was stopped at the same hydrolysis time. As enzymes display various spectra of specificity, the different proteases will result in different types of protein hydrolysates containing peptides with different properties. The degree of hydrolysis (DH %) curves of the smooth hound viscera proteins as a function of time are presented in Fig. 1. All the kinetic curves showed similar evolution, characterized by a high hydrolysis rate during the first 90 min, to be gradually decreased as the reaction time increased. Then, the reaction reached a steady-state phase, mainly owing to the inhibition of the enzymes or to the decrease of the susceptible peptide bonds number. The final DH values of the different hydrolysates after about 10 h min are reported in Table 1. Data showed that, when used at the same ratios, Purafect was the most efficient enzymes (DH = 30%), followed by Esperase (DH = 20.71%) and the endogenous crude enzymes (DH = 14.15%), while Neutrase was the least one (DH = 6.85%). Previous studies reported the preparation of protein hydrolysates from Nile tilapia (Oreochromis niloticus) viscera using Alcalase, and the obtained DH values were between 30.89% and 41.46% [4]. In addition, Ovissipour et al. [6] optimized the production of protein hydrolysate from the viscera of yellowfin tuna (Thunnus albacares) using Alcalase and the results showed DH values more than 53%. Slizyte et al. [28] showed that the hydrolysate obtained from defatted salmon backbones with the action of Protamex during 120 min was 20.9%, while that prepared by the action Protamex + Trypsin without lipid elimination was of 22.6%.

The chemical composition of the freeze-dried VPHs was determined and presented in Table 1. Data revealed that all the hydrolysates exhibited high protein content (more than 83%), based on dry weight matter. This high protein level indicated about the nutritive quality of the samples. Obviously, all the hydrolysates had relatively low lipid levels (2–3%), as they derived all from a defatted raw material. Similarly, Thiansilakul et al. [29] obtained low-fat protein hydrolysate from defatted round scad (Decapterus maruadsi) fish using isopropanol prior to hydrolysis. The ash content was ranged between 7 and 9%, which were higher than the initial ash viscera level (2%). This may be explained by the continuous addition of NaOH during the reaction in order to keep pH at the target value. In fact, the hydrolysates showing the highest DH values (VPH-E and VPH-P) exhibited the highest ash contents (8.94 and 9.7%).

3.2. Fractionation by ultrafiltration membrane system

It is well known that the biological properties of a protein hydrolysate are strongly related to the specificity of the protease used, the degree of hydrolysis, as they affected the peptides length and their amino acid sequences and composition [19]. Several previous works proved that increased DH positively correlated with increased medium and short peptide chains contents, and therefore increased bio-activity. Indeed, medium molecular weight peptides are mainly endowed with great antioxidant activity, while short peptides were most efficient on the ACE inhibition. In this sense, Slizyte et al. [28] demonstrated that the ACE-inhibitory activity of defatted Salmon backbone hydrolysates increased with increasing hydrolysis time, while Nasri et al. [9] proved that a medium DH was suitable to obtain peptides with good antioxidant activity. Thus, in the present study, VPH-P showing the highest DH value (30.1%) was selected to be fractionated by ultrafiltration (UF), because it is mainly composed of the most bioactive peptide sequences.

Membrane technology separation has been widely described to fractionate protein hydrolysates into fractions with specific peptide sizes resulting in the concentration of the most bioactive peptides [12] (Siddik et al., 2021). For instance, Peñaranda-López et al. [30] used UF equipped with three tubular ceramic membranes with different MWCO (50, 10 and 5 kDa) to fractionate lecithin-free egg yolk. Hence, in order to improve its bioactivity, VPH-P was fractionated by UF system using successive membranes with decreased molecular weight cut-offs (MWCO) ranged from 50 to 5 kDa, in a tangential flow filtration system (TFF). All the fractions were recovered and freeze-dried to be then analyzed, as mentioned in the following sections.

3.3. Amino acids composition and free amino acids of VPHs

As the biological activity and nutritional value of a protein hydrolysate are mainly based on its amino acid composition, the amino acid (AA) profiles of the defatted viscera protein hydrolysates were determined and compared to the undigested proteins (Table 2). Data revealed that glycine was the prominent AA found in all the hydrolysates, achieving a value superior than 22%, followed by Ala, Glx, Lys, Asx, Pro and Ser. To note, Cys and Trp were not detected in this study because they were destroyed during the acid hydrolysis.

Table 2

Amino acids composition (%) of freeze-dried VPHs prepared by endogenous or exogenous proteases
AA	UVPs	VPH-EE	VPH-N	VPH-E	VPH-P
Asx	7.54 ± 0.09^b	5.20 ± 0.05^c	5.03 ± 0.03^c	7.11 ± 0.07^b	8.01 ± 0.09^a
Glx	8.90 ± 0.06^a	7.04 ± 0.03^c	6.80 ± 0.0^d	8.54 ± 0.03^b	8.92 ± 0.05^a
Hyp	1.89 ± 0.09^b	3.86 ± 0.19^a	3.03 ± 0.16^a	1.17 ± 0.06^c	0.99 ± 0.05^d
Ser	7.10 ± 0.12^a	5.72 ± 0.08^c	6.12 ± 0.06^b	6.92 ± 0.1 ^a	7.10 ± 0.11^a
Gly	25.57 ± 0.30^c	35.77 ± 0.34^a	33.26 ± 0.17^a	23.10 ± 0.21^b	22.22 ± 0.24^b
Tau	2.53 ± 0.18^c	3.39 ± 0.23^b	4.98 ± 0.32^a	2.44 ± 0.17^c	2.47 ± 0.17^c
His#	1.02 ± 0.18^a	0.53 ± 0.09^b	0.63 ± 0.11^b	0.92 ± 0.16^a	1.00 ± 0.18^a
Thr#	2.62 ± 0.03^c	4.76 ± 0.04^b	5.36 ± 0.02^b	6.38 ± 0.04^a	6.69 ± 0.06^a
Ala*	9.43 ± 0.10^a	9.03 ± 0.07^a	8.49 ± 0.03^b	9.29 ± 0.07^a	9.13 ± 0.09^a
Arg	3.80 ± 0.03^a	3.26 ± 0.04^a	2.81 ± 0.04^b	3.52 ± 0.04^a	3.61 ± 0.04^a
Pro*	6.91 ± 0.17^b	7.57 ± 0.17^a	7.63 ± 0.14^a	6.86 ± 0.15^b	6.88 ± 0.16^b
Tyr*	1.20 ± 0.04^a	0.71 ± 0.02^b	0.58 ± 0.02^b	1.16 ± 0.04^a	1.24 ± 0.04^a
*Val#**	3.56 ± 0.19^a	2.47 ± 0.13^b	2.65 ± 0.16^b	3.66 ± 0.20^a	3.57 ± 0.19^a
*Met#**	1.29 ± 0.07^a	0.97 ± 0.05^b	0.94 ± 0.06^b	1.40 ± 0.08^a	1.40 ± 0.08^a
*Ile#**	2.79 ± 0.10^a	1.25 ± 0.05^c	1.85 ± 0.08^b	2.72 ± 0.11^a	2.72 ± 0.10^a
*Leu#**	3.89 ± 0.13^a	1.66 ± 0.06^c	2.68 ± 0.11^b	4.26 ± 0.16^a	4.09 ± 0.14^a
*Phe#**	2.23 ± 0.10^a	1.37 ± 0.06^b	1.44 ± 0.07^b	2.23 ± 0.1 ^a	2.29 ± 0.11^a
Lys#	7.72 ± 0.12^b	5.46 ± 0.30^c	5.72 ± 0.05^c	8.33 ± 0.11^a	7.65 ± 0.06^b
HAA	31.30	25.02	26.27	31.57	31.34
EAA	25.12	18.46	21.27	29.89	29.42
TAA	100	100	100	100	100
Asx and Glx indicate Asp + Asn and Glu + Gln, respectively. Trp and Cys were not determined.
HAA (*), EAA (#) and TAA indicate hydrophobic, essentail and total amino acids, respectively.

Table 3

Free amino acids (mg/g) of freeze-dried VPHs prepared by endogenous or exogenous proteases
AA	UVPs	VPH-EE	VPH-N	VPH-E	VPH-P
Asx	0.58	2.97	0.56	2.91	4.22
Glu	0.97	5.21	2.60	1.38	9.13
Gln	0.50	1.71	0.71	1.30	3.83
Hyp	0.32	0.50	0.30	1.35	0.37
Ser	0.81	2.24	0.81	3.49	0.61
Gly	0.87	2.54	1.03	0.51	0.93
Tau	5.30	11.11	5.67	5.84	9.67
His	1.02	0.63	0.92	1.68	2.83
Thr	1.10	2.73	1.22	4.08	6.71
Ala	2.20	3.83	2.07	0.88	1.29
Arg	1.87	2.94	2.46	5.44	8.84
Pro	0.84	2.23	0.92	2.08	1.33
Tyr	1.98	2.33	2.11	0.90	1.02
Val	3.33	4.24	3.56	1.12	1.37
Met	2.07	1.40	0.88	7.35	11.76
Ile	2.49	1.11	10.85	11.28	18.44
Leu	9.05	4.09	1.13	2.17	3.95
Phe	6.66	2.90	5.25	1.33	2.25
Trp	2.59	0.67	1.91	3.90	6.67
Lys	2.50	5.24	2.60	6.51	1.16
TAA	47.06	60.60	47.56	65.53	97.75
Asx indicate Asp + Asn. TAA: Total amino acids.
Results are expressed in mg of amino acid per g of freeze-dried sample.

The undigested defatted viscera showed the highest levels of Gly (35.77%) and Hyp (3.86%), compared to the hydrolysates (p < 0.05), proving the presence of high level of connective collagenous tissues in this material [31]. Contrary, all the hydrolysates exhibited higher levels of hydrophobic (31.57–26.27%) and essential (29.89–21.27%) amino acids than the UVPs (25.02% and 18.46% for the HAA and EAA, respectively). The highest contents of EAA and HAA were found in the VPH-E and VPH-P (~ 31% for HAA and ~ 29% for EAA), having the highest DH values, which suggest their high nutritional quality and bioactivity. In fact, the amount of EAA is an important indicator about the nutritional quality of a protein mixture. Hassan et al. [32] reported similar EAA contents for pepsin- and endogenous enzymes alkali- treated Pangasius visceral protein hydrolysates. Furthermore, it has been demonstrated that the presence of hydrophobic residues is responsible for the increase of the antioxidant potential [33] and the ACE-inhibitory activity [19, 34]. Similar amount of hydrophobic amino acids (35.6%) was obtained from the Flavourzyme hydrolysate of Argentine croaker (Umbrina canosai) [35].

Among the all the hydrolysates, VPH-P and VPH-EE contained the highest levels of His (p < 0.05), the imidazole-containing amino acid, which may expect the chelating and lipid radical-trapping ability of these hydrolysates. Hassan et al. [32] reported that the His-rich hydrolysates exhibited the highest DPPH and ABTS scavenging abilities. Furthermore, both UVPs and VPHs contained a great amount of Taurine (2-amino ethanesulfonic acid) (2.44–4.98%), which is one of the most abundant free amino acids in mammalian cells [36]. It has been demonstrated that Tau may be used to protect against oxidative stress [37].

Thus, all over these data lead to suggest that the studied VPHs represent a good source of biologically active peptides with great nutritional value that may be used in functional food formulations. Thus, this fraction was thereafter fractionated by ultrafiltration in order to obtain the most active peptides.

As a protein hydrolysate is a mixture of peptides and free amino acids that exhibited many advantages as nutraceuticals or in functional foods, the free amino acid (FAA) contents of all the VPHs were estimated, as illustrated in Table. 3. Data showed that the amounts of FAA varied from 47 to 97 mg/g of viscera protein hydrolysate powder and positively correlated with the DH value (from 6 to 30%). In fact, VPH-P having the highest DH showed the highest FAA content, while the lowest FAA value was observed in the UVPs. The predominant FAA found in all the samples was Tau (> 5 mg/g) where its value increased after hydrolysis.

Similar FAA evolution was observed in Herring (Clupea harengus) filleting co-products hydrolysates, where increased DH correlated with increased FAA contents, varying from 44.41 to 77.28% (DH) and from 25.31 to 51.04 mg/g (FAA) [38]. In addition, Wu et al. [39] reported that the hydrolysis of mackerel (Scomber austriasicus) for 25 h resulted in increased FAA content from ~ 250 to ~ 1300 mg/ 100 ml as a function of the hydrolysis time and the FAA levels in the hydrolysates obtained with protease N were much higher than those obtained by autolysis.

3.4. Amino acids composition of the membrane fractions

Membrane separation by UF was done to separate the viscera hydrolysate from Purafect (VPH-P) based on its MWCO of > 50, <50 and > 10, <10 and > 5 and < 5 kDa, to produce the fractions FI, FII, FIII and FIV, respectively, with enhanced bioactivity. The amino acids (AA) composition of the resulting membrane fractions was determined and the results are shown in Table 4.Gly (25–29%), Ala (8–10%), Glx (7–8%), Asx (6–7%), Lys (6–7%), Pro (6–7%) and Ser (6–7%) were the most predominant amino acids in all the membrane fractions, similarly to the initial hydrolysate (before fractionation). However, fractionation resulted in increased levels of Ser, Tau, Thr, His, Ala, Val, Met, Leu, Ile, Phe and Lys in the < 5 kDa fraction when compared to the other fractions. In contrast, the < 5 kDa peptide fraction had less contents of Gly and Hyp, compared to the initial hydrolysate and other fractions. Overall, the total HAA and EAA levels increased after membrane separation, reaching 31% (excluding Trp) and 28%, respectively, in the FIV fraction. In this sense, Wang et al. [40] reported that the permeate with a MWCO lower than 5 kDa had higher surface hydrophobicity than the hydrolysate. The presence of high level of hydrophobic amino acids in the low molecular weight fraction indicated about its expected antioxidant potential. In fact, hydrophobic residues are commonly known by their strong antioxidant effect [41]. For instance, Saidi et al. [14] indicated that the NF retentate (1–4 kDa), recovered from the fractionation of tuna dark muscle protein by-product hydrolysate, and containing 30.8% HAA of the total AA, exhibited the highest superoxide radical and reducing power activities, while the NF permeate (< 1 kDa) from the same hydrolysate showed the highest radical scavenging activities. Moreover, FIV contained the highest level of His, compared to the other MWCO fractions (p < 0.05). As His is known by a strong radical scavenging activity, due to the presence of the imidazole ring acting as a proton door [42], it may be suggested that the FIV fraction is the most bioactive one.

Table 4

Amino acids composition (%) of VPH-P fractions obtained after UF process
AA	FI	FII	FIII	FIV
Asx	6.77 ± 0.03^b	7.23 ± 0.03^a	6.03 ± 0.04^c	6.31 ± 0.06^c
Glx	8.14 ± 0.01^a	8.34 ± 0.02^a	7.49 ± 0.01^b	7.92 ± 0.03^ab
Hyp	2.49 ± 0.14^a	2.05 ± 0.11^b	2.69 ± 0.14^a	1.45 ± 0.07^c
Ser	6.31 ± 0.06^b	6.51 ± 0.06^b	6.21 ± 0.07^b	7.08 ± 0.10^a
Gly	28.24 ± 0.10^a	25.51 ± 0.08^b	29.85 ± 0.18^a	25.21 ± 0.22^b
Tau	2.31 ± 0.14^c	3.39 ± 0.21^b	2.13 ± 0.14^c	5.47 ± 0.37^a
His#	0.97 ± 0.18^b	0.91 ± 0.17^b	0.95 ± 0.17^b	1.04 ± 0.18^a
Thr#	4.76 ± 0.01^c	5.40 ± 0.01^b	4.21 ± 0.02^c	6.19 ± 0.04^a
Ala*	9.11 ± 0.02^b	9.46 ± 0.02^b	8.95 ± 0.04^c	10.14 ± 0.07^a
Arg	3.40 ± 0.06^a	3.50 ± 0.06^a	3.66 ± 0.05^a	3.23 ± 0.04^b
Pro*	7.58 ± 0.13^a	6.30 ± 0.10^b	7.86 ± 0.15^a	6.07 ± 0.13^b
Tyr*	1.12 ± 0.04^a	1.14 ± 0.04^a	1.05 ± 0.04^a	1.17 ± 0.04^a
*Val#**	3.22 ± 0.19^b	3.64 ± 0.22^a	3.19 ± 0.18^b	3.90 ± 0.21^a
*Met#**	1.00 ± 0.06^c	1.27 ± 0.08^b	1.08 ± 0.06^c	1.51 ± 0.08^a
*Ile#**	2.47 ± 0.11^b	1.34 ± 0.06^c	2.53 ± 0.11^b	2.89 ± 0.11^a
*Leu#**	3.28 ± 0.14^b	2.22 ± 0.10^c	3.31 ± 0.13^b	3.99 ± 0.14^a
*Phe#**	1.88 ± 0.10^b	1.98 ± 0.11^b	1.80 ± 0.09^b	2.31 ± 0.11^a
Lys#	6.93 ± 0.55^a	7.21 ± 0.57^a	7.02 ± 0.35^a	7.01 ± 0.13^a
HAA	29.66	28.01	29.76	31.22
EAA	24.51	24.53	24.09	28.18
TAA	100	100	100	100
FI, FII, FIII and FIV indicate fractions of peptides with MW more than 50 kDa, between 50 and 10 kDa, between 10 and 5 kDa and below 5 kDa, respectively.
# Asx and Glx indicate Asp + Asn and Glu + Gln, respectively. Trp and Cys were not determined.
HAA (*), EAA (#) and TAA indicate hydrophobic, essentail and total amino acids, respectively.

3.5. RP-HPLC profiles of the hydrolysates and the VPH-P fractions

Besides the AA composition and the free AAs, the RP-HPLC analysis of the different hydrolysates and membrane fractions was also done to determine their hydrophobic character, as illustrated in Fig. 2. The profiles of the VPHs showed the presence of several peaks with different hydrophobic / hydrophilic character. As compared to the UVPs, new peaks appeared after protein hydrolysis, proving the heterogeneous composition of the hydrolysates which differ as a function of the enzyme specificity. Of particular, the last eluted peak detected in the UVPs after 30 min of elution time was gradually reduced after hydrolysis, to be mainly absent in VPH-N. This decrease may be due to the breaking down of the last-eluting proteins to generate new other peptides with different hydrophobic behaviors. The VPH-N and VPH-EE, having the lowest DH values, showed the highest content of hydrophilic peptides, mainly eluted between 5 and 25 min, contrary to the other hydrolysates that showed different hydrophobic character. In this context, Rao et al. [43] reported that the neutral proteases show high affinity for hydrophobic AAs and they are advantageous for controlling their reactivity during the production of food hydrolysates with a low degree of hydrolysis, while the alkaline proteases are characterized by their broad specificity, and therefore the generation of peptides with different hydrophobic properties.

On the other hand, the fractions recovered from the ultrafiltration of VPH-P, were also characterized by RP-HPLC. As UF membrane fractionation allowed the separation of peptides according to their MW, they would exhibit different hydrophobic/hydrophilic characters. The chromatograms showed similar elution profiles, but with different peaks’ intensities. As shown, FIV showed the highest intensity of the highest hydrophobic peptides, which were mainly eluted at high concentration of acetonitrile, higher than 25%.

3.6. Evaluation of the antioxidant activities

3.6.1. Antioxidant activities of the hydrolysates

The antioxidant activities of the different hydrolysates were assessed in terms of the reducing power, ferrous ion chelating activity and β-carotene bleaching inhibition. The results are presented in Fig. 3 (a, b, c).

The reducing power (RP) of the different hydrolysates was evaluated at different concentrations and compared to the undigested proteins and BHA, as negative and positive controls, respectively as shown in Fig. 3a. The reducing power of all the samples increased with increasing concentration to reach a steady state level at high concentrations. Nevertheless, all the samples showed lower reducing power activities than BHA at the tested concentrations. At low concentrations (< 3 mg/ml), except for the VPH-P, no significant differences were observed between the different hydrolysates, while their power increased to reach their maximum at levels above 4 mg/ml. The UVPs and VPH-N showed the lowest reducing power levels (OD₇₀₀ ~ 1 at 5 mg/ml). However, VPH-P reached the highest value (OD₇₀₀ = 3) since a concentration of 2 mg/ml, which indicated that this hydrolysate had a strong ability to reduce ferric ions into their ferrous form. As expected, the highest RP absorbance was obtained in the hydrolysate having the highest DH value (VPH-P with a DH of 30%). In this sense, Wang et al. [44] reported that, at a concentration of 1.35 mg/ml, the Neutrase-treated Mackerel (Pneumatophorus japonicus) viscera hydrolysate, showing the highest DH value (27.96%), exhibited the highest RP absorbance of 0.5.

The β-carotene bleaching inhibition test is based on the protection of β-carotene from discoloration induced by its thermal oxidation by the action of linoleic acid peroxyl radicals, in an emulsified model system. The β-carotene protection levels of the VPHs are reported in Fig. 3b. All the hydrolysates protected the β-carotene from discoloration in a dose-dependent way. In addition, the activities positively correlated with the DH value. In fact, the VPH-P reached an 80% ferrous chelating activity at 1 mg/ml, which is the strongest activity, against 43 and 50% in VPH-EE and VPH-N, respectively, at the same concentration. You et al. [45] reported that the hydrolysate prepared by Protamex from loach (Misgurnus anguillicaudatus) showed the strongest hydroxyl radical scavenging activity (55.0%) at DH 28%, DPPH radical scavenging activity (92.2%) at DH 23%, and the highest reducing power (1.17) at a DH of 33%. The capacity of the hydrolysates on the β-carotene color protection can be hindered by the presence of strong free-radical scavenger peptides in the emulsion mixture. Nevertheless, the UVPs were deprived from any activity at the tested concentrations.

Moreover, the ferrous metal chelating capacity of the UVPs and VPHs was evaluated based on the inhibition of the purple color that resulted from the formation of the Fe²⁺-ferrozine complex. As shown in Fig. 3c, all the samples exhibited a dose-dependent chelating activity, but with different potentialities. Except the VPH-N where the activity did not exceed 60% at 4 mg/ml, a little difference in the activities was observed between all the other hydrolysates. In fact, at 0.5 mg/ml, the chelating activities of VPH-EE, VPH-E and VPH-P were of 74, 81 and 90%, respectively. Of particular, above 1 mg/ml, the Purafect- hydrolysate exhibited a similar activity to EDTA, the positive control, showing values close to 100%. However, the undigested proteins were deprived of any activity, whatever the concentration tested. Murthy et al. [46] reported that the small size croaker fish digested with the crude tuna visceral protease at 4.36% was found to give a protein hydrolysate with the best DPPH free radical scavenging activity, ferric reducing antioxidant power and metal chelating activity.

The findings of the present study correlated with the AA composition, showing that the Purafect-hydrolysate had the highest HAA and His levels, known by their strong antioxidant potential [19, 32]. In addition, it has been reported that acid and basic amino acids with carboxyl and amino groups in the side chains are thought to play an important role in chelating metal ions [47]. Hence, the VPH-P was selected to be fractionated by UF and the activities of the resulted fractions were evaluated.

3.6.2. Antioxidant activities of the membrane fractions

The antioxidant activities of the membrane fractions after VPH-P fractionation were evaluated and the results are presented in Fig. 3 (d, e, f). The ferrous chelating activity and the β-carotene bleaching inhibition were expressed as the half maximal inhibitory concentration (IC₅₀) that inhibits 50% of oxidation, while the reducing power value was expressed at a concentration of 1 mg/ml.

Concerning the RP, data showed that the activity increased as the molecular weight of the peptide fractions decreased. Obviously, FIV containing the < 5 kDa peptides exhibited the highest value (OD₇₀₀ = 1.35) at 1 mg/ml, while at the same concentration, the absorbance was only equal to 0.4 in FI. The high reducing potential observed in FIV may be explained by the presence of peptides gifted by an electron donation capacity. He et al. [48] reported that the all membrane ultrafiltration fractions exhibited DPPH and superoxide scavenging properties, but only the < 1 kDa peptides showed ferric reducing antioxidant power.

Furthermore, to inhibit 50% of linoleic acid peroxyl radicals, a concentration of 271 µg/ml of FIV peptides was enough. Statistical analysis showed that there was significant difference of IC₅₀ between FIV and all the other fractions (p < 0.05), showing very close IC₅₀ values (Fig. 3e). In fact, FI, FII and FIII showed IC₅₀ values between 388 and 464 µg/ml. Even these values are statistically similar (p > 0.05), their level increased with increasing the MW of peptides. The chelating activity of the different MWCO fractions was also expressed in IC₅₀. As expected, FIV containing the lowest MW peptides, showed the lowest IC₅₀ value of 146 µg/ml, which tended to be significantly increased (p < 0.05) to reach 275.11 µg/ml in the FI fraction. In this sense, Ren et al. [49] observed that peptides with MW below 3000 Da, recovered after the UF of grass carp muscle hydrolysate, possessed the strongest hydroxyl radical scavenging ability and lipid inhibition activity, in which more than 57% antioxidant activity was recorded. Hence, FIV peptides can be considered as a promising antioxidant agents. Also, Abou-Diab et al. [13] demonstrated that the enzymatic hydrolysis of hemoglobin under regulated pH condition coupled to electrodialysis with bipolar membranes give bioactive peptides with antibacterial and antioxidant interest.

These findings are in accordance with the amino acids composition of the different fractions showing that the most active fraction FIV was the richest one in HAA and imidazole-ring-containing AA, among all the other samples. It has been reported that the antioxidant activity of Histidine-containing peptides from mussel sauce has been attributed to the hydrogen-donating, lipid peroxyl radical trapping and/or the metal ion-chelating ability of the imidazole group [42]. In addition, Mundi and Aluko [50] showed that the radical scavenging activity for < 1 and 5 − 10 kDa peptide fractions was due to their high contents of aliphatic and aromatic amino acids, compared to the 1–3 and 3–5 kDa membrane fractions. Moreover, Saidi et al. [14] reported that the tuna dark muscle by-product hydrolysate and its membrane fractions, containing the highest level of HAA, are the most able to interact with lipid molecules and lipid-derived radicals through proton donation.

3.8. Antibacterial properties

The antibacterial activities of the defatted viscera hydrolysates as well as the UF membrane fractions were evaluated using the agar diffusion assay against five Gram + and Gram– bacteria by measuring, in mm, the clear zone of bacteria growth inhibition found around the well (Tables 5 and 6). In this test, the antimicrobial effect increases in accordance with the diameter of the zone of inhibition formed. All the bacteria tested were inhibited by, at least one sample. Evidently, different degrees of bacteria growth inhibition capacities were observed, where the VPH-P was found the only hydrolysate able to act against all the bacteria tested and to inhibit specifically S. aureus. The strain M. luteus was the most sensitive against all the hydrolysates, showing large diameters of clear inhibition zones, exceeding 2 cm for the VPH-EE (24.5 mm), VPH-E (22 mm) and VPH-P (23.25 cm). However, S. aureus was the most resistant one, showing a weak clear inhibition zone of 8 mm by the action of VPH-P used at 20 mg/ml. Furthermore, the untreated proteins were found to exhibit inhibitory activity against S. typhi (16 mm), K. pneumoniae (16 mm) and M. luteus (10.5 mm). However, all the obtained antibacterial activities were markedly lower than the Gentamicin disks, used as positive control.

Table 5

Antibacterial activity of defatted VPHs tested at 20 mg/ml
	UVPs	VPH-EE	VPH-N	VPH-E	VPH-P	Gentamicin
E. coli	-	-	-	8.0 ± 1.4^a	8.5 ± 0.7^a	29
K. pneumoniae	16 ± 1.41^a	16.75 ± 0.35^a	-	13.5 ± 0.7^b	14 ± 1.41^ab	22
S. typhi	16 ± 1.41^c	-	18.5 ± 0.7^b	-	20 ± 0.0^a	30
M. luteus	10.5 ± 0.7^b	24.5 ± 0.7^a	10.75 ± 1.06^b	22 ± 1.41^a	23.25 ± 0.35^a	34
S. aureus	-	-	-	-	8.0 ± 0.2	37
Results are expressed in mm of the growth inhibition zone. The gentamicin was used in the form of discs of 30 µg.
a,b,c Different letters in the same line indicate significant differences (p ≤ 0.05).

Table 6

Antibacterial activity of UF fractions of VPH-P tested at 20 mg/ml
	FI	FII	FIII	FIV
K. pneumoniae	24.5 ± 0.7^b	26.25 ± 1.06^b	22.25 ± 1.06^c	33.25 ± 1.06^a
M. luteus	19.25 ± 1.06^d	29 ± 0.0^b	26 ± 1.4^c	35 ± 0.0^a
S. typhi	33.75 ± 1.76^b	37.25 ± 1.06^a	31.10 ± 1.62^c	33.25 ± 1.06^b
Results are expressed in mm of the growth inhibition zone. a,b,c Different letters in the same line indicate significant differences (p ≤ 0.05).

In the same context, Wald et al. [5] demonstrated that the highest antibacterial activity against Flavobacterium psychrophilum and Renibacterium salmoninarum was obtained with the Trout viscera hydrolysate having a DH value of 30%. Moreover, Jang et al. [51] demonstrated that all the examined antioxidant peptides were found to inhibit all the pathogenic bacteria tested (E. coli, Pseudomonas aeruginosa, S. typhimurium, S. aureus, Bacillus cereus and Listeria monocytogenes). In addition, Di Bernardini et al. [52] reported that previous works on antimicrobial peptides showed that their activity was influenced by the presence of hydrophobic amino acids.

Thereafter, only the three most sensitive bacteria to the VPH-P action (S. typhi, K. pneumoniae and M. luteus) were selected to be tested against the MWCO fractions, as shown in Table 6. All the bacteria tested were inhibited by all the fractions, but with different potentialities. In fact, the best inhibition values were recorded with FIV with clear zone diameters superior to 3 cm against all the bacteria, where the S. typhi was the most sensitive. The inhibition potentialities of the MWCO fractions reflected their differences in terms of size, composition and sequences. In fact, it has been reprted by Ennaas, et al. [53] that hydrophobic and cationic peptides purified from Atlantic mackerel (Scomber scombrus) by-products exhibited the highest antibacterial activity against Listeria innocua and E. coli strains. In addition, the mackerel was used by Offret et al. [54] to produce antimicrobial peptides characterized by their high Leu and Ile content and positively charged amino acid residues. However, Beaulieu et al. [55] demonstrated that the > 10 kDa peptide fraction from Saccharina longicruris trypsin-hydrolysate exhibited the highest activity against S. aureus bacterium. Therefore, the overall results showed the potential use of the VPH fractions as good food-preservatives to prevent bacterial growth.

After selecting the best defatting method, viscera from smooth hound (M. mustelus) were used as raw material for the production of four protein hydrolysates using three microbial enzymes and the endogenous crude extract (autolysate). Their total and free AA contents and hydrophobic properties were determined and correlated to the final DH value obtained in each hydrolysate. In addition, their antioxidant, ACE-inhibitory and antimicrobial activities revealed the high potentiality of the Purafect-hydrolysate. Therefore, it was selected to be fractionated by ultra-filtration and the recovered MWCO fractions were characterized. The results showed that the last fraction contacting the lowest MW peptides (FIV) exhibited the best bio-activities with the lowest IC₅₀ values. The differences in bioactivities detected in the tested fractions are mainly due to the differences in the AA composition, size and sequences. So, the results of the present study demonstrated the high potential of FIV from the VPH-P to be used a promising natural source of bioactive peptides for further biotechnology applications.

Acknowledgements

This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia.

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Proteolysis coupled with membrane separation for the isolation of bioactive peptides from defatted smooth hound byproduct proteins

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Abstract

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1. Introduction

2. Materials and methods

2.1. Raw material preparation and defatting

2.2. Enzymes

2.3. Protein hydrolysates preparation

2.4. Physic-chemical analysis

2.5. UF fractionation

2.6. Amino acids composition

2.7. Free amino acids content

2.8. RP-HPLC analysis

2.9. Antioxidant activities assessment

2.9.1. Reducing power assay

2.9.2. Iron chelating activity

2.9.3. β-carotene bleaching protection

2.10. Antibacterial activity

2.11. Statistical analysis

3. Results and discussion

3.1. Preparation of protein hydrolysates from defatted smooth hound viscera

3.2. Fractionation by ultrafiltration membrane system

3.3. Amino acids composition and free amino acids of VPHs

3.4. Amino acids composition of the membrane fractions

3.5. RP-HPLC profiles of the hydrolysates and the VPH-P fractions

3.6. Evaluation of the antioxidant activities

3.6.1. Antioxidant activities of the hydrolysates

3.6.2. Antioxidant activities of the membrane fractions

3.8. Antibacterial properties

4. Conclusion

Declarations

References

Supplementary Files

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