Effect of pH on the Antimicrobial Activity and Peptide Population of Pepsin Hydrolysates Derived from Bovine and Porcine Hemoglobins

Abstract

Slaughterhouse blood is considered a valuable by-product of the meat industry that is mainly derived from the slaughter of cattle and pigs. Its cruor fraction is mainly constituted of hemoglobin, which has been widely described as an important source of several antimicrobial peptides that can be released by hydrolysis with pepsin. Despite the high sequence similarity between bovine (Hb-B) and porcine (Hb-P) hemoglobins, the release of antimicrobial peptides may be affected by varying hydrolysis conditions. This study presents, for the first time, the impact of pH (pH 2, 3, 4, and 5) on the antimicrobial peptide population of hydrolysates derived from the main hemoglobin sources by using pepsin (bovine and porcine). In the same way, their antibacterial, antifungal, and antiyeast activities are compared, as well as their type of mechanism of action (static or cidal effect). These characteristics have not been described previously in the literature. A total of 29 peptides from Hb-B and 35 peptides for Hb-P with potential antimicrobial peptides were identified by reversed-phase ultrahigh-performance liquid chromatography with tandem mass spectrometry (RP-UPLC–MS/MS) and bioinformatics tools. Similarities in the peptide population between Hb-B and Hb-P hydrolysates were observed by PCA, mainly in the enzymatic hydrolysis at pH 2 and pH 3. Both bovine and porcine hydrolysates produced at pH 2 and pH 3 showed antibacterial activity against L. ivanovii (MIC = 0.62–1.25 mg·mL–1) and antiyeast activity against R. mucilaginosa (0.31–1.25 mg·mL–1). Conversely, only antifungal activity against M. racemosus in the porcine hydrolysate at pH 3 was observed (1.25 mg·mL–1). This study confirms the similarities of Hb-B and Hb-P in terms of peptide profiles and antimicrobial activity, which indicates they are suitable sources of antimicrobial peptides with antibacterial, antifungal, and antiyeast activities that could be employed in the preservation of food products.

Impact of Pulsed Electric Fields and pH on Enzyme Inactivation and Bioactivities of Peptic Hydrolysates Produced from Bovine and Porcine Hemoglobin

“…Figure 2 shows the workflow of the present study. Hb-B and Hb-P hydrolysates were produced according to Sanchez-Reinoso et al [ 17 ] with slight modifications. Briefly, 0.5% ( w / w ) solutions (1 L) of Hb-B and Hb-P were prepared with Milli-Q water via overnight stirring (150 rpm) at 4 °C.…”

“…The molar concentration of peptides was determined by Equation (2), which was based on the UV peak areas recovered from RP-UPLC chromatograms at 214 nm [ 54 ]. where C pep (µM) is the concentration of peptides, A 214 (AU min) is the RP-UPLC peak area of the absorbance at 214 nm, L (cm) is the path length of the UV cell (1 cm), V inj (μL) is the injection volume (1 μL), f is the flow rate (500 μL/min), K cell is the cell constant (which was previously determined to be 0.06 via calibration using pure peptide solutions of neokyotorphin (TSKYR) at known concentrations, assessed using the same parameter of analysis) [ 17 ], and ε 214 (1/M cm) is the molar extinction coefficient at 214 nm, determined for each peptide according to Kuipers and Gruppen [ 55 ]. Co-elution of peptides was observed in some RP-UPLC peaks.…”

“…Listeria ivanovii HP B28 (Gram + bacteria) and Escherichia coli MP 4100 (Gram − bacteria) were assessed for antibacterial activity. Escherichia coli was chosen since it is a common foodborne pathogen, while Listeria ivanovii was selected because it is a non-pathogenic strain, which can give an idea about the prevention of listeriosis (an issue for some foods) [ 17 ]. Antifungal and anti-yeast activities were evaluated for Mucor racemosus LMA-722 (filamentous fungal strain) and Rhodotorula mucilaginosa 27,173 (yeast strain), respectively.…”

“…The agar-well diffusion assay was performed as described in detail in our previous study [ 17 ]. Briefly, the culture media were inoculated with the target strains.…”

“…In this context, pepsin has shown better performance for hemoglobin hydrolysis in comparison to other enzymes such as trypsin, papain, and alcalase, leading to a higher production of bioactive peptides [ 3 , 13 , 14 , 16 ]. More than 30 antimicrobial peptides have been identified in peptic hydrolysates of bovine hemoglobin (Hb-B); among them, antibacterial activity against both Gram + and Gram − strains, as well as antifungal ( Candida albicans , Aspergillus niger , Mucor racemosus , Penicillium crustosum , and Paecilomyces spp) and anti-yeast ( Rhodotorula mucilaginosa ) activities have been reported [ 17 ]. In contrast, antimicrobial peptides derived from porcine hemoglobin (Hb-P) have been studied to a much lesser extent, but their antibacterial ( Micrococcus luteus , Escherichia coli , Salmonella Newport , and Listeria ivanovii ) antifungal ( Mucor racemosus ), and anti-yeast ( Rhodotorula mucilaginosa ) activities have also been demonstrated in crude peptic hydrolysates [ 17 , 18 ].…”