A novel antimicrobial peptide obtained by enzyme synthesis potentially useful as natural bio-preservative to enhance the shelf-life of foods

Abstract

Peptides are polymers containing from two to few dozen amino acids with a molecular weight limit of 6,000 Da, and relevant to the health and nutrition. Antibacterial peptides have acquired significance because of the microbial increasing resistance to conventional antibiotics, so that they are becoming a promising alternative to them (Bersi et al., 2018).The aim of this work is to compare the ability of both soluble antiacanthain and granulosain, the pre-purified proteolytic extracts obtained from the latex of Bromelia antiacantha (Bertol.) and Solanum granuloso leprosum, respectively; for the synthesis of N-α-CBZ-Tyr-Val-OH (Z-YV), a novel antibacterial dipeptide, in a media formed by 50 % v/v ethyl acetate in 0.1 M Tris ? ClH buffer pH 8.Antiacanthain and granulosain, the pre-purified proteolytic extracts from the latex of Bromelia antiacantha (Bertol.) and Solanum granuloso leprosum, respectively, were obtained according to Vallés et al., 2004 and 2008; and lyophilized for later immobilization.The reaction of synthesis were carried out in a 50 % v/v biphasic system, consisting of an aqueous phase (0.1 M Tris?HCl buffer pH 8) containing 0.075 mg/mL of soluble antiacanthain or 0.064 mg/mL of soluble granulosain, 20 mM 2-mercaptoethanol, 7.55 mM of both Val-OH and triethyl ammonium (TEA), and an organic phase (ethyl acetate) containing 17.25 mM of Z-Y-pNO. The reaction was conducted at 40 °C in a GFL Shaking Incubator Orbital Motion (Model 3031, Germany) at 200 rpm. Aliquots (0.1 mL) were taken from the aqueous and organic phase at different times during 24 h and mixed with 0.1 mL of 1 % v/v trifluoroacetic acid (TFA) for quenching the reaction. The reaction components were analyzed by RP-HPLC and the synthetized peptide was purified by means of C18 cartridge (Merck), lyophilized and elucidated by mass spectrometry (MS).After optimization, 72 % and 60 % product yields (Z-YV) were obtained using antiacanthain and granulosain, respectively.Antibacterial activity of Z-YV was tested by means of three independent trials, which were done by duplicate, against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 in batch culture of Müller-Hinton broth, using Z-YV (0 - 50 g/mL), at 37 ºC and 180 rpm. According to Kruskal Wallis test, the specific growth rates of S. aureus and E. coli in batch culture of Müller-Hinton broth alone (control) and with the addition of Z-YV showed significant differences (p ≤ 0.05) within studied range. Then, Z-YV is a promising natural bio-preservative to enhance the shelf-life of foods.