RESUMO
Growing consumer awareness of the potential negative health effects of synthetic antibiotics has prompted the search for more natural preservatives that can improve the safety and quality of food. In this study we report the enzymatic synthesis of N-α-[Carbobenzyloxy]-Ile-Gln (Z-IQ) which is the precursor of Ile-Gln (IQ), a new antibacterial dipeptide, using an aqueous-organic biphasic system formed by 50% (v/v) ethyl acetate in 0.1 M Tris - HCl buffer pH 8. A partially purified proteolytic extract from the fruits of Solanum granuloso leprosum, named granulosain, proved to be a robust biocatalyst for the synthesis of Z-IQ, eliciting 71 ± 0.10% maximal peptide yield in the above described conditions. After cleaving and purifying IQ dipeptide, antimicrobial activity was assayed against Staphylococcus aureus ATCC 25923, Staphylococcus hominis A17771, and Staphylococcus aureus C00195, and MIC values between 118 ± 0.01 µg/mL and 133.7 ± 0.05 µg/mL were obtained. In addition, IQ showed MIC of 82.4 ± 0.01 µg/mL and 85.0 ± 0.00 µg/mL against Escherichia coli ATCC 25922 and Escherichia coli A17683, respectively. IQ did not show inhibitory activity against single-drug resistance (SDR) strains, such as Klebsiella oxytoca A19438 (SDR) and Pseudomonas aeruginosa C00213 (SDR), and against multidrug-resistant Enterococcus faecalis I00125 (MDR). IQ also caused growth inhibition of Helicobacter pylori NCTC 11638 and three wild-type H. pylori strains, which are sensitive to AML, MTZ, LEV and CLA (H. pylori 659), resistant to LEV (H. pylori 661 SDR), and resistant to MTZ (H. pylori 662 SDR). Finally, this study contributes with a new dipeptide (IQ) that can be used as an antimicrobial agent for food preservation or as a safe ingredient of functional foods.
RESUMO
Antiacanthain and granulosain are the partially purified proteolytic extracts from the South American native fruits of Bromelia antiacantha (Bertol. ) and Solanum granuloso leprosum, respectively. The aim of this work was to compare the ability of both soluble and immobilized antiacanthain and granulosain f or the synthesis of Z-Tyr-Val-OH, a novel antibacterial dipeptide, in different reaction systems formed by almost anhydrous organic solvents (Xw: 1 × 10-5) and several percentages of immiscible organic solvents in 100 mM Tris(hydroxymethyl)aminomethane hydrochloride buffer pH 8.0. Soluble antiacanthain in half of the 24 different organic biphasic media showed higher catalytic potential than in 100 mM Tris(hydroxymethyl)aminomethane hydrolchloride buffer pH 8.0. Soluble granulosain showed lower catalytic potential in all liquid-liquid biphasic media than in the same buffer. However, 50% (v/v) ethyl ethanoate in 100 mM Tris(hydroxymethyl)aminomethane hydrolchloride buffer pH 8.0 allowed to express the highest catalytic potential of both soluble enzymes. In 50% v/v ethyl ethanoate, soluble antiacanthain and granulosain catalyzed the synthesis of Z-Tyr-Val-OH with 72 ± 0.15 and 60 ± 0.10% maximal peptide yields, respectively. Multi-point immobilization in glyoxyl-silica did not lead to better peptide yields than soluble enzymes, in that liquid-liquid biphasic medium under the same reaction conditions. Soluble and glyoxyl-silica immobilized antiacanthain in almost anhydrous ethyl ethanoate (Xw: 1 × 10-5) were able to retain 17.3 and 45% of the initial proteolytic activity of antiacanthain in 100 mM Tris hydrolchloride buffer pH 8.0, respectively, at 40°C under agitation (200 rpm). Soluble and glyoxyl-silica immobilized granulosain were inactivated under the same reaction conditions. Glyoxyl-silica immobilized antiacanthain showed to be a robust biocatalyst in almost anhydrous ethyl ethanoate (Xw: 1 × 10-5), eliciting the best peptide yield (75 ± 0.13%). The synthesis reaction of Z-Tyr-Val-OH could not proceed when soluble antiacanthain was used under the same conditions. Both peptidases only catalyzed the synthesis reaction under kinetic control, using activated acyl donor substrates. Finally, this work reports a novel broad-spectrum antibacterial peptide that significantly decreased (p ≤ 0.05) the specific growth rates of Gram positive and Gram negative microorganisms at very low concentrations (≥15 and 35 µg/ml, respectively); contributing with a new safe food preservative of applying for different food systems.
RESUMO
Helicobacter pylori is a gram-negative, helix-shaped, and microaerophilic bacteria that colonizes the human gastric mucosa, causing chronic infections, gastritis, peptic ulcer, lymphomas associated with lymphoid mucosa tissue, and gastric cancer. H. pylori is considered a Type 1 human carcinogen by WHO. The prevalence of the infection is estimated in more than half of the world population. Treatment of H. pylori infection includes antibiotics and proton pump inhibitors, but the increasing antibiotic resistance promotes the research of novel, more effective, and natural antibacterial compounds. The aim of this work was to study the effect of the partially purified proteolytic extract (RAP) of the fruits from Solanum granuloso-leprosum (Dunal), a South American native plant, and a purified fraction named granulosain I, against H. pylori, to obtain natural food additives for the production of anti-H. pylori functional foods. Furthermore, granulosain I and RAP could be used as natural adjuncts to conventional therapies. Granulosain I and RAP antibacterial activity was evaluated as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against H. pylori NCTC 11638 (reference strain) and twelve H. pylori wild strains, using a microdilution plating technique (Clinical and Laboratory Standards Institute). All the strains tested were susceptible to granulosain I with MIC from 156.25 to 312.5 µg/mL and MBC from 312.5 to 625 µg/mL, respectively. Besides, all the strains tested were susceptible to the RAP with MIC from 312.5 to 625 µg/mL and MBC from 625 to 1,250 µg/mL, respectively. The effect of granulosain I and RAP on the transcription of H. pylori genes encoding pathogenic factors, omp18, ureA, and flaA, with respect to a housekeeping gene (16S rRNA), was evaluated by RT-PCR technique. The band intensity between pathogenic factors and control gene was correlated under treated or untreated conditions, using the ImageJ program. Granulosain I and RAP significantly decreased the expression of pathogenic factors: omp18, ureA, and flaA. The combined inhibitory effect of granulosain I or RAP and an antibiotic such as, amoxicillin (AML, 10 µg), clarithromycin (CLA, 15 µg), levofloxacin (LEV, 5 µg), and metronidazole (MTZ, 5 µg) was evaluated, using the agar diffusion technique. Granulosain I and RAP showed significant synergistic effect on AML, CLA, and LEV, but no significant effect on MTZ was observed. Besides, granulosain I and RAP did not show toxicological effects at the concentrations studied. Finally, granulosain I and RAP could be used as safe natural food additives and as adjuvants for conventional therapies against H. pylori.