Identification of colupulone and lupulone as the main contributors to the antibacterial activity of hop extracts using activity-guided fractionation and metabolome analysis.

Hop is widely used in beer brewing and as a medicinal product. The present study comprehensively analyzed the main molecular determinants of the antibacterial activity of hop extracts. Minimum inhibitory concentrations (MIC) against Bacillus subtilis between 31.25 and 250 µg/mL were found in the ethanolic extracts of five hop varieties for beer brewing, but not in the tea hop sample. Activity-guided fractionation revealed the highest antibacterial activity for lupulone and adlupulone (MIC 0.98 µg/mL). Metabolome profiling and subsequent multistep statistical analysis detected 33 metabolites out of 1826 features to be associated with the antibacterial activity including humulone, adhumulone, colupulone, lupulone, and adlupulone. Xanthohumol, the three humulone- and three lupulone congeners were quantified in the hop extracts by a validated ultrahigh-performance liquid chromatography-mass spectrometry method. Considering concentrations and MICs, colupulone and lupulone were identified as major contributors to the antibacterial activity of hop extract with the highest antibacterial activity values (concentration/MIC) of 1.59 and 2.56.

Stability and Activity of the Antimicrobial Peptide Leg1 in Solution and on Meat and Its Optimized Generation from Chickpea Storage Protein.

The antimicrobial peptide Leg1 (RIKTVTSFDLPALRFLKL) from chickpea legumin is active against spoilage bacteria, yeast, and mold. The present study tested its effectiveness under food storage conditions and examined options to obtain a food-grade agent. The minimum inhibitory concentration (MIC) of Leg1 against E. coli (62.5 µM) proved stable over seven days at 20 °C or 4 °C. It was not influenced by reduced pH (5.0 vs. 6.8), which is relevant in food such as meat. An incubation temperature of 20 °C vs. 37 °C reduced the MIC to 15.6/7.8 µM against E. coli/B. subtilis. With a minimum bactericidal concentration in meat of 125/15.6 µM against E. coli/B. subtilis, Leg1 is equivalently effective as nisin and 5000-82,000 times more active than sodium benzoate, potassium sorbate, or sodium nitrite. Replacing the counter-ion trifluoroacetate derived from peptide synthesis by the more natural alternatives acetate or chloride did not impair the activity of Leg1. As an alternative to chemical synthesis, an optimized protocol for chymotryptic hydrolysis was developed, increasing the yield from chickpea legumin by a factor of 30 compared to the standard procedure. The present results indicate that food-grade Leg1 could possibly be applicable for food preservation.

Antioxidative, Antifungal and Additive Activity of the Antimicrobial Peptides Leg1 and Leg2 from Chickpea.

The fight against food waste benefits from novel agents inhibiting spoilage. The present study investigated the preservative potential of the antimicrobial peptides Leg1 (RIKTVTSFDLPALRFLKL) and Leg2 (RIKTVTSFDLPALRWLKL) recently identified in chickpea legumin hydrolysates. Checkerboard assays revealed strong additive antimicrobial effects of Leg1/Leg2 with sodium benzoate against Escherichia coli and Bacillus subtilis with fractional inhibitory concentrations of 0.625 and 0.75. Additionally, Leg1/Leg2 displayed antifungal activity with minimum inhibitory concentrations of 500/250 µM against Saccharomyces cerevisiae and 250/125 µM against Zygosaccharomyces bailii. In contrast, no cytotoxic effects were observed against human Caco-2 cells at concentrations below 2000 µM (Leg1) and 1000 µM (Leg2). Particularly Leg2 showed antioxidative activity by radical scavenging and reducing mechanisms (maximally 91.5/86.3% compared to 91.2/94.7% for the control ascorbic acid). The present results demonstrate that Leg1/Leg2 have the potential to be applied as preservatives protecting food and other products against bacterial, fungal and oxidative spoilage.

Generation of antimicrobial peptides Leg1 and Leg2 from chickpea storage protein, active against food spoilage bacteria and foodborne pathogens.

Contamination with bacteria leads to food waste and foodborne diseases with severe consequences for the environment and human health. Aiming to reduce food spoilage and infection, the present study developed novel highly active food-grade antimicrobial peptides affecting a wide range of bacteria. After extraction from chickpea, the storage protein legumin was hydrolyzed by the digestive protease chymotrypsin. Subsequent analysis by ultrahigh-performance micro-liquid chromatography-triple quadrupole time-of-flight tandem mass spectrometry determined the resulting peptide profiles. Virtual screening identified 21 potential antimicrobial peptides in the hydrolysates. Among those, the peptides Leg1 (RIKTVTSFDLPALRFLKL) and Leg2 (RIKTVTSFDLPALRWLKL) exhibited antimicrobial activity against 16 different bacteria, including pathogens, spoilage-causing bacteria and two antibiotic-resistant strains. Leg1/Leg2 showed minimum inhibitory concentrations (MIC) down to 15.6 µmol/L and were thus 10-1,000-fold more active compared to conventional food preservatives. Moreover, Leg1 and Leg2 showed bactericidal activity in contrast to the bacteriostatic activity of conventional preservatives.

Analysis of Chemically Labile Glycation Adducts in Seed Proteins: Case Study of Methylglyoxal-Derived Hydroimidazolone 1 (MG-H1).

Seeds represent the major source of food protein, impacting on both human nutrition and animal feeding. Therefore, seed quality needs to be appropriately addressed in the context of viability and food safety. Indeed, long-term and inappropriate storage of seeds might result in enhancement of protein glycation, which might affect their quality and longevity. Glycation of seed proteins can be probed by exhaustive acid hydrolysis and quantification of the glycation adduct Nɛ-(carboxymethyl)lysine (CML) by liquid chromatography-mass spectrometry (LC-MS). This approach, however, does not allow analysis of thermally and chemically labile glycation adducts, like glyoxal-, methylglyoxal- and 3-deoxyglucosone-derived hydroimidazolones. Although enzymatic hydrolysis might be a good solution in this context, it requires aqueous conditions, which cannot ensure reconstitution of seed protein isolates. Because of this, the complete profiles of seed advanced glycation end products (AGEs) are not characterized so far. Therefore, here we propose the approach, giving access to quantitative solubilization of seed proteins in presence of sodium dodecyl sulfate (SDS) and their quantitative enzymatic hydrolysis prior to removal of SDS by reversed phase solid phase extraction (RP-SPE). Using methylglyoxal-derived hydroimidazolone 1 (MG-H1) as a case example, we demonstrate the applicability of this method for reliable and sensitive LC-MS-based quantification of chemically labile AGEs and its compatibility with bioassays.