Microscopic and metabolomics analysis of the anti-Listeria activity of natural and engineered cruzioseptins.

Listeria monocytogenes is a human opportunistic foodborne pathogen that produces life-threatening infections with a high mortality rate. The control of Listeria in the food production environment and effective clinical management of human listeriosis are challenging due to the emergence of antibiotic resistance. Hence we evaluate the in vitro anti-Listeria activity of two synthetic cruzioseptins reproducing their natural sequences CZS-9, and CZS-12, and one engineered sequence based on CZS-1, named [K4K15]CZS-1. The assessment of the in vitro potential of cruzioseptins, highlighted the promising antibacterial effect of [K4K15]CZS-1 in very low concentrations (0.91 µM) and its thermal stability at high-temperature conditions, is compatible with the food industry. Microscopic and metabolomic analyses suggest cruzioseptin induces anti-Listeria bioactivity through membrane disruption and changes in the intracellular metabolome. We also report that [K4K15]CZS-1 is not resistant to peptidases/proteases emphasizing a key advantage for their use as a food preservative. However, there is a need for further structural and functional optimisations for the potential clinical application as an antibiotic. In conclusion, [K4K15]CZS-1 stand out as membrane-active peptides with the ability to induce shifts in the bacteria metabolome and inspire the development of strategies for the prevention of L. monocytogenes emergence and dissemination.

Biorefinery of brewery spent grain to obtain bioproducts with high value-added in the market.

The brewery industry is under economic and environmental pressure to minimize residual management costs, particularly brewery spent grain (BSG), which accounts for 80-85% (w/w) of the total by-products generated. BSG is a lignocellulosic material primarily composed of carbohydrates, proteins and lipids. Developing a biorefinery model for conversion of BSG into value-added products is a plausible idea. Previous work optimized the pretreatment of BSG with the ionic liquid [N1112OH][Gly] and further release of fermentable sugar-containing solutions by enzymatic hydrolysis, using an enzymatic cocktail obtained by solid-state fermentation of BSG with Aspergillus brasiliensis CECT 2700 and Trichoderma reesei CECT 2414. The current work ends the biorefinery process, studying the fermentation of these culture media with two LAB strains, Lactobacillus pentosus CECT 4023 and Lactobacillus plantarum CECT 221, from which the production of organic acids, bacteriocins, and microbial biosurfactants (mBS) was obtained. In addition to the bacteriocin activity observed, a mass balance of the whole biorefinery process quantified the production of 106.4 g lactic acid and 6.76 g mBS with L. plantarum and 116.1 g lactic acid and 4.65 g mBS with L. pentosus from 1 kg of dry BSG. Thus, BSG shows a great potential for waste valorization, playing a major role in the implementation of biomass biorefineries in circular bioeconomy.

Biorefinery of Brewery Spent Grain by Solid-State Fermentation and Ionic Liquids.

Novel environmentally friendly pretreatments have been developed in recent years to improve biomass fractionation. Solid-state fermentation (SSF) and treatment with ionic liquids show low environmental impact and can be used in biorefinery of biomass. In this work, these processes were assessed with brewery spent grain (BSG). First, BSG was used as a substrate to produce cellulases and xylanases by SSF with the fungi Aspergillus brasiliensis CECT 2700 and Trichoderma reesei CECT 2414. Then, BSG was pretreated with the ionic liquid [N1112OH][Gly] and hydrolyzed with the crude enzymatic extracts. Results showed that SSF of BSG with A. brasiliensis achieved the highest enzyme production; meanwhile, the pretreatment with ionic liquids allowed glucan and xylan fractions to increase and reduce the lignin content. In addition, a mixture of the extracts from both fungi in a ratio of 2.5:0.5 Aspergillus/Trichoderma (v/v) efficiently hydrolyzed the BSG previously treated with the ionic liquid [N1112OH][Gly], reaching saccharification percentages of 80.68%, 54.29%, and 19.58% for glucan, xylan, and arabinan, respectively. In conclusion, the results demonstrated that the BSG biorefinery process developed in this work is an effective way to obtain fermentable sugar-containing solutions, which can be used to produce value-added products.

Pan-genomic and comparative analysis of Pediococcus pentosaceus focused on the in silico assessment of pediocin-like bacteriocins.

Bacteriocins are antimicrobial peptides produced by different species of bacteria, especially the Gram-positive lactic acid bacteria (LAB). Pediococcus pentosaceus is widely applied in the industry and stands out as Bacteriocin-Like Inhibitory Substances (BLIS) producer known to inhibit pathogens commonly considered a concern in the food industries. This study aimed to perform in silico comparisons of P. pentosaceus genomes available in the public GenBank database focusing on their pediocin-like bacteriocins repertoire. The pan-genome analysis evidenced a temporal signal in the pattern of gene gain and loss, supporting the hypothesis that the complete genetic repertoire of this group of bacteria is still uncovered. Thirteen bacteriocin genes from Class II and III were predicted in the accessory genome. Four pediocin-like bacteriocins (54% of the detected bacteriocin repertoire) and their accompanying immunity genes are highlighted; penocin A, coagulin A, pediocin PA-1, and plantaricin 423. Additionally, in silico, modeling of the pediocin-like bacteriocins revealed different configurations of the helix motif compared to other physically determined pediocin-like structures. Comparative and phylogenomic analyses support the hypothesis that a dynamic mechanism of bacteriocin acquisition and purging is not dependent on the bacterial isolation source origin. Synteny analysis revealed that while coagulin A, pediocin PA-1, and Plantaricin 423 loci are associated with insertion sequences mainly from the IS30 family and are likely of plasmid origin, penocin A lies in a conserved chromosomal locus. The results presented here provide insights into the unique pediocin-like bacteriocin peptide fold, genomic diversity, and the evolution of the bacteriocin genetic repertoire of P. pentosaceus, shedding new insights into the role of these biomolecules for application in inhibiting bacterial pathogens, and suggesting that prospecting and sequencing new strains is still an alternative to mining for new probiotic compounds.

Fed-batch production of vanillin by Bacillus aryabhattai BA03.

Bacillus aryabhattai BA03, a strain isolated in our laboratory, has interesting properties related to the production of natural aromas and flavors. Specifically, we have found that it was able to produce vanillin from ferulic acid (FA). Furthermore, this strain produces high amounts of 4-vinylguaiacol in only 14h, this being the only intermediate metabolite observed in the process. FA is an inexpensive feedstock for the production of natural value-added compounds when extracted from lignocellulosic wastes. In this study, we optimized the operational conditions (temperature, pH and agitation), medium composition and bioconversion technology (batch or fed-batch) to produce vanillin. In a fed-batch process conducted with just one additional supplementation after 24h, the maximal concentration of vanillin (147.1±0.9mg/L) was observed after 216h (QV=0.681mg/Lh; YV/fFA=0.082mg/mg) after degrading 90.3% FA. In view of our data, we postulate that Bacillus aryabhattai BA03 carries out a decarboxylation of ferulic acid as a metabolic pathway.

Ferulic acid transformation into the main vanilla aroma compounds by Amycolatopsis sp. ATCC 39116.

The wild strain Amycolatopsis sp. ATCC 39116 was explored in ferulic acid-based media to produce naturally the aroma components of the cured vanilla pod, namely vanillin,vanillic acid, and vanillyl alcohol. Other phenolic compounds(4-vinyl guaiacol, guaiacol, and protocatechuic acid) were also evaluated. The influence of medium composition,fermentation technology (batch or fed-batch), supplementation with vanillic acid, and inoculum concentration on ferulic acid biotransformation were evaluated. The results postulate the initial concentration of cell mass as the variable with the strongest impact on ferulic acid metabolization under the studied conditions. The highest amounts of vanillin and vanillic acid were achieved at intermediate values of cell mass.Vanillyl alcohol and protocatechuic acid were more closely linked to high cell mass concentrations. Conversely, 4-vinyl guaiacol reached its highest amount at the lowest amount of cell mass. Guaiacol was not detected in any case. Therefore,the initial cell concentration must be considered a critical parameter when using Amycolaptosis sp. ATCC 39116 for the production of vanillin and related compounds.