Dual antibacterial mechanism of [K4K15]CZS-1 against Salmonella Typhimurium: a membrane active and intracellular-targeting antimicrobial peptide.

Salmonella genus is a leading cause of food-borne infections with strong public health impact and economic ramifications. The development of antimicrobial resistance added complexity to this scenario and turned the antibiotic drug discovery into a highly important challenge. The screening of peptides has served as a successful discovery platform to design new antibiotic candidates. Motivated by this, the antimicrobial and cytotoxic properties of three cruzioseptins against Salmonella Typhimurium and RAW 264.7 murine macrophage cells, respectively, were investigated. [K4K15]CZS-1 was the most potent antimicrobial peptide identified in the screening step with a minimum inhibitory concentration (MIC) of 16 µg/mL (7.26 µM) and moderate cytotoxicity. From a structural point of view, in vitro and in silico techniques evidenced that [K4K15]CZS-1 is a α-helical cationic antimicrobial peptide. In order to capture mechanistic details and fully decipher their antibacterial action, we adopted a multidimensional approach, including spectroscopy, electron microscopy and omics analysis. In general lines, [K4K15]CZS-1 caused membrane damage, intracellular alterations in Salmonella and modulated metabolic pathways, such as the tricarboxylic acid (TCA) cycle, fatty acid biosynthesis, and lipid metabolism. Overall, these findings provide deeper insights into the antibacterial properties and multidimensional mode of action of [K4K15]CZS-1 against Salmonella Typhimurium. In summary, this study represents a first step toward the screening of membrane-acting and intracellular-targeting peptides as potential bio-preservatives to prevent foodborne outbreaks caused by Salmonella.

Enhanced production of N-acetyl-glucosaminidase by marine Aeromonas caviae CHZ306 in bioreactor.

N-Acetyl-glucosaminidases (GlcNAcases) are exoenzymes found in a wide range of living organisms, which have gained great attention in the treatment of disorders related to diabetes, Alzheimer's, Tay-Sachs', and Sandhoff's diseases; the control of phytopathogens; and the synthesis of bioactive GlcNAc-containing products. Aiming at future industrial applications, in this study, GlcNAcase production by marine Aeromonas caviae CHZ306 was enhanced first in shake flasks in terms of medium composition and then in bench-scale stirred-tank bioreactor in terms of physicochemical conditions. Stoichiometric balance between the bioavailability of carbon and nitrogen in the formulated culture medium, as well as the use of additional carbon and nitrogen sources, played a central role in improving the bioprocess, considerably increasing the enzyme productivity. The optimal cultivation medium was composed of colloidal α-chitin, corn steep liquor, peptone A, and mineral salts, in a 5.2 C:N ratio. Optimization of pH, temperature, colloidal α-chitin concentration, and kLa conditions further increased GlcNAcase productivity. Under optimized conditions in bioreactor (i.e., 34 °C, pH 8 and kLa 55.2 h-1), GlcNAcase activity achieved 173.4 U.L-1 after 12 h of cultivation, and productivity no less than 14.45 U.L-1.h-1 corresponding to a 370-fold enhancement compared to basal conditions.

Evaluating the potential of Pediococcus pentosaceus as a biocontrol agent against tenuazonic acid-producing Alternaria alternata on livestock feeds.

AIMS: This study aims to demonstrate the potential of the lactic acid bacteria (LAB) Pediococcus pentosaceus LBM18 against the mycotoxin-producing Alternaria alternata TEF-1A and highlight its application as an effective grain silage inoculant to control mycotoxin contamination. METHODS AND RESULTS: The antifungal properties of Ped. pentosaceus lyophilized (PPL) were assessed by evaluating its effect on A. alternata TEF-1A grown in a corn silage-based medium, which included morphological changes by Scanning Electron Microscopy (SEM) observations, growth rate, conidia production assays, and inhibition of Tenuazonic acid (TeA) production by high-performance liquid chromatography (HPLC-MS/MS) analyses. Furthermore, TeA biosynthesis was monitored for changes at the molecular level by PKS gene expression. The growth and sporulation processes of A. alternata TEF-1A were affected by Ped. pentosaceus LBM18 in a concentration-dependent manner. Moreover, a significant inhibition of TeA production (74.3%) and the transcription level of the PKS gene (42.9%) was observed. CONCLUSIONS: Ped. pentosaceus is one of the promising LAB to be applied as an inoculant for corn silage preservation, aiming to inhibit mycotoxigenic fungi growth and their mycotoxin production. SIGNIFICANCE AND IMPACT OF THE STUDY: Ped. pentosaceus could be used as an inoculant to reduce fungal and mycotoxins contamination in grain silage production.

Bacteriocin-like inhibitory substances production by Enterococcus faecium 135 in co-culture with Ligilactobacillus salivarius and Limosilactobacillus reuteri.

The use of lactic acid bacteria (LAB) and probiotic cultures in the breeding of animals such as poultry and swine are quite common. It is known that those strains can produce bacteriocins when grown in pure culture. However, the production of bacteriocin using co-culture of microorganisms has not been much studied so far. The present study contributes with innovation in this area by embracing the production of bacteriocin-like inhibitory substances (BLIS) by a newly isolated strain of Enterococcus faecium 135. Additionally, the co-cultivation of this strain with Ligilactobacillus salivarius and Limosilactobacillus reuteri was also investigated. The antimicrobial activity of the produced BLIS was evaluated against Listeria monocytogenes, Listeria innocua, Salmonella enterica, and Salmonella enterica serovar Typhimurium using two methods: turbidimetric and agar diffusion. In addition, the presence of enterocin genes was also evaluated. The BLIS produced showed a bacteriostatic effect against the bio-indicator strains, and the highest antimicrobial activities expressed by arbitrary units per mL (AU/mL) were obtained against L. monocytogenes in monoculture (12,800 AU/mL), followed by the co-culture of E. faecium with Limosilactobacillus reuteri (400 AU/mL). After concentration with ammonium sulfate, the antimicrobial activity raised to 25,600 AU/mL. Assays to determine the proteinaceous nature of the BLIS showed susceptibility to trypsin and antimicrobial activity until 90 °C. Finally, analysis of the presence of structural genes of enterocins revealed that four enterocin genes were present in E. faecium 135. These results suggest that BLIS produced by E. faecium 135 has potential to be a bacteriocin and, after purification, could potentially be used as an antimicrobial agent in animal breeding.

Pediocin PA-1 production by Pediococcus pentosaceus ET34 using non-detoxified hemicellulose hydrolysate obtained from hydrothermal pretreatment of sugarcane bagasse.

Listeria monocytogenes is one of the foodborne pathogens of most concern for food safety. To limit its presence in foods, bacteriocins have been proposed as natural bio-preservatives. Herein, a bacteriocin was produced on hemicellulose hydrolysate of sugarcane bagasse by Pediococcus pentosaceous ET34, whose genome sequencing revealed an operon with 100% similarity to that of pediocin PA-1. ET34 grown on hydrolysate-containing medium led to an increase in the expression of PA-1 genes and a non-optimized purification step sequence resulted in a yield of 0.8 mg·L-1 of pure pediocin (purity > 95%). Culture conditions were optimized according to a central composite design using temperature and hydrolysate % as independent variables and validated in 3-L Erlenmeyers. Finally, a process for scaled-up implementation by sugar-ethanol industry was proposed, considering green chemistry and biorefinery concepts. This work stands up as an approach addressing a future proper sugarcane bagasse valorisation for pediocin production.

Glutaminase-free L-asparaginase production by Leucosporidium muscorum isolated from Antarctic marine-sediment.

L-asparaginase (ASNase) is an essential drug in the treatment of acute lymphoblastic leukemia (ALL). Commercial bacterial ASNases increase patient survival, but the consequent immunological reactions remain a challenge. Yeasts ASNase is closer to human congeners and could lead to lower side effects. Among 134 yeast strains isolated from marine-sediments in King George Island, Antarctica, nine were L-asparaginase producing yeasts and glutaminase-free. Leucosporidium muscorum CRM 1648 yielded the highest ASNase activity (490.41 U.L-1) and volumetric productivity (5.12 U.L-1 h-1). Sucrose, yeast extract and proline were the best carbon and nitrogen sources to support growth and ASNase production. A full factorial design analysis pointed the optimum media condition for yeast growth and ASNase yield: 20 g L-1 sucrose, 15 g L-1 yeast extract and 20 g L-1 proline, which resulted in 4582.5 U L-1 and 63.64 U L-1 h-1 of ASNase and volumetric productivity, respectively. Analysis of temperature, pH, inoculum and addition of seawater indicated the best condition for ASNase production by this yeast: 12-15 °C, pH 5.5-6.5 and seawater >25% (v/v). Inoculum concentration seems not to interfere. This work is pioneer on the production of ASNase by cold-adapted yeasts, highlighting the potential of these microbial resources as a source of glutaminase-free L-asparaginase for commercial purposes.

Buffalo milk increases viability and resistance of probiotic bacteria in dairy beverages under in vitro simulated gastrointestinal conditions.

Probiotic dairy beverages prepared from buffalo and cow milks with different levels of whey (0, 25, and 50%) were evaluated for kinetic fermentation parameters, protein and fat contents, post-acidification profile, viability of Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus acidophilus during 21 d of refrigerated storage, and resistance to in vitro gastrointestinal conditions. Progressive acidification that occurred during storage of all dairy products was reduced in the presence of whey. Lactic acid bacteria showed viable cell counts at the end of shelf life, with the highest values (7.33 to 8.83 log cfu/mL) detected in buffalo dairy products. Compared with fermented cow milk products, those made with buffalo milk showed better bacterial viability during in vitro simulated gastrointestinal digestion, which suggests a beneficial protective effect on human microbiome.

Antibacterial and antifungal activity of crude and freeze-dried bacteriocin-like inhibitory substance produced by Pediococcus pentosaceus.

Pediococcus pentosaceus LBM 18 has shown potential as producer of an antibacterial and antifungal bacteriocin-like inhibitory substance (BLIS). BLIS inhibited the growth of spoilage bacteria belonging to Lactobacillus, Enterococcus and Listeria genera with higher activity than Nisaplin used as control. It gave rise to inhibition halos with diameters from 9.70 to 20.00 mm, with Lactobacillus sakei being the most sensitive strain (13.50-20.00 mm). It also effectively suppressed the growth of fungi isolated from corn grain silage for up to 25 days and impaired morphology of colonies by likely affecting fungal membranes. These results point out that P. pentosaceus BLIS may be used as a new promising alternative to conventional antibacterial and antifungal substances, with potential applications in agriculture and food industry as a natural bio-controlling agent. Moreover, cytotoxicity and cell death induction tests demonstrated cytotoxicity and toxicity of BLIS to human colon adenocarcinoma Caco-2cells but not to peripheral blood mononuclear cells, with suggests possible applications of BLIS also in medical-pharmaceutical applications.