A new and promiscuous α/ß hydrolase from Acinetobacter tandoii DSM 14970 T inactivates the mycotoxin ochratoxin A.

The presence of ochratoxin A (OTA) in food and feed represents a serious concern since it raises severe health implications. Bacterial strains of the Acinetobacter genus hydrolyse the amide bond of OTA yielding non-toxic OTα and L-ß-phenylalanine; in particular, the carboxypeptidase PJ15_1540 from Acinetobacter sp. neg1 has been identified as an OTA-degrading enzyme. Here, we describe the ability to transform OTA of cell-free protein extracts from Acinetobacter tandoii DSM 14970 T, a strain isolated from sludge plants, and also report on the finding of a new and promiscuous α/ß hydrolase (ABH), with close homologs highly distributed within the Acinetobacter genus. ABH from A. tandoii (AtABH) exhibited amidase activity against OTA and OTB mycotoxins, as well as against several carboxypeptidase substrates. The predicted structure of AtABH reveals an α/ß hydrolase core composed of a parallel, six-stranded ß-sheet, with a large cap domain similar to the marine esterase EprEst. Further biochemical analyses of AtABH reveal that it is an efficient esterase with a similar specificity profile as EprEst. Molecular docking studies rendered a consistent OTA-binding mode. We proposed a potential procedure for preparing new OTA-degrading enzymes starting from promiscuous α/ß hydrolases based on our results. KEY POINTS: • AtABH is a promiscuous αß hydrolase with both esterase and amidohydrolase activities • AtABH hydrolyses the amide bond of ochratoxin A rendering nontoxic OTα • Promiscuous αß hydrolases are a possible source of new OTA-degrading enzymes.

Ferulic Acid Esterase Producing Lactobacillus johnsonii from Goat Feces as Corn Silage Inoculants.

Ferulic acid esterase (FAE+)-producing lactobacilli are being studied as silage inoculants due to their potential of increasing forage fiber digestibility. In this work, three FAE+ Lactobacillus (L.) johnsonii strains were isolated from caprine feces and characterized according to their potential probiotic characteristics and as silage inoculants. Limosilactobacillus fermentum CRL1446, a human probiotic isolated from goat cheese, was also included in the experiments as a potential silage inoculant. FAE activity quantification, probiotic characterization, and growth in maize aqueous extract indicated that L. johnsonii ETC187 might have a better inoculant and probiotic aptitude. Nevertheless, results in whole-corn mini silos indicated that, although acid detergent fiber (ADF) was significantly reduced by this strain (3% compared with the uninoculated (UN) group), L. johnsonii ETC150 and CRL1446 not only induced similar ADF reduction but also reduced dry matter (DM) loss (by 7.3% and 6.5%, respectively) compared with the UN group. Additionally, CRL1446 increased in vitro DM degradability by 10%. All treatments reduced gas losses when compared with the UN group. The potential probiotic features of these strains, as well as their beneficial impact on corn fermentation shown in this study, encourage further studies as enhancers in animal production.

Spray dried lactobacilli maintain viability and feruloyl esterase activity during prolonged storage and under gastrointestinal tract conditions.

The use of lactobacilli with feruloyl esterase (FE) activity in the development of functional foods has gained considerable interest in recent years. Microencapsulation of FE-producing bacteria to facilitate their incorporation into food is a challenge. The aim of this study was to evaluate survival and maintenance of FE activity during storage at 4 °C and under simulated gastrointestinal tract (GIT) conditions of microcapsules of FE-producing Lactobacillus (Lb.) strains obtained by spray drying. Lb. fermentum CRL1446 and Lb. johnsonii CRL1231 powders maintained viability at concentrations ≥ 106 CFU/g (minimum probiotic dose) when stored at 4 °C for 12 months. Lb. acidophilus CRL1014 powders were only able to maintain ≥ 106 CFU/g during 4 months of storage. FE activity was conserved in three microencapsulated strains evaluated, an increase of specific activity being observed until month 12 of storage. Powders of the three strains incubated under GIT conditions maintained their viability (≥ 106 CFU/g), but specific FE activity was only detected in Lb. fermentum and Lb. johnsonii powders (0.80-0.83 and 0.21-0.56 U/mg, respectively). CRL1446 and CRL1231 microcapsules were able to resist prolonged storage and GIT conditions, retaining FE activity and preserving their probiotic potential and could be incorporated into functional foods.

Specific Strains of Lactic Acid Bacteria Differentially Modulate the Profile of Adipokines In Vitro.

Obesity induces local/systemic inflammation accompanied by increases in macrophage infiltration into adipose tissue and production of inflammatory cytokines, chemokines, and hormones. Previous studies have shown that probiotics could improve the intestinal dysbiosis induced by metabolic diseases such as obesity, diabetes, and metabolic syndrome. Microorganisms could (directly or indirectly) affect adipokine levels due to their capacity to induce translocation of several intestinal microbial antigens into systemic circulation, which could lead to metabolic endotoxemia or produce immunomodulation in different organs. The aim of the present study was to select non-inflammatory lactic acid bacteria (LAB) strains with the capacity to modulate adipokine secretion by the adipose tissue. We wish to elucidate the role of potential probiotic strains in the regulation of the cross talking between immune cells such as macrophages and adipose cells. Mouse macrophage cell line RAW 264.7 was used for evaluating the ability of 14 LAB strains to induce cytokine production. The LAB strains were chosen based on their previously studied beneficial properties in health. Then, in murine adipocyte culture and macrophage-adipocyte coculture, we determined the ability of these strains to induce cytokines and leptin secretion. Tumor necrosis factor alpha, interleukin 6 (IL-6), IL-10, monocyte chemoattractant protein-1, and leptin levels were measured in cell supernatants. We also performed the detection and quantification of leptin receptor (Ob-Rb) expression in macrophage cell lines stimulated by these LAB strains. Differential secretion profile of cytokines in macrophage cells induced by LAB strains was observed. Also, the levels of Ob-Rb expression diverged among different LAB strains. In LAB-stimulated coculture cells (adipocytes and macrophages), we observed differential production of leptin and cytokines. Furthermore, we detected lower production levels in single culture than cocultured cells. The principal component analysis showed an association between the four clusters of strains established according to their inflammatory profiles and leptin adipocyte production and leptin receptor expression in macrophages. We conclude that coculture is the most appropriate system for selecting strains with the ability to modulate adipokine secretion. The use of microorganisms with low and medium inflammatory properties and ability to modulate leptin levels could be a strategy for the treatment of some metabolic diseases associated with dysregulation of immune response.

The secreted esterase of Propionibacterium freudenreichii has a major role in cheese lipolysis.

Free fatty acids are important flavor compounds in cheese. Propionibacterium freudenreichii is the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants of P. freudenreichii CIRM-BIA1(T) for each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of the pf279 gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of the pf774 gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of the pf279 gene sequence, leading to a premature stop codon, whereas they harbored a pf774 gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase in P. freudenreichii and a key agent of Swiss cheese lipolysis.