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The hydrolysis of plant glucosinolates by myrosinases (thioglucosidases) originates metabolites with chemopreventive properties. In this study, the ability to hydrolyze the glucosinolate sinigrin by cultures or protein extracts of Lactiplantibacillus plantarum WCFS1 was assayed. This strain possesses myrosinase-like activity as sinigrin was partly hydrolyzed by induced cultures but not by protein extracts. The 11 glycoside hydrolase GH1 family proteins, annotated as 6-phospho-β-glucosidases, were the proteins most similar to plant myrosinases. The activity of these proteins was assayed against sinigrin and synthetic glucosides. As expected, none of the proteins assayed possessed myrosinase activity against sinigrin or the synthetic β-thio-glucoside derivative or against the β-glucoside. However, all 11 proteins were active on the phosphorylated-β-glucoside derivative. Moreover, only eight of these proteins were active on phospho-β-thioglucose. These results supported that, in L. plantarum WCFS1, glucosinolates may undergo previous phosphorylation, and GH1 proteins are the glycosidases involved in the hydrolysis of phosphorylated glucosinolates., This work was financially supported by grants AGL2017-84614-C2-1 R and AGL2017-84614-C2-2 R funded by MCIN/AEI/10.13039/501100011033 and by ERDF A way of making Europe. Ana Sánchez-Arroyo is a recipient of the FPI contract PRE2018-083862 funded by MCIN/AEI/10.13039/501100011033 by ESF Investing in your future.

The production, biochemical characterization, and carbohydrate specificity of LacA β-galactosidase (locus lp_3469) belonging to the glycoside hydrolase family 42 from the probiotic organism Lactobacillus plantarum WCFS1 are addressed. The β-d-galactosidase activity was maximal in the pH range of 4.0-7.0 and at 30-37 °C. High hydrolysis capacity toward the β(1 → 4) linkages between galactose and glucose (lactose) or fructose (lactulose) was found. High efficiency toward galactosyl derivative formation was observed when lactose and glycerol, xylitol, or erythritol were used. Galactosyl derivatives of xylitol were characterized for the first time as 3-O-β-d-galactopyranosyl-xylitol and 1-O-β-d-galactopyranosyl-xylitol, displaying high preference of LacA β-galactosidase for the transfer of galactosyl residues from lactose to the C1 or C3 hydroxyl group of xylitol. These results indicate the feasibility of using LacA β-galactosidase for the synthesis of different galactosyl-polyols, which could be promising candidates for beneficial and appealing functional and technological applications such as novel prebiotics or hypocaloric sweeteners., This work has been financed by the Spanish Ministry of Economy, Industry and Competitiveness (Projects AGL2017- 84614-C2-1-R and AGL2017-84614-C2-2-R).

This work addresses the high-yield and fast enzymatic production of theanderose, a naturally occurring carbohydrate, also known as isomaltosucrose, whose chemical structure determined by NMR is α-d-glucopyranosyl-(1 → 6)-α-d-glucopyranosyl-(1 → 2)-ß-d-fructofuranose. The ability of isomaltose to act as an acceptor in the Bacillus subtilis CECT 39 levansucrase-catalyzed transfructosylation reaction to efficiently produce theanderose in the presence of sucrose as a donor is described by using four different sucrose:isomaltose concentration ratios. The maximum theanderose concentration ranged from 122.4 to 130.4 g L-1, was obtained after only 1 h and at a moderate temperature (37 °C), leading to high productivity (109.7-130.4 g L-1h-1) and yield (up to 37.3%) values. The enzymatic synthesis was highly regiospecific, since no other detectable acceptor reaction products were formed. The development of efficient and cost-effective procedures for the biosynthesis of unexplored but appealing oligosaccharides as potential sweeteners, such as theanderose, could help to expand its potential applications which are currently limited by their low availability., This work has been funded by Optibiotix Health plc. L.R-A. thanks the Spanish Research Council (CSIC) and the Spanish Ministry of Economy and Competitiveness for a Juan de la Cierva contract.

Lactobacillus plantarum is a lactic acid bacteria that can be found in numerous fermented foods. Esterases from L. plantarum exert a fundamental role in food aroma. In the present study, the gene lp-2631 encoding a putative esterase was cloned and expressed in Escherichia coli BL21 (DE3) and the overproduced Lp-2631 protein has been biochemically characterized. Lp-2631 exhibited optimal esterase activity at 20 °C and more than 90% of maximal activity at 5 °C, being the first cold-active esterase described in a lactic acid bacteria. Lp-2631 exhibited 40% of its maximal activity after 2 h of incubation at 65 °C. Lp-2631 also showed marked activity in the presence of compounds commonly found in food fermentations, such as NaCl, ethanol, or lactic acid. The results suggest that Lp-2631 might be a useful esterase to be used in food fermentations. © 2014 American Chemical Society., This work was financially supported by grants AGL2011-22745 and BFU2010-17929 (MINECO), S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid), and RM2012-00004 (INIA). M. Esteban-Torres is a recipient of a JAE predoctoral fellowship from the CSIC.

A novel strategy for the construction of disposable amperometric affinity biosensors is described in this work. The approach uses a recombinant bacterial penicillin binding protein (PBP) tagged by an N-terminal hexahistidine tail which was immobilized onto Co2+−tetradentate nitrilotriacetic acid (NTA)-modified screen-printed carbon electrodes (SPCEs). The biosensor was employed for the specific detection and quantification of β-lactam antibiotics residues in milk, which was accomplished by means of a direct competitive assay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling. The amperometric response measured at −0.20 V versus the Ag pseudoreference electrode of the SPCE upon the addition of H2O2 in the presence of hydroquinone (HQ) as redox mediator was used as the transduction signal. The developed affinity sensor allowed limits of detection to be obtained in the low part-per-billion level for the antibiotics tested in untreated milk samples. Moreover, the biosensor exhibited a good selectivity against other antibiotics residues frequently detected in milk and dairy products. The analysis time was of approximately 30 min., The financial support of the Spanish Ministerio de Economía y Competitividad Research Projects, CTQ2012-34238, and the AVANSENS Program from the Comunidad de Madrid (S2009PPQ-1642) are gratefully acknowledged. Felipe Conzuelo acknowledges an FPU fellowship from the Spanish Ministry of Education.

In this study, the hypothesis that food-derived opioid peptides besides β-casomorphin 7 might modulate the production of mucin via a direct action on epithelial goblet cells was investigated in HT29-MTX cells used as a model of human colonic epithelium. Seven milk whey or casein peptides, a human milk peptide, and a wheat gluten-derived peptide with proved or probable ability to bind μ- or δ-opioid receptors were tested on the cell culture. Significantly increased secretion of mucins was found after exposure to six of the assayed peptides, besides the previously described β-casomorphin 7, as measured by an enzyme-linked lectin assay (ELLA). Human β-casomorphin 5 and α-lactorphin were selected to study the expression of mucin 5AC gene (MUC5AC), the HT29-MTX major secreted mucin gene. α-Lactorphin showed increased expression of MUC5AC from 4 to 24 h (up to 1.6-fold over basal level expression), although differences were statistically different only after 24 h of exposure. However, this increased expression of MUC5AC did not reach significance after cell treatment with human β-casomorphin 5. In conclusion, six food-derived peptides have been identifed with described or probable opioid activity that induce mucin secretion in HT29-MTX cells. Concretely, α-lactorphin is able to up-regulate the expression of the major secreted mucin gene encoded by these cells. © 2012 American Chemical Society., This work was supported by Projects AGL2008-01713, AGL2011-26643, and Consolider-Ingenio FUN-C-Food CSD 2007-063 from Ministerio de Economía y Competitividad. We are participants in the FA1005COST Action INFOGEST on food digestion. D.M.-M. acknowledges CSIC for a JAE Program fellowship.

A recombinant tannase from Lactobacillus plantarum, overexpressed in Escherichia coli, was purified in a single step by metal chelate affinity chromatography on poorly activated nickel supports. It was possible to obtain 0.9 g of a pure enzyme by using only 20 mL of chromatographic support. The pure enzyme was immobilized and stabilized by multipoint covalent immobilization on highly activated glyoxyl agarose. Derivatives obtained by multipoint and multisubunit immobilization were 500- and 1000-fold more stable than both the soluble enzyme and the one-point-immobilized enzyme in experiments of thermal and cosolvent inactivation, respectively. In addition, up to 70 mg of pure enzyme was immobilized on 1 g of wet support. The hydrolysis of tannic acid was optimized by using the new immobilized tannase derivative. The optimal reaction conditions were 30% diglyme at pH 5.0 and 4 C. Under these conditions, it was possible to obtain 47.5 mM gallic acid from 5 mM tannic acid as substrate. The product was pure as proved by HPLC. On the other hand, the immobilized biocatalyst preserved >95% of its initial activity after 1 month of incubation under the optimal reaction conditions, This work was supported by Grants AGL2008-01052, AGL-2009-07625, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD(CICYT),RM2008-00002 (INIA), and S-0505/AGR/000153 (CAM). J.A.C. is the recipient of a predoctoral fellowship from the I3P-CSIC Program and FPI-MEC, and G.F.-L. and L.B. are recipients of Ramon y Cajal postdoctoral contracts.

Tannase is an enzyme with important biotechnological applications in the food industry. Previous studies have identified the tannase encoding gene in Lactobacillus plantarum and also have reported the description of the purification of recombinant L. plantarum tannase through a protocol involving several chromatographic steps. Here, we describe the high-yield production of pure recombinant tannase (17 mg/L) by a one-step affinity procedure. The purified recombinant tannase exhibits optimal activity at pH 7 and 40 C. Addition of Ca2þ to the reaction mixture greatly increased tannase activity. The enzymatic activity of tannase was assayed against 18 simple phenolic acid esters. Only esters derived from gallic acid and protocatechuic acid were hydrolyzed. In addition, tannase activity was also assayed against the tannins tannic acid, gallocatechin gallate, and epigallocatechin gallate. Despite L. plantarum tannase representing a novel family of tannases, which shows no significant similarity to tannases from fungal sources, both families of enzymes shared similar substrate specificity range. The physicochemical characteristics exhibited by L. plantarum recombinant tannase make it an adequate alternative to the currently used fungal tannases, This work was supported by grants AGL2005-00470, AGL2008-01052, BFU2007-67404/BMC, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD, and Consolider INGENIO 2007 Factoria de Crystalización (CICYT), RM2008-00002 (INIA), and S-0505/AGR/000153 (CAM). H. Rodríguez and J. A. Curiel were recipients of predoctoral fellowships from the I3P-CSIC Program and FPI-MEC, respectively.

Nitroreductases reduce nitroaromatic compounds and other oxidants in living organisms, having interesting implications in environmental and human health. A putative nitrobenzoate reductase encoding gene (lp_0050) was recently annotated in the completed DNA sequence of lactic acid bacterium Lactobacillus plantarum WCFS1 strain. In this research, this L. plantarum gene was cloned and expressed, and the corresponding protein (PnbA) was biochemically characterized. This L. plantarum PnbA reductase is a 216 amino acid residue FMN-flavoprotein, which exhibits 23% identity with Pseudomonas putida and Ralstonia eutropha nitroreductases and, We thank the MICINN (Spanish government) for support of this research through Grants AGL2006-02414 and AGL2008-01052. H.G. and J.A.C. thank CSIC and MICINN for a JAE predoctoral and a FPI predoctoral fellowship, respectively.

The potential to produce volatile phenols from hydroxycinnamic acids was investigated for lactic acid bacteria (LAB) isolated from Spanish grape must and wine. A PCR assay was developed for the detection of LAB that potentially produce volatile phenols. Synthetic degenerate oligonucleotides for the specific detection of the pdc gene encoding a phenolic acid decarboxylase were designed. The pdc PCR assay amplifies a 321 bp DNA fragment from phenolic acid decarboxylase. The pdc PCR method was applied to 85 strains belonging to the 6 main wine LAB species. Lactobacillus plantarum, Lactobacillus brevis, and Pediococcus pentosaceus strains produce a positive response in the pdc PCR assay, whereas Oenococcus oeni, Lactobacillus hilgardii, and Leuconostoc mesenteroides strains did not produce the expected PCR product. The production of vinyl and ethyl derivatives from hydroxycinnamic acids in culture media was determined by high-performance liquid chromatography. A relationship was found between pdc PCR amplification and volatile phenol production, so that the LAB strains that gave a positive pdc PCR response produce volatile phenols, whereas strains that did not produce a PCR amplicon did not produce volatile phenols. The proposed method could be useful for a preliminary identification of LAB strains able to produce volatile phenols in wine, We thank I. Vaquero for help with the PCR experiments. We greatly appreciate the technical assistance of M. V. Santamaría and A. Gómez.