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This study describes the molecular identification, biochemical characterization, and stabilization of three recombinant AlfA, AlfB, and AlfC fucosidases from Lacticaseibacillus rhamnosus INIA P603. Even though previous studies revealed the presence of fucosidase activity in L. rhamnosus extracts, the identification of the fucosidases, their physicochemical properties, and the substrate spectrum remained unknown. Although the presence of alfB is not common in strains of L. rhamnosus, fucosidases from L. rhamnosus INIA P603 were selected because this strain exhibited higher fucosidase activity in culture and the complete set of fucosidases. A high yield of purified recombinant AlfA, AlfB, and AlfC fucosidases was obtained (8, 12, and 18 mg, respectively). AlfA, AlfB, and AlfC showed their optimal activities at pH 5.0 and 4.0 at 60 °C, 40 °C, and 50 °C, respectively. Unlike 3-fucosyllactose, all three recombinant fucosidases were able to hydrolyze 2′-fucosyllactose (2′-FL), and their activities were improved through their immobilization on agarose supports. Nevertheless, immobilized AlfB exhibited the highest hydrolysis, releasing 39.6 µmol of fucose mg enzyme−1 min−1. Only the immobilized AlfB was able to synthetize 2′-FL. In conclusion, the enzymatic properties elucidated in this study support the potential ability of fucosidases from L. rhamnosus INIA P603 to hydrolyze fucosylated substrates as well as justifying interest for further research into AlfB for its application to catalyze the synthesis of fucosylated prebiotics., This research was funded by the Spanish Ministry of Science and Innovation grants PID2020-119630RB-I00 and RYC2019-026368-I.

5 Pág., Some lactic acid bacteria (LAB) strains have the ability to synthesize riboflavin, a trait linked to the presence of ribG, ribB, ribA and ribH genes in the rib operon. Multiple sequence alignments of these genes showed that these sequences are not identical in different LAB species, so primers designed to detect these genes in one species do not always work with others. Therefore, we designed degenerate primers based on sequences from Lactococcus lactis MG1363, Levilactobacillus brevis ATCC 367 and Limosilactobacillus fermentum IFO3956, and established optimal PCR conditions for the detection of rib genes in different LAB species. Simultaneously, we selected riboflavin-producing LAB strains from our bacterial collection belonging to the species L. brevis, L. fermentum, L. lactis, Leuconostoc mesenteroides and Lactiplantibacillus plantarum, and we were able to detect ribG, ribB, ribA and ribH genes in these strains by PCR using the designed primers. Thus, the development of degenerate primers and optimal PCR conditions for the detection of ribG, ribB, ribA and ribH genes in LAB allowed the detection and the selection of potential riboflavin-producing strains of different species, which could be good candidates for the development of riboflavin-enriched functional foods., This work was supported by the project PID2020-11960RB-I00 from the Spanish Ministry of Science and Innovation .

12 Pág. Departamento de Tecnología de Alimentos​​, The genetic engineering of bacteria for food applications has biosafety requirements, including the use of non-antibiotic selectable markers. These can be gene-encoding bacteriocin immunity proteins, such as nisI and pedB, which require the use of promoters to ensure optimal expression. Our aim was to search for promoters for the expression of pediocin (pedB) and nisin (nisI) immunity genes, which could allow the selection of a wide variety of transformed lactic acid bacteria (LAB) and bifidobacteria strains. Eight promoters from LAB or bifidobacteria were initially studied using evoglow-Pp1 as the reporter gene in Lactococcus lactis NZ9000, resulting in the selection of P32, P3N, PTuR and PEF-P, which exhibited a strong constitutive expression. These promoters were further tested for the expression of the food-grade selectable markers pedB and nisI in agar diffusion assays with pediocin and nisin, respectively. The results obtained demonstrated that both the PTuR and PEF-P promoters allowed a good level of expression of nisI and pedB in the LAB and bifidobacteria strains tested. A suitable concentration of nisin or pediocin could be established for the selection of the strains transformed with vectors harbouring the combination of the selected promoters and markers nisI and pedB, and this was successfully applied to different strains of LAB and bifidobacteria. Therefore, PTuR and PEF-P promoters are excellent candidates for the expression of nisI and/or pedB as selectable markers in LAB and bifidobacteria, and they are suitable for use in food grade vectors to allow the selection of genetically engineered strains. KEY POINTS: • Food-grade vectors require non-antibiotic selectable markers such as pedB and nisI. • Eight promoters from LAB or bifidobacteria were initially tested in L. lactis NZ9000. • PTuR and PEF-P efficiently drove the expression of pedB and nisI in LAB and bifidobacteria., Funding This study was funded by the project PID2020-11960RB-I00 from the Spanish Ministry of Science, Innovation and Universities

6 Pág., Biochanin A and formononetin are methylated isoflavones, abundant in legumes, which can be converted in genistein and daidzein by means of an O-demethylation reaction. Our objective was to transform these methylated isoflavones into higher biological activity and bioavailability compounds, such as daidzein and genistein. Prior screening, using 10 lactic acid bacteria (LAB) and 3 bifidobacteria strains, revealed that only 7 lactobacilli strains were able to transform biochanin A into genistein, while none of the strains demethylated formononetin. As the dmt734 gene from Bifidobacterium breve INIA P734 was putatively annotated as O-demethylase, it was cloned in a vector expressing antibiotic resistance (pNZ:TuR.dmt734) as well as in a food grade vector (pLEB590.dmt734), which were subsequently transformed in LAB and bifidobacteria O-demethylase lacking strains. Those recombinant strains harbouring pNZ:TuR.dmt734, or pLEB590.dmt734, managed to transform biochanin A into genistein, but did not transform formononetin into daidzein. These results confirm the demethylation specificity of isoflavones and the biotechnological interest of B. breve INIA P734 O-demethylase, as well as the food grade strains harbouring O-demethylase activity, which would be of value for the development of fermented vegetable-based foods enriched in genistein., This work was supported by projects RTA2017-00002-00-00 and PID2020-11960RB-I00 from the Spanish Ministry of Science, Innovation and Universities. We are grateful to the ICTAN (Institute of Food Science, Technology and Nutrition, Madrid, Spain) Analysis Services Unit for providing chromatography and mass spectrometry facilities.

Among vegetable functional products, soy-based foods stand out for their high content in protein and flavonoids,mainly isoflavones. The fermentation of soy beverages with selected bacterial strains is a way to obtain a product,enriched in isoflavone aglycones, alongside with other isoflavone derivates and other bioactive flavonoids. Two bacterial strains with high activity on the transformation of these compounds, Limosilactobacillus mucosae INIA P508 and Bifidobacterium pseudocatenulatum INIA P815, were tested for the fermentation of soy beverage. Both strains produced high concentration of the aglycones daidzein and genistein, in addition to a significant increase (P < 0.01) of glycitein, O-desmethylangolensin, 6′′ -hydroxy-O-desmethylangolensin, tetrahydrodaidzein and other flavonoid aglycones such as kaempferol, naringenin and luteolin. Two alternatives to conserve the developed functional beverages were explored, cold storage and thermal treatment. Refrigeration did not affect the concentration of isoflavones, even observing an increase in aglycones. Similarly, the thermal treatment and further room temperature storage did not reduce the amounts of daidzein and genistein. Therefore, both strains could be used for the development of soy beverages enriched in bioactive isoflavones and other bioactive flavonoids which would maintain their concentrations in refrigeration or at room temperature after heat treatment.

Second Edition., Lactobacillus plantarum (currently Lactiplantibacillus plantarum) is the main bacterial species associated to olive processing. During the last years, numerous studies have been carried out on the metabolism of phenolics compounds by L. plantarum strains, due to the great importance of these compounds in the health beneficial properties of olive products. Among the olive phenolic acids studied, some hydroxycinnamic acids (caffeic, m-coumaric, o-coumaric, p-coumaric, ferulic, and sinapic acids) as well as some hydroxybenzoic acids (gallic and protocatechuic acids) were metabolized by L. plantarum. The decarboxylase and reductase enzymes responsible for these biochemical reactions have been identified, recombinantly produced, and biochemically characterized. Oleuropein, the main secoiridoid glucoside present in olives, is hydrolyzed by L. plantarum by the sequential action of unidentified enzymes, giving elenoic acid and hydroxytyrosol as end-products. It has been also studied the response of L. plantarum to some olive-related compounds (olive oil, p-coumaric acid, gallic acid, and oleuropein) by genome-scale transcriptomic signature and physiological analysis.

Avocado peel is a by-product obtained in high amounts in the food industry with no further applications despite its richness in bioactive compounds. In this context, an efficient "green" microwave assisted extraction (MAE) was optimized to maximize the extraction of bioactive polyphenols. Moreover, the phenolic composition of the developed green avocado extract was characterized by HPLC coupled to MS analysers and the potential applications for the food industry were studied assaying different bioactivities. Thus, the matrix metalloproteinases inhibition, the antioxidant capacity and the antimicrobial activity against gram-positive and gram-negative bacteria, yeast and mold were tested. The results pointed out both, high matrix metalloproteinases inhibitory capacity and antioxidant activity of avocado peel MAE extract. These findings suggest the potential food industry applications of this extract as natural food preservative, functional food ingredient or nutraceuticals with antioxidant and anti-aging activities.

The use of non-Saccharomyces strains in aerated conditions has proven effective for alcohol content reduction in wine during lab-scale fermentation. The process has been scaled up to 20 L batches, in order to produce lower alcohol wines amenable to sensory analysis. Sequential instead of simultaneous inoculation was chosen to prevent oxygen exposure of Saccharomyces cerevisiae during fermentation, since previous results indicated that this would result in increased acetic acid production. In addition, an adaptation step was included to facilitate non-Saccharomyces implantation in natural must. Wines elaborated with Torulaspora delbrueckii or Metschnikowia pulcherrima in aerated conditions contained less alcohol than control wine (S. cerevisiae, non-aerated). Sensory and aroma analysis revealed that the quality of mixed fermentations was affected by the high levels of some yeast amino acid related byproducts, which suggests that further progress requires a careful selection of non-Saccharomyces strains and the use of specific N-nutrients., This work was supported by grants MINECO/FEDER RTC-2014-2186-2 (SOUNDWINE) developed in collaboration with Agrovin S.A., and AGL2015-63629-R, and EU project YeSVitE. (7FP-IRSES-GA n° 612441). JAC and PSN are the recipients of a MINECO Formación Postdoctoral contract from the Spanish Government.

The respiratory metabolism of yeast species alternative to Saccharomyces cerevisiae has been explored in recent years as a tool to reduce ethanol content in grape wine. The efficacy of this strategy has been previously proven for mixed cultures of non-Saccharomyces and S. cerevisiae strains. In this work, we perform a transcriptomic analysis of the Crabtree-negative yeast Kluyveromyces lactis under tightly controlled growth conditions in order to better understand physiology of non-Saccharomyces yeasts during the fermentation of grape must under aerated conditions. Transcriptional changes in K. lactis are mainly driven by oxygen limitation, iron requirement, and oxidative stress. Oxidative stress appears as a consequence of the hypoxic conditions achieved by K. lactis once oxygen supply is no longer sufficient to sustain fully respiratory metabolism. This species copes with low oxygen and iron availability by repressing iron consuming pathways and activating iron transport mechanisms. Most of the physiological and transcriptomic features of K. lactis in aerobic wine fermentation are not shared with the Crabtree-positive yeast S. cerevisiae., This work was supported by the Spanish Government trough MINECO and FEDER (AGL2015-63629-R) funds: MINECO AGL2015-63629-R grants, MINECO training contract for A.J. Rodrigues, and MINECO Formación Postdoctoral contract for J.A. Curiel.

The use of non-Saccharomyces species as starter cultures together with Saccharomyces cerevisiae is becoming a common practice in the oenological industry to produce wines that respond to new market demands. In this context, microbial interactions with these non-Saccharomyces species must be considered for a rational design of yeast starter combinations. Previously, transcriptional responses of S. cerevisiae to short-term co-cultivation with Torulaspora delbrueckii, Candida sake, or Hanseniaspora uvarum was compared. An activation of sugar consumption and glycolysis, membrane and cell wall biogenesis, and nitrogen utilization was observed, suggesting a metabolic boost of S. cerevisiae in response to competing yeasts. In the present study, the transcription profile of S. cerevisiae was analyzed after 3 h of cell contact with Metschnikowia pulcherrima. Results show an over-expression of the gluco-fermentative pathway much stronger than with the other species. Moreover, a great repression of the respiration pathway has been found in response to Metschnikowia. Our hypothesis is that there is a direct interaction stress response (DISR) between S. cerevisiae and the other yeast species that, under excess sugar conditions, induces transcription of the hexose transporters, triggering glucose flow to fermentation and inhibiting respiration, leading to an increase in both, metabolic flow and population dynamics., This work was funded by the Spanish Government through grant AGL2015-63629-R (MINECO/FEDER UE). JT was funded by FGCSIC by the COMFUTURO program. JAC was funded by MINECO Formación Postdoctoral contract (FPDI-2013-15520). AM is funded by MINECO Formación Predoctoral grant (BES-2016-077557).

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license.-- This article belongs to the Section Natural Products., A novel β-galactosidase from Lactobacillus plantarum (LPG) was over-expressed in E. coli and purified via a single chromatographic step by using lowly activated IMAC (immobilized metal for affinity chromatography) supports. The pure enzyme exhibited a high hydrolytic activity of 491 IU/mL towards o-nitrophenyl β-D-galactopyranoside. This value was conserved in the presence of different divalent cations and was quite resistant to the inhibition effects of different carbohydrates. The pure multimeric enzyme was stabilized by multipoint and multisubunit covalent attachment on glyoxyl-agarose. The glyoxyl-LPG immobilized preparation was over 20-fold more stable than the soluble enzyme or the one-point CNBr-LPG immobilized preparation at 50°C. This β-galactosidase was successfully used in the hydrolysis of lactose and lactulose and formation of different oligosaccharides was detected. High production of galacto-oligosaccharides (35%) and oligosaccharides derived from lactulose (30%) was found and, for the first time, a new oligosaccharide derived from lactulose, tentatively identified as 3′-galactosyl lactulose, has been described., This work has been sponsored by Consolider INGENIO 2010 CSD2007-00063 FUNC-FOOD(CICYT), the Spanish Ministry of Science and Innovation (Project CTQ2009-07568), CSIC(Intramural Project 200980I133) and S2009/AGR-1469 (ALIBIRD) (CAM)., We acknowledge the support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

The effects of air classification and lactic acid bacteria fermentation on the reduction of anti-nutritional factors (vicine and convicine, trypsin inhibitor activity, condensed tannins and phytic acid) and in vitro protein and starch digestibility of faba bean flour were studied. Free amino acid (FAA) profile analysis was also carried out. Air classification allowed the separation of the flour into protein and starch rich fractions, showing different chemical compositions and microstructures. Lactobacillus plantarum growth and acidification in faba bean flour and its fractions were assessed. The anti-nutritional compounds were separated mostly to the fine protein-rich fraction. Fermentation caused the decrease of vicine and convicine contents by more than 91% and significantly reduced trypsin inhibitor activity and condensed tannins (by more than 40% in the protein-rich fraction). No significant (P>0.05) variation was observed for total phenols and phytic acid content. Fermentation increased the amount of FAA, especially of the essential amino acids and γ-aminobutyric acid, enhanced the in vitro protein digestibility and significantly lowered the hydrolysis index. This work showed that the combination of air classification and fermentation improved nutritional functionality of faba bean flour which could be utilized in various food applications.

The growing interest of the winemaking industry on the use of non-Saccharomyces starters has prompted several studies about the physiological features of this diverse group of microorganisms. The fact that the proposed use of these new starters will almost invariably involve either simultaneous or sequential inoculation with Saccharomyces cerevisiae has also driven the attention to the potential biological interactions between different starters during wine fermentation. Our current understanding is that alternative yeast starters will affect wine features by both direct and indirect mechanisms (through metabolic or other types of interactions with S. cerevisiae). There are still few studies addressing the question of yeast–yeast interactions in winemaking by a transcriptomic approach. In a previous report, we revealed early responses of S. cerevisiae and Torulaspora delbrueckii to the presence of each other under anaerobic conditions, mainly the overexpression of genes related with sugar consumption and cell proliferation. We have now studied the response, under aerobic conditions, of S. cerevisiae to other two non-Saccharomyces species, Hanseniaspora uvarum and Candida sake, keeping T. delbrueckii as a reference; and always focusing on the early stages of the interaction. Results point to some common features of the way S. cerevisiae modifies its transcriptome in front of other yeast species, namely activation of glucose and nitrogen metabolism, being the later specific for aerobic conditions., This work was supported by grant AGL2015-63629-R (MINECO/FEDER, UE), YeSVitE consortium (EU project, 7FP-IRSES-GA no. 612441), JC is the recipient of a MINECO Formación Postdoctoral contract from the Spanish Government.

This study describes the identification and characterization of a novel recombinant Leuconostoc mesenteroides glycosidase (BgLm1). Since the protein encoded by LEUM_0847 gene was annotated as putative β-glucosidase, characterization procedures were done using p-nitrophenyl-β- d -glucopyranoside as substrate. A high yield of purified recombinant BgLm1 was obtained (12 mg/L). The enzyme showed an optimal activity at pH 6.0 and 40 °C and preserved 65% of residual activity after 48 h of incubation at 25 °C. Ca2+ and Mn2+ ions greatly increased the β-glucosidase activity. Moreover, BgLm1 demonstrated β-galactosidase and β-fucosidase activities. Kinetic parameters of BgLm1 revealed its low affinity to p-nitrophenyl-β- d -glucopyranoside (Km of 9.93 mmol/L). Then, although LEUM_0847 gene was annotated as a β-glucosidase, our results suggest that BgLm1can be indeed considered as a β-galactosidase since high hydrolysis using skimmed milk (lactose) as natural substrate and high affinity (Km of 0.56 mmol/L) and specific constant (2254 mmol/L−1 s−1) to p-nitrophenyl-β- d -galactopyranoside were observed. In conclusion, the enzymatic properties observed in this study, support the interest of BgLm1 for food industrial applications.

In this work, the influence of bioprocessing on the nutritional quality and health effects of wheat bran of different particle sizes (750, 400, 160, 50 μm) was evaluated. Bioprocessing was carried out by a 24 h-fermentation using Lactobacillus brevis E95612 and Kazachstania exigua C81116 as starters, with or without the addition of an enzyme mixture with specific carbohydrase activities. Bioprocessing clearly affected the microstructure and chemical and nutritional features of wheat bran depending on the particle size. Bioprocessing significantly improved the antioxidant and phytase activities (up to 3.7 fold, respectively), peptides and total free amino acids and content (up to 40%) and the in vitro digestibility of proteins. The antioxidant power and nutritional indexes were higher for the bioprocessed brans compared to the native, mainly in bran having smaller particle size. In every case, the addition of the enzymes further improved the positive effect of the microbial fermentation. Industrial relevance Wheat bran is a source of nutritionally important compounds such as dietary fibers, minerals, vitamins and phenolic acids. Commonly, processing of bran has mostly been performed for technological purposes, to facilitate its use as a DF rich ingredient in foods improving its structural and organoleptic features. The bioprocessing technology here applied offers a tool to enhance also the nutritional value of wheat bran, especially of finer particle size. As a result, bioprocessed wheat bran showed higher potential compared to the native bran, and qualified as a suitable ingredient for food preparations.

Wheat flour, which was rendered gluten-free by sourdough lactic acid bacteria fermentation and fungal proteases, was used for manufacturing experimental gluten-free pasta (E-GFp), according to a traditional process with low temperature drying cycle. Chemical, technological, structural, nutritional and sensory features were characterized and compared with those of commercial gluten-free (C-GFp) and durum wheat pasta (C-DWp). As shown through immunological analyses, the residual concentration of gluten of the hydrolyzed wheat flour was below 10 ppm. E-GFp showed rapid water uptake and shorter optimal cooking time compared to the other pastas. Despite the absence of the gluten network, the supplementation with pre-gelatinized rice flour allowed structural properties of E-GFp, which were comparable to those of C-GFp. The in vitro protein digestibility of E-GFp resulted the highest. Probably due to proteolysis during sourdough fermentation; chemical scores, essential amino acid profile, biological value and nutritional index of E-GFp were higher than those of C-DWp. The hydrolysis index (HI) of E-GFp was ca. 30% lower than that found for C-GFp. As shown by sensory analysis, the characteristic of E-GFp were acceptable. The manufacture of E-GFp should be promising to expand the choice of gluten-free foods, which combine sensory and nutritional properties.

Lactobacillus plantarum is a lactic acid bacterium that can degrade food tannins by the successive action of tannase and gallate decarboxylase enzymes. In the L. plantarum genome, the gene encoding the catalytic subunit of gallate decarboxylase (lpdC, or lp_2945) is only 6.5 kb distant from the gene encoding inducible tannase (L. plantarum tanB [tanB], or lp_2956). This genomic context suggests concomitant activity and regulation of both enzymatic activities. Reverse transcription analysis revealed that subunits B (lpdB, or lp_0271) and D (lpdD, or lp_0272) of the gallate decarboxylase are cotranscribed, whereas subunit C (lpdC, or lp_2945) is cotranscribed with a gene encoding a transport protein (gacP, or lp_2943). In contrast, the tannase gene is transcribed as a monocistronic mRNA. Investigation of knockout mutations of genes located in this chromosomal region indicated that only mutants of the gallate decarboxylase (subunits B and C), tannase, GacP transport protein, and TanR transcriptional regulator (lp_2942) genes exhibited altered tannin metabolism. The expression profile of genes involved in tannin metabolism was also analyzed in these mutants in the presence of methyl gallate and gallic acid. It is noteworthy that inactivation of tanR suppresses the induction of all genes overexpressed in the presence of methyl gallate and gallic acid. This transcriptional regulator was also induced in the presence of other phenolic compounds, such as kaempferol and myricetin. This study complements the catalog of L. plantarum expression profiles responsive to phenolic compounds, which enable this bacterium to adapt to a plant food environment., N. Jiménez is a recipient of an FPI Fellowship from MINECO. This work was supported by grants AGL2014-52911-R, AGL2011-22745 (MINECO), and RM2008-00002 (Instituto Nacional de Investigación Agraria y Alimentaria).

Food phenolics exhibit a wide range of beneficial health effects due to their antioxidant properties. Natural phenolic compounds often occur as glycosides, esters, or polymers on which their bioactivity is reduced. Some food phenolics are transformed by the microbiota present during fermentation. This conversion is often essential for absorption and modulates the biological activity of these dietary compounds. Lactobacillus plantarum is the species most frequently used to ferment plant food products where phenolic compounds are abundant. The knowledge of the metabolism of phenolic compounds in L. plantarum is of interest as this bacterium possesses relevant enzymatic activities to obtain bioactive compounds. Complex phenolics are hydrolyzed by L. plantarum to simpler and more biologically active compounds. The complete enzymatic pathways for the biodegradation of polymeric hydroxybenzoic- and hydoxycinnamic-derived compounds are described. The adaptative behavior of L. plantarum under stress induced by phenolics also modulates traits beneficial for its gastrointestinal survival.

[Background] Aerobic fermentation of grape must, leading to respiro-fermentative metabolism of sugars, has been proposed as way of reducing alcohol content in wines. Two factors limit the usefulness of Saccharomyces cerevisiae for this application, the Crabtree effect, and excess volatile acidity under aerobic conditions. This work aimed to explore the impact on ethanol acetate production of different S. cerevisiae strains deleted for genes previously related with the Crabtree phenotype. [Results] Recombinant strains were constructed on a wine industrial genetic background, FX10. All yeast strains, including FX10, showed respiro-fermentative metabolism in natural grape must under aerobic conditions, as well as a concomitant reduction in ethanol yield. This indicates that the Crabtree effect is not a major constrain for reaching relevant respiration levels in grape must. Indeed, only minor differences in ethanol yield were observed between the original and some of the recombinant strains. In contrast, some yeast strains showed a relevant reduction of acetic acid production. This was identified as a positive feature for the feasibility of alcohol level reduction by respiration. Reduced acetic acid production was confirmed by a thorough analysis of these and some additional deletion strains (involving genes HXK2, PYK1, REG1, PDE2 and PDC1). Some recombinant yeasts showed altered production of glycerol and pyruvate derived metabolites. [Conclusions] REG1 and PDC1 deletion strains showed a strong reduction of acetic acid yield in aerobic fermentations. Since REG1 defective strains may be obtained by non-GMO approaches, these gene modifications show good promise to help reducing ethanol content in wines., This work was supported in part by the Spanish Government trough MINECO and FEDER funds: MINECO AGL2015-63629-R, MINECO training contract for AJR, and MINECO Formación Postdoctoral contract for JAC. The authors are also grateful Junta de Andalucía (P10_AGR6544) and to Laffort SA (Bordeaux-France) for financial support and for providing the S. cerevisie FX10 strain.

Rising sugar content in grape must, and the concomitant increase in alcohol levels in wine, are some of the main challenges affecting the winemaking industry nowadays. Among the several alternative solutions currently under study, the use of nonconventional yeasts during fermentation holds good promise for contributing to relieve this problem. Non-Saccharomyces wine yeast species comprise a high number or species, so encompassing a wider physiological diversity than Saccharomyces cerevisiae. Indeed, the current oenological interest of these microorganisms was initially triggered by their potential positive contribution to the sensorial complexity of quality wines, through the production of aroma and other sensory-active compounds. This diversity also involves ethanol yield on sugar, one of the most invariant metabolic traits of S. cerevisiae. This review gathers recent research on non-Saccharomyces yeasts, aiming to produce wines with lower alcohol content than those from pure Saccharomyces starters. Critical aspects discussed include the selection of suitable yeast strains (considering there is a noticeable intra-species diversity for ethanol yield, as shown for other fermentation traits), identification of key environmental parameters influencing ethanol yields (including the use of controlled oxygenation conditions), and managing mixed fermentations, by either the sequential or simultaneous inoculation of S. cerevisiae and non-Saccharomyces starter cultures. The feasibility, at the industrial level, of using non-Saccharomyces yeasts for reducing alcohol levels in wine will require an improved understanding of the metabolism of these alternative yeast species, as well as of the interactions between different yeast starters during the fermentation of grape must., Experimental work by researchers from ICVV is supported by the Spanish Government trough MINECO and FEDER funds: MINECO AGL2012-32064 and AGL2015-63629-R grants, INIA RM2012-00007-00-00 grant, MINECO RTC-2014-2186-2 grant, MINECO training contract for AR, MINECO Formación Postdoctoral contract for JC. Experimental work by the authors from Polytechnic University of Marche is supported by Ricerca Scientifica di Ateneo RSA2015.

The occurrence of in vitro production of biogenic amines in bacteria isolated from ice-preserved sardine and mackerel was studied. Biogenic amine production was investigated by means of amino acid decarboxylation by growth on decarboxylase differential medium, biogenic amine detection by thinlayer chromatography (TLC) and decarboxylase gene detection by PCR. Decarboxylase medium overestimate the number of biogenic amine-producer strains, as the production of amine was confirmed by TLC in only five out the 17 presumptive strains. On the producer strains, PCR was used to confirm the presence of the genes encoding the amino acid decarboxylase responsible for the synthesis of these amines. Moreover, biogenic amine-producer bacteria were molecularly identified by sequencing their 16S rRNA. Form sardine, enterobacteria producing simultaneously several biogenic amines were isolated. A Kluyvera intermedia strain producing histamine, putrescine and cadaverine, and an Enterobacter asburiae strain producing only the diamines cadaverine and putrescine were identified. From mackerel, lactic acid bacteria from the Enterococcus durans species producing tyramine were isolated. This study constitutes the first description of the presence on these putatively harmful species on ice-preserved sardine and mackerel., This work was supported by grants RM2008-00002 (Instituto Nacional de Investigación Agraria y Alimentaria), AGL2008-01052, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (Comisión Interministerial de Ciencia y Tecnología), S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid), Tunisian Ministry of High Education and Scientific Research and Programa de Cooperación Interuniversitaria e Investigación Científica (AECI), Ministerio de Asuntos Exteriores y Cooperación, Spain (B/019335/08). We are grateful to M. V. Santamaría and J. M. Barcenilla. J. A. Curiel and G. Landeta are recipients of predoctoral fellowships from the I3P-CSIC and CONACyT, respectively.

This article reports for the first time on the capacity of a wine Lactobacillus plantarum strain to alter the oxidative coloration of (þ)-catechin and (-)-epicatechin hydroethanolic wine model solutions in the presence of Fe2þ as catalyst. The time course of color development and pigment formation in the solutions was tracked over 42 days. The pigments formed were characterized as xanthylium structures regardless of the flavanol isomer present in the solution. The solutions supplied with Lactobacillus plantarum RM71 were oxidized at a slower rate, and consequently, its final color was less than that in the controls. The formation of both (þ)-catechin and (-)-epicatechin-derived xanthylium pigments was also delayed over time in the presence of the bacterium compared to their respective cell-free controls. The delaying effects provided by L. plantarum on the oxidative coloration and the generation of xanthylium-derived pigments were more pronounced for the (-)-epicatechin than for the (þ)-catechin model solutions. In view of these results and given that L. plantarum is naturally present in winemaking and generally recognized as a safe microorganism, the potential application of this bacterium as an antibrowning agent for wine is now opened

The broad number of health benefits which can be obtained from the long-term consumption of olive oil are attributed mainly to its phenolic fraction. Many olive oil phenolics have been studied deeply since their discovery due to their bioactivity properties, such as Hydroxytyrosol. Similarly, in the last decade, the special attention of researchers has been addressed to Oleocanthal (OC). This olive oil phenolic compound has recently emerged as a potential therapeutic agent against a variety of diseases, including cancer, inflammation, and neurodegenerative and cardiovascular diseases. Recently, different underlying mechanisms of OC against these diseases have been explored. This review summarizes the current literature on OC to date, and focuses on its promising bioactivities against different disease-targets., This work was supported by the project of Spanish Ministry of Science and Innovation (AGL2015-67995-C3-2-R).

Integrated amperometric biosensors for the determination of l-malic and l-lactic acids were developed by coimmobilization of the enzymes l-malate dehydrogenase (MDH) and diaphorase (DP), or l-lactate oxidase (LOX) and horseradish peroxidase (HRP), respectively, together with the redox mediator tetrathiafulvalene (TTF), on a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode by using a dialysis membrane. The electrochemical oxidation of TTF at +100 mV (vs. Ag/AgCl), and the reduction of TTF+ at -50 mV were used for the monitoring of the enzyme reactions involved in l-malic and l-lactic acid determinations, respectively. Experimental variables concerning the biosensors composition and the detection conditions were optimized for each biosensor. Good relative standard deviation values were obtained in both cases for the measurements carried out with the same biosensor, with no need of cleaning or pretreatment of the bioelectrodes surface, and with different biosensors constructed in the same manner. After 7 days of continuous use, the MDH/DP biosensor still exhibited 90% of the original sensitivity, while the LOX/HRP biosensor yielded a 91% of the original response after 5 days. Calibration graphs for l-malic and l-lactic were obtained with linear ranges of 5.2 × 10-7 to 2.0 × 10-5 and 4.2 × 10-7 to 2.0 × 10-5 M, respectively. The calculated detection limits were 5.2 × 10-7 and 4.2 × 10-7 M, respectively. The biosensors exhibited a high selectivity with no significant interferences. They were applied to monitor malolactic fermentation (MLF) induced by inoculation of Lactobacillus plantarum CECT 748T into a synthetic wine. Samples collected during MLF were assayed for l-malic and l-lactic acids, and the results obtained with the biosensors exhibited a very good correlation when plotted against those obtained by using commercial enzymatic kits. © 2010 Elsevier B.V. All rights reserved., The financial support of the Spanish Ministerio de Educación y Ciencia Research Project CTQ2006-02743BQU

The potential to degrade 15 food phenolic acids was investigated for several Lactobacillus brevis strains isolated from different sources. All the strains analysed in this study showed a similar metabolism on phenolic acids. Among the cinnamic acids assayed, only p-coumaric, ferulic and caffeic acids were metabolized by the L. brevis strains. These acids were decarboxylated to produce their corresponding vinyl derivatives. Contrarily to the results previously reported on Lactobacillus plantarum, the L. brevis strains analysed in this study were unable to subsequently reduce or metabolize these vinyl derivatives. In L. brevis, vinyl phenol, vinyl catechol, and vinyl guaiacol were the final metabolic products from p-coumaric, caffeic or ferulic acids, respectively. From the benzoic acids analysed, and similarly to L. plantarum strains, only gallic and protocatechuic acids were modified by L. brevis strains. Both acids were decarboxylated to pyrogallol and catechol, respectively. Currently, the enzymes involved in the metabolism of phenolic acids in L. brevis remain uncharacterized, This work was supported by Grants AGL2005-00470, AGL2008- 01052, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (CICYT), RM2008-00002 (INIA), and S-0505/AGR/000153 (CAM). We are grateful to M. V. Santamaría, H. Rodríguez, and J. A. Curiel were recipients of predoctoral fellowships from the I3P-CSIC Program and FPI-MEC, respectively. J. M. Landete was a recipient of a postdoctoral fellowship from the MEC.

In recent years, the exquisite stereoselectivity and high efficiency of carbohydrate-processing enzymes have been exploited for many biotechnological applications, including flavor enhancement in foods. In particular, much attention has been focused on the use of b-glucosidases for the enzymatic hydrolysis of flavorless glycoconjugates present in juices and wine beverages for the release aroma volatiles. With the aim to analyze a novel glycosidase with potential applications food industry we have produced and structurally characterized the Bgl glycosidase from the food lactic acid bacterium Lactobacillus plantarum. For that purpose, we have cloned and heterologously expressed the bgl gene (lp_3629) in Escherichia coli. The recombinant protein containing an amino terminal His6 tag (Bgl) has been produced in a soluble form. Purified recombinant enzyme shows galactosidase activity against 4-nitrophenyl b-D-galactopyranoside but not glucosidase activity. Analytical size-exclusion gel filtration chromatography reveals that Bgl behaves in solution as a mixture of monomeric and a high-molecular weight assembly. Purified Bgl has been crystallized by the hanging-drop vapor-diffusion method at 18 C. Diffraction data have been collected at ESRF to a resolution of 2.4 Å. The crystals belong to the space group C2 with unit-cell parameters a = 196.7, b = 191.7, c = 105.9, b = 102.7 . The structure refinement is in progress, JMM thanks the Ministerio de Educación y Ciencia for a research grant (BFU2007-67404/BMC) and ‘‘Factoría de Cristalización” Consolider- Ingenio 2010 in support of his research and the ESRF (Grenoble, France) for provision of synchrotron radiation facilities. This work was also supported by Grants AGL2008-01052 and CSD2007-00063 FUN-C-FOOD (MICINN), RM2008-00002 (INIA), and S-0505/AGR/000153 ALIBIRD (CAM). J.A. Curiel is a recipient of a FPI predoctoral fellowship from the MICINN.

Phenolic compounds are important constituents of food products of plant origin. These compounds are directly related to sensory characteristics of foods such as flavour, astringency, and colour. In addition, the presence of phenolic compounds on the diet is beneficial to health due to their chemopreventive activities against carcinogenesis and mutagenesis, mainly due to their antioxidant activities. Lactic acid bacteria (LAB) are autochthonous microbiota of raw vegetables. To get desirable properties on fermented plant-derived food products, LAB has to be adapted to the characteristics of the plant raw materials where phenolic compounds are abundant. Lactobacillus plantarum is the commercial starter most frequently used in the fermentation of food products of plant origin. However, scarce information is still available on the influence of phenolic compounds on the growth and viability of L. plantarum and other LAB species. Moreover, metabolic pathways of biosynthesis or degradation of phenolic compounds in LAB have not been completely described. Results obtained in L. plantarum showed that L. plantarum was able to degrade some food phenolic compounds giving compounds influencing food aroma as well as compounds presenting increased antioxidant activity. Recently, several L. plantarum proteins involved in the metabolism of phenolic compounds have been genetically and biochemically characterized. The aim of this review is to give a complete and updated overview of the current knowledge among LAB and food phenolics interaction, which could facilitate the possible application of selected bacteria or their enzymes in the elaboration of food products with improved characteristics., This work was supported by grants AGL2005-00470, AGL2008-01052, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (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. J. M. Landete was a recipient of a postdoctoral fellowship from the MEC.

It was previously reported that cell cultures from Lactobacillus plantarum CECT 748T were able to decarboxylate phenolic acids, such as p-coumaric, m-coumaric, caffeic, ferulic, gallic, and protocatechuic acid. The p-coumaric acid decarboxylase (PDC) from this strain has been overexpressed and purified. This PDC differs at its C-terminal end when compared to the previously reported PDC from L. plantarum LPCHL2. Because the C-terminal region of PDC is involved in enzymatic activity, especially in substrate activity, it was decided to biochemically characterize the PDC from L. plantarum CECT 748T. Contrarily to L. plantarum LPCHL2 PDC, the recombinant PDC from L. plantarum CECT 748T is a heat-labile enzyme, showing optimal activity at 22 °C. This PDC is able to decarboxylate exclusively the hydroxycinnamic acids p-coumaric, caffeic, and ferulic acids. Kinetic analysis showed that the enzyme has a 14-fold higher KM value for p-coumaric and caffeic acids than for ferulic acid. PDC catalyzes the formation of the corresponding 4-vinyl derivatives (vinylphenol and vinylguaiacol) from p-coumaric and ferulic acids, respectively, which are valuable food additives that have been approved as flavoring agents. The biochemical characteristics showed by L. plantarum PDC should be taken into account for its potential use in the food-processing industry, The technical assistance of M. V. Santamaría is greatly appreciated.

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License.-- Rizzello et al., [Background]: There is an increasing interest toward the use of legumes in food industry, mainly due to the quality of their protein fraction. Many legumes are cultivated and consumed around the world, but few data is available regarding the chemical or technological characteristics, and especially on their suitability to be fermented. Nevertheless, sourdough fermentation with selected lactic acid bacteria has been recognized as the most efficient tool to improve some nutritional and functional properties. This study investigated the presence of lunasin-like polypeptides in nineteen traditional Italian legumes, exploiting the potential of the fermentation with selected lactic acid bacteria to increase the native concentration. An integrated approach based on chemical, immunological and ex vivo (human adenocarcinoma Caco-2 cell cultures) analyses was used to show the physiological potential of the lunasin-like polypeptides., [Results]: Italian legume varieties, belonging to Phaseulus vulgaris, Cicer arietinum, Lathyrus sativus, Lens culinaris and Pisum sativum species, were milled and flours were chemically characterized and subjected to sourdough fermentation with selected Lactobacillus plantarum C48 and Lactobacillus brevis AM7, expressing different peptidase activities. Extracts from legume doughs (unfermented) and sourdoughs were subjected to western blot analysis, using an anti-lunasin primary antibody. Despite the absence of lunasin, different immunoreactive polypeptide bands were found. The number and the intensity of lunasin-like polypeptides increased during sourdough fermentation, as the consequence of the proteolysis of the native proteins carried out by the selected lactic acid bacteria. A marked inhibitory effect on the proliferation of human adenocarcinoma Caco-2 cells was observed using extracts from legume sourdoughs. In particular, sourdoughs from Fagiolo di Lamon, Cece dell’Alta Valle di Misa, and Pisello riccio di Sannicola flours were the most active, showing a decrease of Caco-2 cells viability up to 70 %. The over-expression of Caco-2 filaggrin and involucrin genes was also induced. Nine lunasin-like polypeptides, having similarity to lunasin, were identified., [Conclusions]: The features of the sourdough fermented legume flours suggested the use for the manufacture of novel functional foods and/or pharmaceuticals preparations.

Background: Myrtle (Myrtus communis L.) is a medicinal and aromatic plant belonging to Myrtaceae family, which is largely diffused in the Mediterranean areas and mainly cultivated in Tunisia and Italy. To the best of our knowledge, no studies have already considered the use of the lactic acid fermentation to enhance the functional features of M. communis. This study aimed at using a selected lactic acid bacterium for increasing the antioxidant features of myrtle berries, with the perspective of producing a functional ingredient, dietary supplement or pharmaceutical preparation. The antioxidant activity was preliminarily evaluated through in vitro assays, further confirmed through ex vivo analysis on murine fibroblasts, and the profile of phenol compounds was characterized. Results: Myrtle berries homogenate, containing yeast extract (0.4%, wt/vol), was fermented with Lactobacillus plantarum C2, previously selected from plant matrix. Chemically acidified homogenate, without bacterial inoculum and incubated under the same conditions, was used as the control. Compared to the control, fermented myrtle homogenate exhibited a marked antioxidant activity in vitro. The radical scavenging activity towards DPPH increased by 30%, and the inhibition of linoleic acid peroxidation was twice. The increased antioxidant activity was confirmed using Balb 3 T3 mouse fibroblasts, after inducing oxidative stress, and determining cell viability and radical scavenging activity through MTT and DCFH-DA assays, respectively. The lactic acid fermentation allowed increased concentrations of total phenols, flavonoids and anthocyanins, which were 5–10 times higher than those found for the non-fermented and chemically acidified control. As shown by HPLC analysis, the main increases were found for gallic and ellagic acids, and flavonols (myricetin and quercetin). The release of these antioxidant compounds would be strictly related to the esterase activities of L. plantarum. Conclusions: The lactic acid fermentation of myrtle berries is a suitable tool for novel applications as functional food dietary supplements or pharmaceutical preparations.

This study aimed at describing the main chemical and technology features of eight Iranian wheat flours collected from industrial and artisanal mills. Their suitability for bread making was investigated using autochthonous sourdough starters. Chemical analyses showed high concentration of fibers and ash, and technology aptitude for making breads. As shown through 2-DE analyses, gliadin and glutenin subunits were abundant and varied among the flours. According to the back slopping procedure, type I sourdoughs were prepared from Iranian flours, and lactic acid bacteria were typed and identified. Strains of Pediococcus pentosaceus, Weissella cibaria, Weissella confusa, and Leuconostoc citreum were the most abundant. Based on the kinetics of growth and acidification, quotient of fermentation and concentration of total free amino acids, lactic acid bacteria were selected and used as sourdough mixed starters for bread making. Compared to spontaneous fermentation, sourdoughs fermented with selected and mixed starters favored the increase of the concentrations of organic acids and total free amino acids, the most suitable quotient of fermentation, and the most intense phytase and antioxidant activities. Although the high concentration of fibers, selected and mixed starters improved the textural features of the breads. This study might had contribute to the exploitation of the potential of Iranian wheat flours and to extend the use of sourdough, showing positive technology, nutritional and, probably, economic repercussions.

This study aimed at evaluating the composition of nineteen traditional Italian legumes and at investigating the potential of the sourdough fermentation with selected lactic acid bacteria to improve the nutritional and functional features. Traditional Italian legumes, all with product certifications and belonging to Phaseolus vulgaris, Cicer arietinum, Lathyrus sativus, Lens culinaris and Pisum sativum species, were used in this study. Seeds were milled, and flours were analyzed for proximate composition and subjected to sourdough fermentation at 30°C for 24h. Lactobacillus plantarum C48 and Lactobacillus brevis AM7 were used as selected starters. Compared to control doughs, without bacterial inoculum, the concentrations of free amino acids (FAA), soluble fibres, and total phenols increased for all legume sourdoughs. Raffinose decreased of up to ca. 64%. During sourdough fermentation, the level of GABA markedly increased and reached values up to 624mg/kg. Condensed tannins decreased. At the same time, almost all legume sourdoughs showed increases of the antioxidant and phytase activities. As shown by PCA analysis based on data of total FAA, GABA, raffinose, soluble/insoluble dietary fibre, condensed tannins and antioxidant and phytase activities, all legume sourdoughs were clearly differentiated from control doughs. The traditional Italian legumes are bio-diverse, and all showed high levels of nutritional elements and suitability for optimal sourdough fermentation. Legume flours subjected to sourdough fermentation would be suitable to be used alone or better in mixture with cereals, and as gluten-free ingredients for making novel and healthy foods.

In situ extractive fermentation was used to produce 4-vinyl derivatives from hydroxycinnamic acids extracted from corn cobs by recombinant Escherichia coli cells expressing Lactobacillus plantarum phenolic acid descarboxylase (PAD) gene. This microorganism mainly produced 4-vinylphenol (4VP) from p-coumaric acid (p-CA). In the first study , we observed that the concentrations of 4VP are higher than 1g/L which had a negative impact on decarboxylation of p-CA to 4VP by recombinant E. coli cells. Because of this, and in order to improve the downstream process, a two-phase aqueous-organic solvent system was developed. The results of the extractive fermentation indicated that it was possible to use hydrolyzates as aqueous phase to bioproduce 4VP, and recover simultaneously the product in the organic phase containing hexane. The detoxification of pre-treated corn cob alkaline hydrolyzate improved 4VP production up to 1003.5mg/L after 24h fermentation (QP=41.813mg/Lh). Additionally, preliminary experiments using cells immobilized in calcium alginate showed to be a good system for the biotransform of p-CA to 4VP in extractive fermentation, although the process hindered partially the recovery of 4VP in the organic phase. © 2014 Elsevier Inc., Spanish Ministry of Science and Innovation for the financial support of this work (CTQ2011-28967 and AGL2011-22745 projects), which has partial financial support from the FEDER funds of the European Union. J.M.S. is grateful for Postdoctoral fellowship (SFRH / BPD / 84440 / 2012) of Fundação para a Ciência e Tecnologia – FCT.

Six Albanian soft and durum wheat cultivars were characterized based on chemical and technological features, showing different attitudes for bread making. Gliadin and glutenin fractions were selectively extracted from flours, and subjected to two-dimensional electrophoresis. Linja 7 and LVS flours showed the best characteristics, and abundance of high molecular weight (HMW)-glutenins. Type I sourdoughs were prepared through back slopping procedure, and the lactic acid bacteria were typed and identified. Lactobacillus plantarum and Leuconostoc mesenteroides were the predominant species. Thirty-eight representative isolates were singly used for sourdough fermentation of soft and durum wheat Albanian flours and their selection was carried out based on growth and acidification, quotient of fermentation, and proteolytic activity. Two different pools of lactic acid bacteria were designed to ferment soft or durum wheat flours. Sourdough fermentation with mixed and selected starters positively affected the quotient of fermentation, concentration of free amino acids, profile of phenolic acids, and antioxidant and phytase activities. This study provided the basis to exploit the potential of wheat Albanian flours based on an integrated approach, which considered the characterization of the flours and the processing conditions.

This study aimed at investigating the suitability of oat flakes for making functional beverages. Different technological options were assayed, including the amount of flakes, the inoculum of the starter and the addition of enzyme preparations. The beverage containing 25% (wt/wt) of oat flakes and fermented with L. plantarum LP09 was considered optimal on the basis of sensory and technological properties. The enzyme addition favored the growth of the starter, shortened the time needed to reach pH. 4.2 to ca. 8. h, and favored a decrease of the quotient of fermentation. Fermentation increased the polyphenols availability and the antioxidant activity (25 and 70% higher, respectively) and decreased the hydrolysis index in vitro. Sensory analyses showed that fermented oat flakes beverage had the typical features of a yogurt-like beverage, enhancing the overall intensity of odor and flavor compared to the non-fermented control. Selection of proper processing and fermentation condition allowed the obtainment of a beverage with better nutritional and sensory properties.

Multilocus sequence typing (MLST) is a typing scheme that involves the determination of the allelic variation at multiple housekeeping loci. This variation is determined by nucleotide sequencing of internal fragments of approximately 500 bp and the resulting data could be stored on central databases held on the Internet. Population and evolutionary biology of microbial species could be determined by the use of available software for MLST data analysis. MLST provide an unambiguous, portable, reproducible, and scalable typing system.

This is an open-access article distributed under the terms of the Creative Commons Attribution License., [Background]:Owing to its antimicrobial properties dietary tannins may alter the functional efficacy of probiotic lactobacilli in the gastrointestinal (GI)-tract influencing their growth, viability and molecular adaptation to the intestinal environment. [Methods and Findings]:The effects of tannic acid on Lactobacillus plantarum WCFS1 were studied by in vitro growth monitoring and visualizing the morphological alteration on the cell wall using transmission electron microscopy. Growth upon tannic acid was characterized by dose-dependent reductions of initial viable counts and extended lag phases. Lag phase-cells growing upon 0.5 mM tannic acid were abnormally shaped and experienced disturbance on the cell wall such as roughness, occasional leakage and release of cell debris, but resumed growth later at tannic acid concentrations high as 2.5 mM. To gain insight on how the response to tannic acid influenced the molecular adaptation of L. plantarum to the GI-tract conditions, gene expression of selected biomarkers for GI-survival was assessed by RT-qPCR on cDNA templates synthetized from mRNA samples obtained from cells treated with 0.5 or 2 mM tannic acid. Tannic acid-dependent gene induction was confirmed for selected genes highly expressed in the gut or with confirmed roles in GI-survival. No differential expression was observed for the pbp2A gene, a biomarker negatively related with GI-survival. However PBP2A was not labeled by Bocillin FL, a fluorescent dye-labeled penicillin V derivative, in the presence of tannic acid which suggests for enhanced GI-survival reportedly associated with the inactivation of this function. [Conclusions]:Probiotic L. plantarum WCFS1 is able to overcome the toxic effects of tannic acid. This dietary constituent modulates molecular traits linked to the adaptation to intestinal environment in ways previously shown to enhance GI-survival. © 2013 Reverón et al., H. Rodriguez and J. A. Curiel acknowledge I3P (CSIC) or FPI (MICINN) PhD grants, respectively. The authors gratefully acknowledge the partial financial support of the CICYT grants AGL2008-001052, AGL2011-22745, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD, the CAM grant S2009/AGR-1469 (ALIBIRD) and the MINECO grant CTM2012-38222-C02-02.

Technological and safety-related properties were analyzed in a coagulase-negative staphylococci (CNS) collection isolated from Spanish dry-cured meat products in order to use them as starter cultures. The highest nitrate reductase and proteolytic activity was showed by Staphylococcus carnosus and Staphylococcus equorum. Only a few strains were able to form biofilms and the presence of the ica gene was analyzed on them. In relation to antibiotic resistance, all S. carnosus and most of the S. equorum strains were sensitive to the antibiotics tested and the presence of the blaZ gene in the β-lactamic resistant strains was studied. Biogenic amines were produced by 25% of the strains analyzed being all the S. carnosus strains tyramine producers. Taking into account the studied properties, two S. equorum strains could be selected as adequate and safe potential starter cultures for the elaboration of meat products. © 2012 Elsevier Ltd., This work was supported by grants RM2008-00002 (Instituto Nacional de Investigación Agraria y Alimentaria), AGL2008-01052, AGL2011-22745, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (Comisión Interministerial de Ciencia y Tecnología), and S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid). We are grateful to M. V. Santamaría and J. M. Barcenilla. J. A. Curiel and G, Landeta are recipients of predoctoral fellowship from the FPI-MEC, and CONACyT, respectively.

A family of restriction enzyme- and ligation-independent cloning vectors has been developed for producing recombinant His-tagged fusion proteins in Escherichia coli. These are based on pURI2 and pURI3 expression vectors which have been previously used for the successful production of recombinant proteins at the milligram scale. The newly designed vectors combines two different promoters (lppp-5 and T7 RNA polymerase Ø10), two different endoprotease recognition sites for the His6-tag removal (enterokinase and tobacco etch virus), different antibiotic selectable markers (ampicillin and erythromycin resistance), and different placements of the His 6-tag (N- and C-terminus). A single gene can be cloned and further expressed in the eight pURI vectors by using six nucleotide primers, avoiding the restriction enzyme and ligation steps. A unique NotI site was introduced to facilitate the selection of the recombinant plasmid. As a case study, the new vectors have been used to clone the gene coding for the phenolic acid decarboxylase from Lactobacillus plantarum. Interestingly, the obtained results revealed markedly different production levels of the target protein, emphasizing the relevance of the cloning strategy on soluble protein production yield. Efficient purification and tag removal steps showed that the affinity tag and the protease cleavage sites functioned properly. The novel family of pURI vectors designed for parallel cloning is a useful and versatile tool for the production and purification of a protein of interest. © 2010 Elsevier Inc. All rights reserved., This work was supported by grants RM2008-00002 (Instituto Nacional de Investigación Agraria y Alimentaría), AGL2008-01052, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (Comisión Interministerial de Ciencia y Tecnología), and S-0505/AGR/000153 and S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid). J.M.M. thanks the Ministerio de Ciencia e Innovación for a research grant (BFU2007-67404/BMC) and “Factoría de Cristalización” Consolider-Ingenio 2010 in support of his research. The technical assistance of M.V. Santamaría is greatly appreciated. J.A. Curiel is a recipient of a predoctoral fellowship from the MEC.

Scope: To gain insight on the mechanisms used by intestinal bacteria to adapt and resist the antimicrobial action of dietary tannins and identify targets for tannic acid in Lactobacillus plantarum. Methods and results: A proteomic analysis of an L. plantarum human isolate exposed to the tannic acid challenge was undertaken. Tannic acid targeted proteins involved in outstanding processes for bacterial stress resistance including cyclopropanation of membrane lipids, stress response at population scale and maintenance of cell shape. To respond to this aggression, tannic acid-misfit cells of L. plantarum challenged with tannic acid reorganized their metabolic capacity to economize energy and express proteins involved in oxidative stress defense and cell wall biogenesis, indicating that the injury incurred by tannic acid was based on oxidative damage and disruption of the cell envelope. The induction of 3-octaprenyl-4-hydroxybenzoate carboxy-lyase, which is sensitive to changes in redox conditions and involved in ubiquinone biosynthesis in other bacteria, suggests for a tannic acid-induced redox imbalance. Conclusion: The results reveal the adaptation of a gastrointestinal isolate of L. plantarum to tannic acid and identify antibacterial targets for this dietary compound. This provides the basis for the selection of tannin-resistant microorganisms and their use to obtain health benefits from tannin-containing diets., This work was supported by grants AGL2005-000470, AGL2008-001052, and Consolider INGENIO 2010 CSD2007- 00063 FUN-C-FOOD (CICYT), and S2009/AGR-1469 (ALIBIRD) (CAM)

Novel heterofunctional glyoxyl-agarose supports were prepared. These supports contain a high concentration of groups (such as quaternary ammonium groups, carboxyl groups, and metal chelates) that are capable of adsorbing proteins, physically or chemically, at neutral pH as well as a high concentration of glyoxyl groups that are unable to immobilize covalently proteins at neutral pH. By using these supports, a two-step immobilization protocol was developed. In the first step, enzymes were adsorbed at pH 7.0 through adsorption of surface regions, which are complementary to the adsorbing groups on the support, and in the second step, the immobilized derivatives were incubated under alkaline conditions to promote an intramolecular multipoint covalent attachment between the glyoxyl groups on the support and the amino groups on the enzyme surface. These new derivatives were compared with those obtained on a monofunctional glyoxyl support at pH 10, in which the region with the greatest number of lysine residues participates in the first immobilization step. In some cases, multipoint immobilization on heterofunctional supports was much more efficient than what was achieved on the monofunctional support. For example, derivatives of tannase from Lactobacillus plantarum on an amino-glyoxyl heterofunctional support were 20-fold more stable than the best derivative on a monofunctional glyoxyl support. Derivatives of lipase from Geobacillus thermocatenulatus (BTL2) on the amino-glyoxyl supports were two times more active and four times more enantioselective than the corresponding monofunctional glyoxyl support derivative, This work has been sponsored by the Spanish Ministry of Science and Innovation (project AGL-2009- 07625) and Intramural projects (2008801058); and the Comunidad Autónoma de Madrid (project S0505/PPQ/03449). We gratefully recognize to Spanish Ministry of Science and Innovation for “Ramón y Cajal” contract to Dr. Fernandez- Lorente