Characterization and stabilization of GluLm and its application to deglycosylate dietary flavonoids and lignans.

This study describes the characterization of the recombinant GH3 aryl-ß-glucosidase "GluLm" from Limosilactobacillus mucosae INIA P508, followed by its immobilization on an agarose support with the aim of developing an efficient application to increase the availability and concentration of flavonoid and lignan aglycones in a vegetal beverage. In previous studies, heterologous GluLm-producing strains demonstrated a great capacity to deglycosylate flavonoids. Nevertheless, the physicochemical properties and substrate spectrum of the enzyme remained unknown up to now. A high production of purified GluLm was achieved (14 mg L-1). GluLm exhibited optimal activity at broad ranges of pH (5.0-8.0) and temperature (25-60°C), as well as high affinity (Km of 0.10 mmol L-1) and specific constant (86554.0 mmol L-1 s-1) against p-nitrophenyl-ß-D-glucopyranoside. Similar to other GH3 ß-glucosidases described in lactic acid bacteria, GluLm exhibited ß-xylosidase, ß-galactosidase, and ß-fucosidase activities. However, this study has revealed for the first time that a GH3 ß-glucosidase is capable to hydrolyze different families of glycosylated phenolics such as flavonoids and secoiridoids. Although it exhibited low thermal stability, immobilization of GluLm improved its thermostability and allowed the development of a beverage based on soybeans and flaxseed extract with high concentration of bioactive isoflavone (daidzein, genistein), lignan (secoisolariciresinol, pinoresinol, and matairesinol), and other flavonoid aglycones. KEY POINTS: • Limosilactobacillus mucosae INIA P508 GluLm was purified and biochemically characterized • Immobilized GluLm efficiently deglycosylated flavonoids and lignans from a vegetal beverage • A viable application to produce vegetal beverages with a high content of aglycones is described.

Promoters for the expression of food-grade selectable markers in lactic acid bacteria and bifidobacteria.

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.

Characterization and stabilization of the α-L-fucosidase set from Lacticaseibacillus rhamnosus INIA P603.

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. KEY POINTS: • Few strains of L. rhamnosus exhibited alfB on their chromosomes. • Fucosidases from L. rhamnosus INIA P603 were characterized and stabilized. • Although all the fucosidases hydrolyzed 2'-FL, only AlfB transfucosylated lactose.

Architecture Insight of Bifidobacterial α-L-Fucosidases.

Fucosylated carbohydrates and glycoproteins from human breast milk are essential for the development of the gut microbiota in early life because they are selectively metabolized by bifidobacteria. In this regard, α-L-fucosidases play a key role in this successful bifidobacterial colonization allowing the utilization of these substrates. Although a considerable number of α-L-fucosidases from bifidobacteria have been identified by computational analysis, only a few of them have been characterized. Hitherto, α-L-fucosidases are classified into three families: GH29, GH95, and GH151, based on their catalytic structure. However, bifidobacterial α-L-fucosidases belonging to a particular family show significant differences in their sequence. Because this fact could underlie distinct phylogenetic evolution, here extensive similarity searches and comparative analyses of the bifidobacterial α-L-fucosidases identified were carried out with the assistance of previous physicochemical studies available. This work reveals four and two paralogue bifidobacterial fucosidase groups within GH29 and GH95 families, respectively. Moreover, Bifidobacterium longum subsp. infantis species exhibited the greatest number of phylogenetic lineages in their fucosidases clustered in every family: GH29, GH95, and GH151. Since α-L-fucosidases phylogenetically descended from other glycosyl hydrolase families, we hypothesized that they could exhibit additional glycosidase activities other than fucosidase, raising the possibility of their application to transfucosylate substrates other than lactose in order to synthesis novel prebiotics.

Aroma profiling of an aerated fermentation of natural grape must with selected yeast strains at pilot scale.

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.

Current Disease-Targets for Oleocanthal as Promising Natural Therapeutic Agent.

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.

Differential Gene Expression by Lactobacillus plantarum WCFS1 in Response to Phenolic Compounds Reveals New Genes Involved in Tannin Degradation.

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. plantarumtanB [tanBLp ], 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.IMPORTANCELactobacillus plantarum is a bacterial species frequently found in the fermentation of vegetables when tannins are present. L. plantarum strains degrade tannins to the less-toxic pyrogallol by the successive action of tannase and gallate decarboxylase enzymes. The genes encoding these enzymes are located close to each other in the chromosome, suggesting concomitant regulation. Proteins involved in tannin metabolism and regulation, such GacP (gallic acid permease) and TanR (tannin transcriptional regulator), were identified by differential gene expression in knockout mutants with mutations in genes from this region. This study provides insights into the highly coordinated mechanisms that enable L. plantarum to adapt to plant food fermentations.

Identification of target genes to control acetate yield during aerobic fermentation with Saccharomyces cerevisiae.

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.

Lactic acid fermentation as a tool to enhance the antioxidant properties of Myrtus communis berries.

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.

Italian legumes: effect of sourdough fermentation on lunasin-like polypeptides.

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.

Iranian wheat flours from rural and industrial mills: Exploitation of the chemical and technology features, and selection of autochthonous sourdough starters for making breads.

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.

Improving Properties of a Novel ß-Galactosidase from Lactobacillus plantarum by Covalent Immobilization.

A novel ß-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 ß-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 ß-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.

Use of fungal proteases and selected sourdough lactic acid bacteria for making wheat bread with an intermediate content of gluten.

This study was aimed at combining the highest degradation of gluten during wheat flour fermentation with good structural and sensory features of the related bread. As estimated by R5-ELISA, the degree of degradation of immune reactive gluten was ca. 28%. Two-dimensional electrophoresis and RP-FPLC analyses showed marked variations of the protein fractions compared to the untreated flour. The comparison was also extended to in vitro effect of the peptic/tryptic-digests towards K562 and T84 cells. The flour with the intermediate content of gluten (ICG) was used for bread making, and compared to whole gluten (WG) bread. The chemical, structural and sensory features of the ICG bread approached those of the bread made with WG flour. The protein digestibility of the ICG bread was higher than that from WG flour. Also the nutritional quality, as estimated by different indexes, was the highest for ICG bread.

Exploitation of Albanian wheat cultivars: characterization of the flours and lactic acid bacteria microbiota, and selection of starters for sourdough fermentation.

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.

Production of recombinant glycosidases fused with Usp45 and SpaX to avoid the purification and immobilization stages.

The elucidation of the physicochemical properties of glycosidases is essential for their subsequent technological application, which may include saccharide hydrolysis processes and oligosaccharide synthesis. As the application of cloning, purification and enzymatic immobilization methods can be time consuming and require a heavy financial investment, this study has validated the recombinant production of the set of Lacticaseibacillus rhamnosus fucosidases fused with Usp45 and SpaX anchored to the cell wall of Lacticaseibacillus cremoris subsp cremoris MG1363, with the aim of avoiding the purification and stabilization steps. The cell debris harboring the anchored AlfA, AlfB and AlfC fucosidases showed activity against p-nitrophenyl α-L-fucopyranoside of 6.11 ±â€¯0.36, 5.81 ±â€¯0.29 and 9.90 ±â€¯0.58 U/mL, respectively, and exhibited better thermal stability at 50 °C than the same enzymes in their soluble state. Furthermore, the anchored AlfC fucosidase transfucosylated different acceptor sugars, achieving fucose equivalent concentrations of 0.94 ±â€¯0.09 mg/mL, 4.11 ±â€¯0.21 mg/mL, and 4.08 ±â€¯0.15 mg/mL of fucosylgalatose, fucosylglucose and fucosylsucrose, respectively.

Riboflavin bio-enrichment of soy beverage by selected roseoflavin-resistant and engineered lactic acid bacteria.

Some lactic acid bacteria (LAB) have the ability to synthesize riboflavin, a trait linked to the presence of ribG, ribB, ribA and ribH genes located in the rib operon. Previous screening of riboflavin producers identified several LAB strains belonging to different species with this ability, but none of them surpassed 0.25 mg/L production of the vitamin. In this study, we explored two strategies to obtain riboflavin-overproducing strains: by roseoflavin selection of mutants, and by the transformation of selected strains with plasmids pNZ:TuR.rib or pNZ:TuB.rib containing the genes ribG, ribB, ribA and ribH from Lactococcus cremoris MG1363. The resulting riboflavin-overproducing strains were able to produce yields between 0.5 and 6 mg/L in culture media and several of them were selected for the fermentation of soy beverages. Riboflavin in bio-enriched soy beverages was evaluated by direct fluorescence measurement and high-performance liquid chromatography-fluorescence analysis. Soy beverages fermented with the recombinant strains Lactococcus cremoris ESI 277 pNZ:TuB.rib and Lactococcus lactis INIA 12 pNZ:TuR.rib showed the highest riboflavin yields (>5 mg/L) after 24 h fermentation. On the other hand, roseoflavin-resistant mutant Limosilactobacillus fermentum INIA P143R2 was able to enrich fermented soy beverages with 1.5 mg/L riboflavin. Riboflavin-overproducing LAB strains constitute a good option for riboflavin enrichment of soy beverages by fermentation and the commercialization of such beverages could be very useful to prevent riboflavin deficiency.

Food phenolics and Lactiplantibacillus plantarum.

Phenolic compounds are important constituents of plant food products. These compounds play a key role in food characteristics such as flavor, astringency and color. Lactic acid bacteria are naturally found in raw vegetables, being Lactiplantibacillus plantarum the most commonly used commercial starter for the fermentation of plant foods. Hence, the metabolism of phenolic compounds of L. plantarum has been a subject of study in recent decades. Such studies confirm that L. plantarum, in addition to presenting catalytic capacity to transform aromatic alcohols and phenolic glycosides, exhibits two main differentiated metabolic routes that allow the biotransformation of dietary hydroxybenzoic and hydroxycinnamic acid-derived compounds. These metabolic pathways lead to the production of new compounds with new biological and organoleptic properties. The described metabolic pathways involve the action of specialized esterases, decarboxylases and reductases that have been identified through genetic analysis and biochemically characterized. The purpose of this review is to provide a comprehensive and up-to-date summary of the current knowledge of the metabolism of food phenolics in L. plantarum.

Uncovering the Lactobacillus plantarum WCFS1 gallate decarboxylase involved in tannin degradation.

Lactobacillus plantarum is a lactic acid bacterium able to degrade tannins by the subsequent action of tannase and gallate decarboxylase enzymes. The gene encoding tannase had previously been identified, whereas the gene encoding gallate decarboxylase is unknown. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of gallic-acid induced L. plantarum extracts showed a 54-kDa protein which was absent in the uninduced cells. This protein was identified as Lp_2945, putatively annotated UbiD. Homology searches identified ubiD-like genes located within three-gene operons which encoded the three subunits of nonoxidative aromatic acid decarboxylases. L. plantarum is the only bacterium in which the lpdC (lp_2945) gene and the lpdB and lpdD (lp_0271 and lp_0272) genes are separated in the chromosome. Combination of extracts from recombinant Escherichia coli cells expressing the lpdB, lpdC, and lpdC genes demonstrated that LpdC is the only protein required to yield gallate decarboxylase activity. However, the disruption of these genes in L. plantarum revealed that the lpdB and lpdC gene products are essential for gallate decarboxylase activity. Similar to L. plantarum tannase, which exhibited activity only in esters derived from gallic and protocatechuic acids, purified His6-LpdC protein from E. coli showed decarboxylase activity against gallic and protocatechuic acids. In contrast to the tannase activity, gallate decarboxylase activity is widely present among lactic acid bacteria. This study constitutes the first genetic characterization of a gallate decarboxylase enzyme and provides new insights into the role of the different subunits of bacterial nonoxidative aromatic acid decarboxylases.

Synthesis of 2-methoxy benzoquinone and 2,6-dimethoxybenzoquinone by selected lactic acid bacteria during sourdough fermentation of wheat germ.

BACKGROUND: In the last decade, several studies described the promising cytotoxic activity of fermented wheat germ towards cancer cell lines and during in vivo clinical trials. Recent data suggested that the antiproliferative, antimetastatic and immunological effects of this preparation are mainly attributed to quinones. This study aimed at exploiting the potential of sourdough lactic acid bacteria fermentation to release 2-methoxy benzoquinone, and 2,6-dimethoxybenzoquinone, which are naturally present in wheat germ as glycosylated and non-physiologically active form. RESULTS: Preliminarily, forty strains of lactic acid bacteria, previously isolated from wheat germ, were in vitro screened based on ß-glucosidase activity. Lactobacillus plantarum LB1 and Lactobacillus rossiae LB5 were selected based on the highest enzyme activity and on technology features. These strains were used in combination to ferment wheat germ. Raw wheat germ, without bacterial inoculum, was subjected to the same incubation and used as the control. The sourdough fermented wheat germ was characterized based on microbiological, physico-chemical and biochemical features. During incubation, the release of the non-glycosylated and physiologically active 2-methoxy benzoquinone, and 2,6-dimethoxybenzoquinone was almost completed during 24 h. Compared to the control, the concentration of the above bioactive compounds increased almost 4 and 6-folds. Both raw wheat germ (control) and sourdough fermented wheat germ were ex vivo assayed for the anti-proliferative activity towards various cell lines of germ cell tumor, colon carcinoma and ovarian carcinoma. While no effect was found for the raw wheat germ, the sourdough fermented preparation markedly and variously affected the human tumor cell lines. The values of IC50 ranged from 0.105 ± 0.005 to 0.556 ± 0.071 mg/ml, with a median value of IC50 of 0.302 mg/ml. CONCLUSIONS: These results are comparable to those found for other well-known pharmaceutical preparations, and may disclose the use of the sourdough fermented wheat germ as an ingredient, nutritional supplement and/or anticancer drug.

Isoflavone Metabolism by Lactic Acid Bacteria and Its Application in the Development of Fermented Soy Food with Beneficial Effects on Human Health.

Isoflavones are phenolic compounds (considered as phytoestrogens) with estrogenic and antioxidant function, which are highly beneficial for human health, especially in the aged population. However, isoflavones in foods are not bioavailable and, therefore, have low biological activity. Additionally, their transformation into bioactive compounds by microorganisms is necessary to obtain bioavailable isoflavones with beneficial effects on human health. Many lactic acid bacteria (LAB) can transform the methylated and glycosylated forms of isoflavones naturally present in foods into more bioavailable aglycones, such as daidzein, genistein and glycitein. In addition, certain LAB strains are capable of transforming isoflavone aglycones into compounds with a greater biological activity, such as dihydrodaidzein (DHD), O-desmethylangolensin (O-DMA), dihydrogenistein (DHG) and 6-hydroxy-O-desmethylangolensin (6-OH-O-DMA). Moreover, Lactococcus garviae 20-92 is able to produce equol. Another strategy in the bioconversion of isoflavones is the heterologous expression of genes from Slackia isoflavoniconvertens DSM22006, which have allowed the production of DHD, DHG, equol and 5-hydroxy-equol in high concentrations by engineered LAB strains. Accordingly, the consequences of isoflavone metabolism by LAB and its application in the development of foods enriched in bioactive isoflavones, as well as health benefits attributed to their consumption, will be addressed in this work.