Bactericidal action of oleuropein extracted from green olives against Lactobacillus plantarum

The phenolic compound oleuropein extracted from green olives was shown to be bactericidal against nine strains of Lactobacillus plantarum isolated from green olive fermentation brines. Heat-treated oleuropein also demonstrated a strong bactericidal effect but not alkali-treated oleuropein, which allowed survival of most of the strains tested. The bactericidal effect was accompanied by changes in the typical bacillary structure and Gram-positive stain of L. plantarum.     

Effect of medium components on bacteriocin production by Lactobacillus plantarum strains ST23LD and ST341LD, isolated from spoiled olive brine

Bacteriocin ST23LD levels of 2930AU/OD were recorded in MRS broth (pH of 6.5) and in the presence of tryptone and yeast extract as sole nitrogen sources. Growth in MRS broth at an initial pH of 6.0 yielded only 1460AU/OD bacteriocin ST23LD. Activities of 5861AU/OD were recorded with maltose (20, 30 and 40 g/l) as sole carbon source and 9036AU/OD with the addition of 2.0-10.0 g/l KH2PO4. Bacteriocin ST341LD levels of 2850 and 2841AU/OD were recorded in MRS broth at an initial pH of 6.0 or 5.5, respectively. Only 709AU/OD was recorded in the same medium with an initial pH of 6.5. Bacteriocin ST341LD production was stimulated by the presence of tryptone. However, glucose at 10 and 40 g/l, or the presence of 5.0 or 10.0 g/l K2HPO4, resulted in a 50% reduction of bacteriocin activity. Glycerol in the growth medium repressed bacteriocin production. No increased bacteriocin production was recorded in medium supplemented with vitamins.     

Biodegradation of different molecular-mass polyphenols derived from olive mill wastewaters by Geotrichum candidum

The decolourisation of fresh and stored olive mill wastewaters (OMW) and the biodegradation of three groups (F1, F2 and F3) of phenolic compounds by Geotrichum candidum were investigated. Separated phenolic compounds derived from natural OMW ultrafiltration using membranes with a cutoff 2and 100 kDa. G. candidum growth on fresh OMW decreased pH and reduced COD and colour of 75% and 65%, respectively. However, on the stored-black OMW a failure of COD and colour removal were observed. G. candidum activity on this later substrate was enhanced by the addition of a carbon source easily metabolised, misleading an improvement of the COD reduction and decolourization that reached 58% and 48%, respectively. Growth of G. candidum in the presence of F2 or F3 polyphenolic fractions induced high decolourisation and depolymerisation of phenolic compounds. Whereas, very week decolourisation and biodegradation were observed with F1 fraction. Moreover, the highest levels of lignin peroxidase (LiP) and manganese peroxidase (MnP) activities were obtained in the presence of F2 fraction. These results showed that increasing of molecular-mass of aromatics led to an increase in levels of depolymerisation, decolourisation and COD removal by G. candidum culture.     

Vitamin and amino acid requirements of Lactobacillus plantarum strains isolated from green olive fermentations

The requirement for essential amino acids and vitamins was determined in wild-type Lactobacillus plantarum strains isolated from green olive fermentation brines. All the strains were found to be auxotrophic with respect to the amino acids but some of them were prototrophic for pyridoxal, p -aminobenzoic acid and/or nicotinic acid. Their growth response to these nutrients was also studied and found to be quite heterogeneous. Nutritional requirement pattern as a criteria for selecting starter cultures is discussed.     

Plasmid profiles and curing of plasmids in Lactobacillus plantarum strains isolated from green olive fermentations

J.L. RUIZ-BARBA, J.C. PIARD AND R. JIMENEZ-DIAZ. 1991. Plasmid profiles of 35 Lactobacillus plantarum strains isolated from different green olive fermentors were obtained. A large number of plasmids in the CCC form (from 5 to 16) were present in all the tested strains as confirmed by a second dimension electrophoresis of DNA. These plasmids, all of which remain cryptic, ranged from 2.0 to 68 kb in size. Novobiocin, sodium dodecyl sulphate and ethidium bromide were used as plasmid-curing agents but only novobiocin induced loss of extrachromosomal DNA at a high frequency in these strains.     

Oxygen utilization by Lactobacillus plantarum

Cell-free extracts of Lactobacillus plantarum contain non-proteinaceous compounds which mimic superoxide dismutase activity. Using the test system in which O ₂ ^– is generated by xanthine oxidase, superoxide dismutase activity is found in cell-free extracts, where proteins are removed by precipitation. This activity is strongly decreased after dialysis of cell-free extracts. Superoxide dismutase activity was also investigated by means of pulse radiolysis. Cell-free extracts of Escherichia coli were also investigated as a comparison, which were known to contain superoxide dismutase. With cell-free extracts of both L. plantarum and E. coli the decay of O ₂ ^– was markedly increased. However, the type of reaction of the O ₂ ^– decay was of first order in the presence of E. coli extracts due to superoxide dismutase(s), and of second order in the presence of L. plantarum extracts, indicating that O ₂ ^– elimination is not an enzymic reaction. Mn²⁺ phosphate(s) might be responsible for the observed elimination of O ₂ ^– . The production of O ₂ ^– is not detectable during NADH-, lactate- or pyruvate oxidase reactions in L. plantarum extracts.     

Plasmid profiles and curing of plasmids in Lactobacillus plantarum strains isolated from green olive fermentations.

Plasmid profiles of 35 Lactobacillus plantarum strains isolated from different green olive fermentors were obtained. A large number of plasmids in the CCC form (from 5 to 16) were present in all the tested strains as confirmed by a second dimension electrophoresis of DNA. These plasmids, all of which remain cryptic, ranged from 2.0 to 68 kb in size. Novobiocin, sodium dodecyl sulphate and ethidium bromide were used as plasmid-curing agents but only novobiocin induced loss of extrachromosomal DNA at a high frequency in these strains.     

Characterization of Rhamnosidases from Lactobacillus plantarum and Lactobacillus acidophilus

Lactobacilli are known to use plant materials as a food source. Many such materials are rich in rhamnose-containing polyphenols, and thus it can be anticipated that lactobacilli will contain rhamnosidases. Therefore, genome sequences of food-grade lactobacilli were screened for putative rhamnosidases. In the genome of Lactobacillus plantarum, two putative rhamnosidase genes (ram1_Lp and ram2_Lp) were identified, while in Lactobacillus acidophilus, one rhamnosidase gene was found (ramA_La). Gene products from all three genes were produced after introduction into Escherichia coli and were then tested for their enzymatic properties. Ram1_Lp, Ram2_Lp, and RamA_La were able to efficiently hydrolyze rutin and other rutinosides, while RamA_La was, in addition, able to cleave naringin, a neohesperidoside. Subsequently, the potential application of Lactobacillus rhamnosidases in food processing was investigated using a single matrix, tomato pulp. Recombinant Ram1_Lp and RamA_La enzymes were shown to remove the rhamnose from rutinosides in this material, but efficient conversion required adjustment of the tomato pulp to pH 6. The potential of Ram1_Lp for fermentation of plant flavonoids was further investigated by expression in the food-grade bacterium Lactococcus lactis. This system was used for fermentation of tomato pulp, with the aim of improving the bioavailability of flavonoids in processed tomato products. While import of flavonoids into L. lactis appeared to be a limiting factor, rhamnose removal was confirmed, indicating that rhamnosidase-producing bacteria may find commercial application, depending on the technological properties of the strains and enzymes.Lactobacilli such as Lactobacillus plantarum have been used for centuries to ferment vegetables such as cabbage, cucumber, and soybean (34). Fruit pulps, for instance, those from tomato, have also been used as a substrate for lactobacilli for the production of probiotic juices (38). Recently, the full genomic sequences of several lactobacilli have become available (1, 22). A number of the plant-based substrates for lactobacilli are rich in rhamnose sugars, which are often conjugated to polyphenols, as in the case of cell wall components and certain flavonoid antioxidants. Utilization of these compounds by lactobacilli would involve α-l-rhamnosidases, which catalyze the hydrolytic release of rhamnose. Plant-pathogenic fungi such as Aspergillus species produce the rhamnosidases when cultured in the presence of naringin, a rhamnosilated flavonoid (24, 26). Bacteria such as Bacillus species have also been shown to use similar enzyme activities for metabolizing bacterial biofilms which contain rhamnose (17, 40).In food processing, rhamnosidases have been applied primarily for debittering of citrus juices. Part of the bitter taste of citrus is caused by naringin (Fig. ​(Fig.1),1), which loses its bitter taste upon removal of the rhamnose (32). More recently, application of rhamnosidases for improving the bioavailability of flavonoids has been described. Human intake of flavonoids has been associated with a reduced risk of coronary heart disease in epidemiological studies (19). Food flavonoids need to be absorbed efficiently from what we eat in order to execute any beneficial function. Absorption occurs primarily in the small intestine (12, 37). Unabsorbed flavonoids will arrive in the colon, where they will be catabolized by the microflora, which is then present in huge quantities. Therefore, it would be desirable for flavonoids to be consumed in a form that is already optimal for absorption in the small intestine prior to their potential degradation. For the flavonoid quercetin, it has been demonstrated that the presence of rhamnoside groups inhibits its absorption about fivefold (20). A number of flavonoids which are present in frequently consumed food commodities, such as tomato and citrus products, often carry rutinoside (6-β-l-rhamnosyl-d-glucose) or neohesperidoside (2-β-l-rhamnosyl-d-glucose) residues (Fig. ​(Fig.1).1). Therefore, removal of the rhamnose groups from such flavonoid rutinosides and neohesperidosides prior to consumption could enhance their intestinal absorption. With this aim, studies were recently carried out toward the application of fungal enzyme preparations as a potential means to selectively remove rhamnoside moieties (16, 30).Open in a separate windowFIG. 1.Chemical structures of rhamnose-containing flavonoids from plants. Relevant carbon atoms in glycoside moieties are numbered. (1) Rutin (quercetin-3-glucoside-1→6-rhamnoside); (2) narirutin (naringenin-7-glucoside-1→6-rhamnoside); (3) naringin (naringenin-7-glucoside-1→2-rhamnoside); (4) p-nitrophenol-rhamnose.In view of the frequent occurrence of lactobacilli on decaying plant material and fermented vegetable substrates, one could anticipate that their genomes carry one or more genes encoding enzymes capable of utilizing rhamnosilated compounds. In the work reported here, we describe the identification of three putative rhamnosidase genes in lactobacillus genomes. We expressed these genes in Escherichia coli and characterized their gene products. The activities of all three lactobacillus rhamnosidases on flavonoids naturally present in tomato pulp were then assessed. One of the L. plantarum genes, which encoded the enzyme with the highest activity and stability in E. coli, was then also expressed in Lactococcus lactis, with the aim of investigating the potential use of such a recombinant organism to improve the bioavailability of fruit flavonoids and thus their efficacy in common foodstuffs.     

Phenoloxidase-producing halotolerant fungi from olive brine wastewater

The main aim of this study was to assess the ability of producing extracellular phenoloxidases (EPO) of fungi isolated from olive brine wastewater (OBW), the effluent from the debittering process of table olives. Five out of twenty isolates displayed EPO-producing ability as assessed by selective agar plate assays and were all halotolerant. Among them, Citeromyces matritensis (syn. Candida globosa) and Aspergillus fumigatus concomitantly produced laccase and Mn-peroxidase (MnP) activities. Candida boidinii and Candida bombi, instead, only produced laccase and Candida diddensiae only released MnP. Both C. matritensis and A. fumigatus, grown in shaken cultures on OBW, maintained their EPO-producing ability and removed phenols by 52.3 and 82.3%, respectively, after 10 d incubation in the non-supplemented effluent. These results might suggest the possible use of these strains in the treatment of other saline phenol-rich effluents, such as pickling and tannery wastewater.     

Citrate metabolism by Lactobacillus plantarum isolated from orange juice

The behaviour of Strains of Lactobacillus plantarum isolated from fermented orange juice and Lact. plantarum DSM 20174 was studied in the presence of citrate. When used as sole carbon source, citrate scarcely supported the growth of the bacteria. It was shown to enhance the growth of Lact. plantarum in glucose media. Under acid conditions (pH 4·0–5·0), 1 mol of citrate yielded 1·7 mol of acetate as sole major final metabolite with release of CO₂ in the gas phase.     

The recovery of polyphenols from olive mill waste using two adsorbing vegetable matrices

Olive mill wastewater (OMW) is considered one of the most pollutive waste materials in the Mediterranean basin. However, its phenolic fraction should be recovered, since it has been shown to have incredible benefits for health. In the present study, the adsorbent and desorbent capacities of Azolla and granular activated carbon (GAC) were investigated. The GAC was found to be more efficient than Azolla in both the adsorption and the desorption of phenols. The total characterization of two powder products obtained from Azolla and GAC desorption is reported, together with their antioxidant and antiradical activities. In the Azolla powder product, total polyphenols were more than twice as numerous as those found in the GAC powder product. The GAC powder contained hydroxytyrosol in concentrations that were 3.5 times higher than those of Azolla. On the other hand, both powder products showed great antiradical activities: the IC?? was found to be 102 mg ml?1 for the Azolla and 199 mg ml?1 for the GAC powders respectively. The oxygen radical absorbance capacity was very high: 4097 μmol TE g?1 Azolla powder product and 1277 μmol TE g?1 of GAC powder products.     

植物乳杆菌细菌素的研究与应用

植物乳杆菌细菌素不仅种类多,产生菌在发酵过程中还可产生良好的保健功效,因此成为研究的热点。本文对植物乳杆菌细菌素的种类、分子结构、抑菌机制及遗传控制做了较为详尽的介绍,并简要介绍了植物乳杆菌细菌素在食品、医药、饲料中的应用,为进一步研究植物乳杆菌细菌素提供了参考。     

Complete DNA sequence and analysis of two cryptic plasmids isolated from Lactobacillus plantarum

The complete nucleotide sequence of two cryptic plasmids isolated from Lactobacillus plantarum strain AS1.2986 has been determined. The smaller plasmid, designated pLP2000, encodes a 37.0kDa Rep protein and has a 17bp sequence repeated 10 times. Sequence analysis of the larger plasmid, designated pLP9000, revealed nine putative open reading frames (ORFs). Based on sequence similarity, ORF1 codes for a putative magnesium transporter protein that shows similarities to CorA from plasmid pCIS3 (Lactococcus lactis). None of the nine ORFs shows similarity to any known Rep protein. Southern blot analysis indicates these two plasmids both replicate via a rolling circle (RC) mechanism.     

One-step purification of metallothionein extracted from two different sources

We describe a one-step purification of hepatic metallothionein from the Amazon fish Colossoma macropomum injected with cadmium and from the copper-loaded metallothionein from the yeast Saccharomyces cerevisiae, performed by affinity chromatography through metal-chelating columns. Yeast metallothionein was purified from Cu2+-loaded resin and eluted by a continuous EDTA gradient whereas hepatic metallothionein extracted from fishes was purified by Ni2+-loaded resin and eluted by a continuous imidazol gradient. Purified metallothioneins were evaluated by SDS-PAGE and characterized by UV spectra of the apo- and Cd2+-loaded protein. This method allowed high purity and yield as well as rapid one-step extraction of both metal-loaded and apoprotein.     

Antimicrobial potential of polyphenols extracted from almond skins

Aims: To evaluate the antimicrobial properties of flavonoid‐rich fractions derived from natural and blanched almond skins, the latter being a by‐product from the almond processing industry. Methods and Results: Almond skin extracts were tested against Gram‐negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens), Gram‐positive bacteria (Listeria monocytogenes, Enterococcus hirae, Staphylococcus aureus, Enterococcus durans) and the yeast Candida albicans. Almond skin fractions were found to have antimicrobial activity against L. monocytogenes and Staph. aureus in the range 250–500 μg ml^?1, natural skins showing antimicrobial potential against the Gram‐negative Salm. enterica. The interactions between three almond skin flavonoids were also evaluated with isobolograms. Conclusions: Pairwise combinations of protocatechuic acid, naringenin and epicatechin showed both synergistic and indifferent interactions against Salm. enterica and Staph. aureus. Antagonism was observed against L. monocytogenes with all combinations tested. Further studies need to be performed to understand the mechanisms responsible for these interactions. Significance and Impact of the Study: Almond skins are a potential source of natural antimicrobials.