Dynamic analysis of <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">bulgaricus</Emphasis> CFL1 physiological characteristics during fermentation

This study aimed at examining and comparing the relevance of various methods in order to discriminate different cellular states of Lactobacillus bulgaricus CFL1 and to improve knowledge on the dynamics of the cellular physiological state during growth and acidification. By using four fluorescent probes combined with multiparametric flow cytometry, membrane integrity, intracellular esterase activity, cellular vitality, membrane depolarization, and intracellular pH were quantified throughout fermentations. Results were compared and correlated with measurements of cultivability, acidification activity (Cinac system), and cellular ability to recover growth in fresh medium (Bioscreen system). The Cinac system and flow cytometry were relevant to distinguish different physiological states throughout growth. Lb. bulgaricus cells maintained their high viability, energetic state, membrane potential, and pH gradient in the late stationary phase, despite the gradual decrease of both cultivability and acidification activity. Viability and membrane integrity were maintained during acidification, at the expense of their cultivability and acidification activity. Finally, this study demonstrated that the physiological state during fermentation was strongly affected by intracellular pH and the pH gradient. The critical pHi of Lb. bulgaricus CFL1 was found to be equal to pH 5.8. Through linear relationships between dpH and cultivability and pHi and acidification activity, pHi and dpH well described the time course of metabolic activity, cultivability, and viability in a single analysis.     

Enhanced production of lactic acid by an adapted strain of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis>

Lactobacillus delbrueckii subsp. lactis strains were developed having increased activity, by gradually acclimatizing the bacteria to acidic conditions over repeated batch culture. Cells from one batch culture were used as the inoculum for the subsequent batch culture and thereby an adapted strain of Lactobacillus was obtained showing improved lactic acid productivity, cell growth and total glucose utilization. Furthermore, the acclimatized cells used significantly less nitrogen for a given level of lactic acid production, which is significant from an industrial point of view. The developed procedure decreases fermentation time and nutrient use, leading to reduced operation costs, while providing a lactic acid yield superior to previously reported methods.     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.     

Synthesis of carotenoids by<Emphasis Type="Italic"> Rhodotorula rubra</Emphasis> GED8 co-cultured with yogurt starter cultures in whey ultrafiltrate

Two cultures, a yeast (Rhodorula rubra GED8) and a yogurt starter (Lactobacillus bulgaricus 2–11+Streptococcus thermophilus 15HA), were selected for associated growth in whey ultrafiltrate (WU) and active synthesis of carotenoids. In associated cultivation with the yogurt culture L bulgaricus 2–11+S. thermophilus 15HA under intensive aeration (1.3 l^–1min^–1 air-flow rate) in WU (45 g lactose l^–1), initial pH 5.5, 30 °C, the lactose-negative strain R. rubra GED8 synthesized large amounts of carotenoids (13.09 mg l^–1 culture fluid). The carotenoid yield was approximately two-fold higher in association with a mixed yogurt culture than in association with pure yogurt bacteria. The major carotenoid pigments comprising the total carotenoids were -carotene (50%), torulene (12.3%) and torularhodin (35.2%). Carotenoids with a high -carotene content were produced by the microbial association 36 h earlier than by Rhodotorula yeast species. No significant differences were notd in the ratio between the pigments synthesized by R. rubra GED8+L. bulgaricus 2–11, R. rubra GED8+S. thermophilus 15HA, and R.rubra GED8+yogurt culture, despite the fact that the total carotenoid concentrations were lower in the mixed cultures with pure yogurt bacteria.     

Cytoprotective Agent in <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis> Extracts

Adenosine 5′-diphosphoribose (ADP-ribose) has been identified as a significant contributor to the anti-cytotoxic activity of Lactobacillus bulgaricus extracts. Although the biological activities associated with the administration of probiotic bacteria and components thereof are sometimes attributed to the peptidoglycans that comprise a substantial portion of the Gram-positive bacterial cell wall, we found that the beta-nicotine adenine dinucleotide (NAD) hydrolysis product ADP-ribose was a significant contributor to the observed anti-cytotoxicity in our L. bulgaricus extracts. The ADP-ribose was isolated, identified, and quantitated by high performance liquid chromatography (HPLC) and by nuclear magnetic resonance (NMR) spectroscopy. ADP-ribose levels as low as 5 mg/L exhibited a measurable inhibition of tumor necrosis factor alpha (TNF-α) mediated cytotoxicity in an in vitro cell assay, whereas the ADP-ribose content of the L. bulgaricus extracts often exceeded 5 mg/g dry weight.     

Fermentation performance of an exopolysaccharide-producing strain of<Emphasis Type="Italic"> Lactobacillus delbrueckii</Emphasis> subsp.<Emphasis Type="Italic"> bulgaricus</Emphasis>

The formation of exopolysaccharide (EPS) and extracellular metabolites was studied in a strain of Lactobacillus delbrueckii subsp. bulgaricus (NCFB 2483), grown under batch culture conditions in a semi-defined medium incorporating lactose and casein hydrolysate. Performance parameters were derived from the fermentation data, and kinetic models were applied in order to describe the production of EPS, extracellular metabolites, and biomass produced. Lactose was split intracellularly, with the resultant galactose being exported from the cell, and the glucose being metabolised further to EPS and lactic acid. Production of EPS, lactate, and galactose was closely growth-associated and followed a pattern of primary kinetics. A marginally lower galactose level relative to the modelled levels throughout most of the time course of the fermentation suggests that not all galactose is exported from the cell, and that a low level of flux to other metabolites, such as EPS, might exist.     

Lactic acid production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing a <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactate dehydrogenase gene

This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2μ-containing yeast-replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92 g glucose/l in approximately 30 h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38 g lactic acid/l and a yield of 0.44 g lactic acid/g glucose. The optimal pH for lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 units/mg protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated by ethanol yields between 0.25–0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased. Electronic Publication     

Autohydrolysed <Emphasis Type="Italic">Tilapia nilotica</Emphasis> Fish Viscera as a Peptone Source in Bacteriocin Production

Fish processing generates large amounts of solid and liquid wastes. Many different by-products have been produced from fish processing wastes. Studies on solubilization of Bolti fish (Tilapia nilotica) viscera by endogenous enzymes at different pHs are described. Hydrolysis reactions were conducted with freshly thawed viscera utilizing an initial temperature gradient and terminated at various time points by heat inactivation of the enzymes. Various peptones obtained from hydrolysed visceral homogenates of Bolti fish residues showed their suitability for promoting the growth of lactic acid bacteria (mainly Lactobacillus sake Lb 706), microorganisms with particularly complex nutritional requirements especially peptidic sources. The assay of several treatments with L. sakei Lb 706, producer of the bacteriocin sakacin A, demonstrated that optimum conditions for biomass and bacteriocin production only imply a brief autohydrolysis at room temperature. The results showed that the Bolti fish hydrolysates gave remarkable results to those found in costly commercial media, specifically recommended for culturing and large-scale production of lactic acid bacteria.     

A novel chimeric prophage vB_LdeS-phiJB from commercial <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">bulgaricus</Emphasis>

Prophage vB_LdeS-phiJB (phiJB) was induced by mitomycin C and UV radiation from the Lactobacillus delbrueckii subsp. bulgaricus SDMCC050201 isolated from a Chinese yoghurt sample. It has an isometric head and a non-contractile tail with 36,969 bp linear double-stranded DNA genome, which is classified into the group a of Lb. delbrueckii phages. The genome of phiJB is highly modular with functionally related genes clustered together. Unexpectedly, there is no similarity of its DNA replication module to any phages that have been reported, while it consists of open-reading frames homologous to the proteins of Lactobacillus strains. Comparative genomic analysis indicated that its late gene clusters, integration/lysogeny modules and DNA replication module derived from different evolutionary ancestors and integrated into a chimera. Our results revealed a novel chimeric phage of commercial Lb. delbrueckii and will broaden the knowledge of phage diversity in the dairy industry.     

Plant regeneration from <Emphasis Type="Italic">Gossypium davidsonii</Emphasis> protoplasts <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis

Protoplasts isolated from wild cotton Gossypium davidsonii were cultured in KM₈P medium supplemented with different phytohormones. The most effective combination was 0.45 μM 2,4-dichlorophenoxyacetic acid, 2.68 μM α-naphthaleneacetic acid and 0.93 μM kinetin and the division percentage at the 8^th day was 30.78 ± 3.04 %. The density of protoplasts at 2–10 × 10⁵ cm⁻³ was suitable for protoplast division and calli formation, with a division percentage of 32.21 ± 3.64 % and a plating efficiency of 9.12 ± 2.61 % at the 40^th day. The optimal osmotic potential was achieved using 0.5 M glucose or 0.1 M glucose plus 0.5 M mannitol. Protoplasts were cultured in three ways, a double-layer culture system, with liquid over solid medium was proved to be the best way. Embryo induction was further increased by addition of 0.14 μM gibberellic acid.     

Kinetic Modelling of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> ssp. <Emphasis Type="Italic">rhamnosus</Emphasis> Growth and Lactic Acid Production in Batch Cultures Under Various Medium Conditions

Enrichment of medium with yeast extract and tryptone increased growth and lactic acid production in batch cultures of Lactobacillus casei ssp. rhamnosus. A reliable kinetic model that explicitly expresses the strong relationship between microbial growth, lactic acid production and medium enrichment is provided and validated using experimental data obtained with six different medium compositions. Revisions requested 2 February 2005 and 26 July 2005; Revisions received 25 July 2005 and 9 September 2005     

Analysis of <Emphasis Type="Italic">ldh</Emphasis> genes in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> BL23: role on lactic acid production

Lactobacillus casei is a lactic acid bacterium that produces L-lactate as the main product of sugar fermentation via L-lactate dehydrogenase (Ldh1) activity. In addition, small amounts of the D-lactate isomer are produced by the activity of a D-hydroxycaproate dehydrogenase (HicD). Ldh1 is the main L-lactate producing enzyme, but mutation of its gene does not eliminate L-lactate synthesis. A survey of the L. casei BL23 draft genome sequence revealed the presence of three additional genes encoding Ldh paralogs. In order to study the contribution of these genes to the global lactate production in this organism, individual, as well as double mutants (ldh1 ldh2, ldh1 ldh3, ldh1 ldh4 and ldh1 hicD) were constructed and lactic acid production was assessed in culture supernatants. ldh2, ldh3 and ldh4 genes play a minor role in lactate production, as their single mutation or a mutation in combination with an ldh1 deletion had a low impact on L-lactate synthesis. A Deltaldh1 mutant displayed an increased production of D-lactate, which was probably synthesized via the activity of HicD, as it was abolished in a Deltaldh1 hicD double mutant. Contrarily to HicD, no Ldh1, Ldh2, Ldh3 or Ldh4 activities could be detected by zymogram assays. In addition, these assays revealed the presence of extra bands exhibiting D-/L-lactate dehydrogenase activity, which could not be attributed to any of the described genes. These results suggest that L. casei BL23 possesses a complex enzymatic system able to reduce pyruvic to lactic acid.     

Applicability of pectate-entrapped <Emphasis Type="Italic">Lactobacillus casei</Emphasis> cells for <Emphasis Type="SmallCaps">l</Emphasis>(+) lactic acid production from whey

Lactic acid is a versatile organic acid, which finds major application in the food, pharmaceuticals, and chemical industries. Microbial fermentation has the advantage that by choosing a strain of lactic acid bacteria producing only one of the isomers, an optically pure product can be obtained. The production of l(+) lactic acid is of significant importance from nutritional viewpoint and finds greater use in food industry. In view of economic significance of immobilization technology over the free-cell system, immobilized preparation of Lactobacillus casei was employed in the present investigation to produce l(+) lactic acid from whey medium. The process conditions for the immobilization of this bacterium using calcium pectate gel were optimized, and the developed cell system was found stable during whey fermentation to lactic acid. A high lactose conversion (94.37%) to lactic acid (32.95 g/l) was achieved with the developed immobilized system. The long-term viability of the pectate-entrapped bacterial cells was tested by reusing the immobilized bacterial biomass, and the entrapped bacterial cells showed no decrease in lactose conversion to lactic acid up to 16 batches, which proved its high stability and potential for commercial application.     

Impact of fermentation pH and temperature on freeze-drying survival and membrane lipid composition of <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> Si3

During the industrial stabilization process, lactic acid bacteria are subjected to several stressful conditions. Tolerance to dehydration differs among lactic acid bacteria and the determining factors remain largely unknown. Lactobacillus coryniformis Si3 prevents spoilage by mold due to production of acids and specific antifungal compounds. This strain could be added as a biopreservative in feed systems, e.g. silage. We studied the survival of Lb. coryniformis Si3 after freeze-drying in a 10% skim milk and 5% sucrose formulation following different fermentation pH values and temperatures. Initially, a response surface methodology was employed to optimize final cell density and growth rate. At optimal pH and temperature (pH 5.5 and 34 °C), the freeze-drying survival of Lb. coryniformis Si3 was 67% (±6%). The influence of temperature or pH stress in late logarithmic phase was dependent upon the nature of the stress applied. Heat stress (42 °C) did not influence freeze-drying survival, whereas mild cold- (26 °C), base- (pH 6.5), and acid- (pH 4.5) stress significantly reduced survival. Freeze-drying survival rates varied fourfold, with the lowest survival following mild cold stress (26 °C) prior to freeze-drying and the highest survival after optimal growth or after mild heat (42 °C) stress. Levels of different membrane fatty acids were analyzed to determine the adaptive response in this strain. Fatty acids changed with altered fermentation conditions and the degree of membrane lipid saturation decreased when the cells were subjected to stress. This study shows the importance of selecting appropriate fermentation conditions to maximize freeze-drying viability of Lb. coryniformis as well as the effects of various unfavorable conditions during growth on freeze-drying survival.     

Factors affecting tyramine production in <Emphasis Type="Italic">Enterococcus durans</Emphasis> IPLA 655

The decarboxylation of tyrosine by certain lactic acid bacteria leads to the undesirable presence of tyramine in fermented foods. Tyramine is the most frequent biogenic amine found in cheese and is also commonly found in other fermented foods and beverages. The tyramine-producing strain Enterococcus durans IPLA 655 was grown in a bioreactor under different conditions to determine the influence of carbon source, tyrosine and tyramine concentrations, and pH on tyramine production. The carbon source appeared to have no significant effect on the production of tyramine. In contrast, tyrosine was necessary for tyramine production, while the presence of tyramine itself in the growth medium inhibited such production. pH showed by far the greatest influence on tyramine synthesis; tyramine was produced in the greatest quantities at pH 5.0, although this was accompanied by a reduced growth rate.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot culture and antimicrobial activity of <Emphasis Type="Italic">Berberis buxifolia</Emphasis> Lam

Berberis buxifolia Lam., known as “Calafate”, is a plant native to Argentina that exhibits antimicrobial activity. This biological activity is attributed to the isoquinoline alkaloid berberine. The aim of this research was to test the antimicrobial properties of different extracts of this species, taking berberine as the reference molecule, and to examine if the expression of bacterial multidrug resistance (MDR) efflux pumps could be responsible for possible resistance mechanisms. To this end, a wild-type and a mutant strain of Staphylococcus aureus with a defective MDR efflux pump were used and the minimum inhibitory concentrations of the extracts were determined. The studies were carried out with infusions of in vivo shoots and “Calafate” commercial tea, as well as with the media derived from shoot cultures incubated with different plant growth regulators (thidiazuron, picloram, and jasmonic acid). As far as antimicrobial activity is concerned, all the extracts tested were significantly more effective than berberine standard. “Calafate” commercial tea and shoot tea had inhibitory concentrations similar to the one observed for ampicillin standard. The media from the shoot cultures, however, were significantly more effective than all the others, particularly the one derived from jasmonic acid, suggesting the presence of compounds that could be acting synergistically with berberine. There were no differences in antimicrobial activity against the wild-type and the mutant S. aureus; no definite conclusions could be drawn concerning the relationship between MDR pumps and possible pathogen resistance to extracts of B. buxifolia.