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.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

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.     

Effect of crab shell size on bio-demineralization with lactic acid-producing bacterium, <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">tolerans</Emphasis> KCTC-3074

Demineralization (DM) from crab shell (CS) waste was carried out using a lactic acid-producing bacterium, Lactobacillus paracasei subsp. tolerans KCTC-3074 for 7 days at 25, 30, and 35°C. DM rates were 89∼92% and slightly affected by temperature. DM was also performed for four particle-sized shell samples (0.84∼3.35, 3.35∼10, 10∼20, and 20∼35 mm) with 10% inoculum, 5% shell, and 10% glucose at 30°C and 180 rpm for 7 days. It was found out that the shell size had a slight effect on the rate of DM. Negative relationships were found between DM and residual dry weight (r² = 0.960), and between DM and pH (r² = 0.906). Conversely, positive relationships were found between DM and medium protein (r² = 0.696), and between DM and total titratable acidity (r² = 0.630).     

Probiotic properties of <Emphasis Type="Italic">Lactobacillus</Emphasis> and <Emphasis Type="Italic">Bifidobacterium</Emphasis> strains isolated from porcine gastrointestinal tract

One strain of Lactobacillus salivarius, two strains of Lactobacillus reuteri and Lactobacillus amylovorus, and two strains of Bifidobacterium thermacidophilum with antagonistic effect against Clostridium perfringens were isolated from porcine gastrointestinal tract. Isolates were assayed for their ability to survive in synthetic gastric juice at pH 2.5 and were examined for their ability to grow on agar plate containing porcine bile extract. There was a large variation in the survival of the isolates in gastric juice and growth in the medium containing 0.3% (w/v) bile. L. salivarius G11 and L. amylovorus S6 adhered to the HT-29 epithelial cell line. Cell-free supernatant of L. amylovorus S6 showed higher antagonistic activity as effective as the antibiotics such as neomycin, chlortetracycline, and oxytetracycline against bacterial pathogens including C. perfringens, Salmonella typhimurium, Staphylococcus aureus, Vibrio cholerae, Edwardsiella tarda, and Aeromonas salmonicida subsp. salmonicida.     

Hyaluronic acid production by recombinant <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

A gene encoding a new xylanase, named xynZG, was cloned by the genome-walking PCR method from the nematophagous fungus Plectosphaerella cucumerina. The genomic DNA sequence of xynZG contains a 780 bp open reading frame separated by two introns with the sizes of 50 and 46 bp. To our knowledge, this would be the first functional gene cloned from P. cucumerina. The 684 bp cDNA was cloned into vector pHBM905B and transformed into Pichia pastoris GS115 to select xylanase-secreting transformants on RBB-xylan containing plate. The optimal secreting time was 3 days at 25°C and enzymatic activities in the culture supernatants reached the maximum level of 362 U ml⁻¹. The molecular mass of the enzyme was estimated to be 19 kDa on SDS-PAGE. The optimal pH and temperature of the purified enzyme is 6 and 40°C, respectively. The purified enzyme is stable at room temperature for at least 10 h. The K _m and V _max values for birchwood xylan are 2.06 mg ml⁻¹ and 0.49 mmol min⁻¹mg⁻¹, respectively. The inhibitory effects of various mental ions were investigated. It is interesting to note that Cu²⁺ ion, which strongly inhibits most other xylanases studied, reduces enzyme activity by only 40%. Furthermore, enzyme activity is unaffected by EDTA even at a concentration of 5 mM.     

Impact of <Emphasis Type="Italic">cry1AC</Emphasis>-carrying <Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis> on leaf bacterial community

The effects of Chinese cabbage (Brassica rapa subsp. pekinensis) carrying cry1AC derived from Bacillus thuringiensis (Bt) on leaf bacterial community were examined by analyzing the horizontal transfer of trans-gene fragments from plants to bacteria. The effect of plant pathogenic bacteria on the gene transfer was also examined using Pseudomonas syringae pathovar. maculicola. The frequency of hygromycin-resistant bacteria did not alter in Bt leaves, though slight increase was observed in Pseudomonas-infected Bt leaves with no statistical significance. The analysis of bacterial community profiles using the denaturing gradient gel electrophoresis (DGGE) fingerprinting indicated that there were slight differences between Bt and control Chinese cabbage, and also that infected tissues were dominated by P. syringae pv. maculicola. However, the cultured bacterial pools were not found to contain any transgene fragments. Thus, no direct evidence of immediate gene transfer from plant to bacteria or acquisition of hygromycin resistance could be observed. Still, long-term monitoring on the possibility of gene transfer is necessary to correctly assess the environmental effects of the Bt crop on bacteria.     

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     

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.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from sugarcane molasses,sugarcane juice and sugar beet juice by <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Lactobacillus delbrueckii was grown on sugarcane molasses, sugarcane juice and sugar beet juice in batch fermentation at pH 6 and at 40°C. After 72 h, the lactic acid from 13% (w/v) sugarcane molasses (119 g total sugar l⁻¹) and sugarcane juice (133 g total sugar l⁻¹) was 107 g l⁻¹ and 120 g l⁻¹, respectively. With 10% (w/v) sugar beet juice (105 g total sugar l⁻¹), 84 g lactic acid l⁻¹ was produced. The optical purities of d-lactic acid from the feedstocks ranged from 97.2 to 98.3%.     

An efficient process for production and purification of hyaluronic acid from <Emphasis Type="Italic">Streptococcus </Emphasis><Emphasis Type="Italic">equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Growth of Streptococcus zooepidemicus in a 10 l bioreactor with 50 g sucrose/l and 10 g casein hydrolysate/l gave 5–6 g hyaluronic acid/l after 24–28 h. Purification of hyaluronic acid gave a recovery of 65% with the final material having an Mr of ∼4 × 10⁶ Da with less than 0.1% protein.     

Improvement of nisin production in pH feed-back controlled,fed-batch culture by <Emphasis Type="Italic">Lactococcus lactis</Emphasis>subsp<Emphasis Type="Italic">. lactis</Emphasis>

Nisin production by Lactococcus lactis subsp. lactisin fed-batch culture was doubled by using a pH feed-back controlled method. Sucrose concentration was controlled at 10 g l^–1 giving 5010 IU nisin ml^–1 compared to 2660 IU nisin ml^–1 in batch culture.     

Characteristics of the bacteriocin produced by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">cremoris</Emphasis> CTC 204 and the effect of this compound on the mesophilic bacteria associated with raw beef

Summary >Screening for the bacteriocin production of strains of lactic acid bacteria from various meat and meat products resulted in the detection of a bacteriocin-producing Lactococcus lactis subsp. cremoris CTC 204, isolated from chicken. The bacteriocin inhibited not only closely related lactic acid bacteria (Lactobacillus helveticus), but also pathogenic microorganisms (Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, and Clostridium perfringens). It was inactivated by α-chymotrypsin, ficin, papain, and pronase E, but not by lipase or pepsin. This compound was heat stable even at autoclaving temperature (121°C for 10min) and was produced during refrigerated storage. It was also active over a wide pH range (2–10), but the highest activity was observed in the lower pH range. The results indicated that dipping raw beef in the bacteriocin produced by strain CTC 204 could contribute to the extension of the shelf life of refrigerated bovine meat.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Influence of immobilization parameters on growth and lactic acid production by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> and <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis> co-immobilized in calcium alginate gel beads

Streptococcus thermophilusand Lactobacillus bulgaricus were co-immobilized in different systems with varying calcium (0.1–1.5M) and alginate (1–2<><>, w/v) concentrations. Highest lactic acid production was 35 g l₁ when both bacteria were in high viscosity beads (1<><>, w/v alginate) hardened in 0.1 M CaCl₂ .The gel bead composition affected size and distribution of entrapped lactic acid bacteria.     

Improved immobilization of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> ATCC 7469 in polyacrylamide gel,preventing cell leakage during lactic acid fermentation

A new procedure for improved immobilization of Lactobacillus rhamnosus ATCC 7469, producing solely l(+)-lactic acid, in polyacrylamide was developed. A series of gels with varied ingredients concentrations and order of addition was prepared and were tested in batch and repeat-batch processes. Our results revealed that the crucial step for successful immobilization was the initial incubation of the cells in pure 10% AA that leads to improved entrapment in the polyacrylamide gel. In contrast, all gels derived from previously prepared stock AA/MBAA released high amount of cells and free biomass was formed. The most efficient immobilization was achieved using gel, containing L. rhamnosus, incubated in 10% AA (acrylamide) and with 1% MBAA (N,N′-methylene-bis-acrylamide) added. This gel possessed optimal permeation characteristics and at the same time, the cells were completely retained in the polymer lattice (0.03 g free biomass/l at 48 h of the batch process). In addition, it yielded highly concentrated lactic acid: the conversion ratio was about 85% without pH-control for initial lactose concentrations of up to 30 g/l. A series of additional immobilization experiments showed the potential of physicochemical interactions between the monomers of acrylamide and the cell surface of L. rhamnosus.     

Physiological comparison of copper toxicity in the lichens <Emphasis Type="Italic">Peltigera rufescens</Emphasis> (Weis) Humb. and <Emphasis Type="Italic">Cladina arbuscula</Emphasis> subsp. <Emphasis Type="Italic">mitis</Emphasis> (Sandst.) Ruoss

Peltigera rufescens (Weis) Humb. with a prokaryotic photobiont Nostoc sp. and Cladina arbuscula subsp. mitis (Sandst.) Ruoss with a eukaryotic photobiont Trebouxia sp. were studied to determine the copper sensitivity of lichens with different algal symbionts. Samples growing on historic copper mine-spoil heaps at Ľubietová–Podlipa, Slovakia were assessed for physiological parameters, including total and intracellular uptake of copper, assimilation pigmentation, activity of photosystem II, ergosterol levels, thiobarbituric acid reactive substances and water-soluble protein content. Our results indicate that P. rufescens was more sensitive to copper exposure than C. arbuscula subsp. mitis.     

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.