Characterization of ethanol fermentation waste and its application to lactic acid production by Lactobacillus paracasei

In this study, an ethanol fermentation waste (EFW) was characterized for use as an alternative to yeast extract for bulk fermentation processes. EFW generated from a commercial plant in which ethanol is produced from cassava/rice/wheat/barley starch mixtures using Saccharomyces cerevisiae was used for lactic acid production by Lactobacillus paracasei. The effects of temperature, pH, and duration on the autolysis of an ethanol fermentation broth (EFB) were also investigated. The distilled EFW (DEFW) contained significant amounts of soluble proteins (2.91 g/l), nitrogen (0.47 g/l), and amino acids (24.1 mg/l). The autolysis of the EFB under optimum conditions released twice as much amino acids than in the DEFW. Batch fermentation in the DEFW increased the final lactic acid concentration, overall lactic acid productivity, and lactic acid yield on glucose by 17, 41, and 14 %, respectively, in comparison with those from comparable fermentation in a lactobacillus growth medium (LGM) that contained 2 g/l yeast extract. Furthermore, the overall lactic acid productivity in the autolyzed then distilled EFW (ADEFW) was 80 and 27 % higher than in the LGM and DEFW, respectively.     

Random amplified polymorphic DNA-PCR based cloning of markers to identify the beer-spoilage strains of Lactobacillus brevis, Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis

AIMS: Beer-spoilage ability of lactic acid bacteria such as Lactobacillus brevis is a strain-dependent phenomenon in which the mechanism has not yet been completely clarified. In order to systematically identify genes that contribute to beer-spoilage, large-scale random amplified polymorphic DNA (RAPD)-based cloning methods was carried out. METHODS AND RESULTS: A systematic RAPD polymerase chain reaction (PCR) analysis using 600 primers was performed on beer-spoilage and on nonspoilage strains of L. brevis. Among 600 primers, three were found to amplify a single locus highly specific to beer-spoilage strains. DNA sequencing of this locus revealed a three-part operon encoding a putative glycosyl transferase, membrane protein and teichoic acid glycosylation protein. PCR analysis of typical beer-spoilage lactic acid bacteria suggested that this locus is highly specific to beer-spoilage strains. CONCLUSION: The cloned markers are highly specific to identify the beer-spoilage strains not only in L. brevis but also in Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper proves that RAPD-PCR is an efficient method for cloning the strain-specific genes from bacteria. The markers described here is one of the most useful tools to identify the beer-spoilage strains of lactic acid bacteria.     

Soybean response to gibberellic acid treatments

Early-maturing soybean lines (Glycine max (L.) Merr.) grown in subtropical areas during short days can produce economic seed yields. However, the plants are short, and many of the pods are produced close to the soil, making commercial harvest difficult. The feasibility of increasing the lengths of the first three internodes by growth regulator application was evaluated at two locations over a 3-year period using separate plots of Amsoy 71 (Maturity Group (MG) II) and Williams (MG III) soybean by spraying each cultivar with 0, 25, or 50 g (a.i.) ha^–1 of gibberellic acid (GA) in 280 L ha^–1 water when the hypocotyl was cracking the soil. The 25 g ha^–1 treatment increased (p<.05) the length of the hypocotyl and first internode 60% and 25% in 1979 and 36% and 17%, respectively, in 1980. Increasing the GA application to 50 g ha^–1 had no additional effect on hypocotyl elongation, but the length of the first internode continued to increase linearly. Gibberellic acid treatment did not stimulate elongation of internodes above the first two, and internodes above the fifth tended to be shorter, causing total plant height at maturity to be the same as untreated plants. The overall effect of GA application at cracking time on harvestable seed yield was about nil, since GA decreased yields by 8–11%, but it increased stem elongation, so a greater portion of seeds were produced above 80 mm that were accessible to commercial combining. The application of GA generally did not affect (p<.05) seed quality. Seed weight was reduced significantly at both locations in 1979 but not in 1980. These data indicate that elongation of the lower stem portion of early-maturing soybean cultivars grown in subtropical areas during short days can be increased significantly by GA to improve commercial harvesting, but the treatments appear to have little net effect on seed yield.     

Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides.

The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a alpha2beta2gamma2 composition. The Km for the three main substrates were 1.6 mm for 1,2-propanediol, 5.5 mm for 1,2-ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 micro m. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 degrees C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2-propanediol metabolism pathway.     

Characterization of the protein-synthesis dependent adaptive acid tolerance response in Lactobacillus acidophilus

Exposure of L. acidophilus CRL 639 cells to sublethal adaptive acid conditions (pH 5.0 for 60 min) was found to confer protection against subsequent exposure to lethal pH (pH 3.0). Adaptation, which only occurred in complex media, was dependent on de novo protein synthesis and was inhibited by amino acid analogues. There was no modification in the protein synthesis rate during adaptation, but the protein degradation rate decreased. Synthesis of acid stress proteins may increase the stability of pre-existing proteins. By 2D-PAGE, induction of nine acid stress proteins and repression of several housekeeping proteins was observed. Putative heat shock proteins DnaK, DnaJ, GrpE, GroES and GroEL (70, 43, 24, 10 and 55 kDa, respectively) were among the proteins whose synthesis was induced in response to acid adaptation.     

Characterisation of the diol dehydratase pdu operon of Lactobacillus collinoides

Addition of copper (0.5-5 mM) or cadmium (1-5 mM) to the white rot fungus Pleurotus ostreatus cultivated in liquid nitrogen-limited medium for 12 days increased the activity of laccase. The addition of 2 mM Cd led to an 18.5-fold increase of activity, 1 mM Cu increased the activity eight-fold. When added earlier than 12 days, the activation of laccase was delayed (Cu) or decreased (Cd). Ag, Hg, Pb, Zn, and H(2)O(2) decreased laccase activity. To study the effect on native enzymes, purified laccase was incubated with Cd, Cu, and Hg. The addition of Hg decreased the activity of laccase immediately and reduced the temporal stability of the enzyme, while the addition of Cu (0.05-50 mM) increased both enzyme activity and stability. Laccase extracted at different stages of straw colonisation differed in its response to heavy metals.     

The effect of chlorogenic, gallic and quinic acids on the growth of spoilage strains of Lactobacillus collinoides and Lactobacillus brevis

The naturally occurring complex organic acids, chlorogenic acid, gallic acid and quinic acid, at concentrations of 100, 500 and 1000 mg l^-1 were evaluated for effects on the growth of three spoilage strains of Lactobacillus collinoides and one of Lact. brevis in acid tomato broth containing 5% (v/v) ethanol at pH 4.8. During early stages of growth, all the complex acids at each concentration stimulated growth of Lact. collinoides but not of Lact. brevis. During stationary phase, chlorogenic and gallic acids produced greater cell densities of all strains, whereas quinic acid generally had less effect. The presence of these complex acids in fruit products may increase the requirement for added preservative in order to prevent spoilage by certain strains of lactic acid bacteria.     

The response of primary cultured adult mouse sensory neurons to ethanol, propanol, acetaldehyde and acrolein treatments

Primary cultures of adult mouse sensory neurons maintained for 8 days in vitro (8 div), in both the presence of non-neuronal cell (NNC) outgrowth and in NNC-reduced cultures, were exposed to doses of ethanol, propanol, acetaldehyde and acrolein. The effects on cell viability were monitored: LD50's of 600 microM acrolein and 100 mM propanol were obtained after 24 h exposures and after 48 h with 1 mM acetaldehyde and 500 mM ethanol. Morphological effects were evident by scanning electron microscopy with sub-acute doses for each agent, using both lower concentrations and shorter exposures. Membrane pitting of the perikaryon and a reduction in the proportion of neurons bearing neurites were common signs of toxic insult. The neurites of treated cells were thicker and more irregular than those of untreated cells; this proved a good indicator of specific neurotoxicity rather than merely a cytotoxic response. Fetal calf serum in the medium lessened the response of neurons to ethanol treatments. Comparison with other in vitro studies suggests these primary cultures are a more sensitive system than established cell lines of neuronal origin for use in neurotoxicity testing.     

The response of primary cultured adult mouse sensory neurons to ethanol,propanol, acetaldehyde and acrolein treatments

Primary cultures of adult mouse sensory neurons maintained for 8 days in vitro (8 div), in both the presence of non-neuronal cell (NNC) outgrowth and in NNC-reduced cultures, were exposed to doses of ethanol, propanol, acetaldehyde and acrolein. The effects on cell viability were monitored: LD50’s of 600 μM acrolein and 100 mM propanol were obtained after 24 h exposures and after 48 h with 1 mM acetaldehyde and 500 mM ethanol. Morphological effects were evident by scanning electron microscopy with sub-acute doses for each agent, using both lower concentrations and shorter exposures. Membrane pitting of the perikaryon and a reduction in the proportion of neurons bearing neurites were common signs of toxic insult. The neurites of treated cells were thicker and more irregular than those of untreated cells; this proved a good indicator of specific neurotoxicity rather than merely a cytotoxic response. Fetal calf serum in the medium lessened the response of neurons to ethanol treatments. Comparison with other in vitro studies suggests these primary cultures are a more sensitive system than established cell lines of neuronal origin for use in neurotoxicity testing.     

Alterations in fatty acid composition and trehalose concentration ofSaccharomyces brewing strains in response to heat and ethanol shock

Summary The effects of heat and ethanol shock on fatty acid composition and intracellular trehalose concentration of lager and ale brewing yeasts were examined. Exposure of cells to heat shock at 37°C or 10% (v/v) ethanol for 60 min resulted in a significant increase in the ratio of the total unsaturated to saturated fatty acyl residues and the intracellular trehalose concentration of cells. A similar increase in the amount of unsaturated fatty acids was observed in cells after 24 h of fermentation of 16°P (degree Plato) or 25°P wort, at which time more than 2% (v/v) ethanol was present in the growth medium. These results suggest that unsaturated fatty acids and high concentrations of intracellular trehalose may protect the cells from the inhibitory effects of heat and ethanol shock.     

UV-inducible proteins and UV-induced cross-protection against acid,ethanol, H2O2 or heat treatments in Lactococcus lactis subsp. lactis

The relationship between UV-irradiation-induced tolerance to different environmental stresses and change in protein synthesis was examined in Lactococcus lactis subsp lactis IL1403. The results showed that preirradiation of cultures of L. lactis subsp. lactis with UV_254nm light led to increased tolerance of usually lethal challenges to acid (pH 4.0), ethanol (20%, v/v), H₂O₂ (15 mM), or heat (52° C). This suggests that there is an overlapping regulation between the UV-induced pathway and the other stress responses. Whole-cell protein extracts from UV-treated (100 J/m²) and untreated cultures were compared using two-dimensional polyacrylamide gel electrophoresis. At least 14 polypeptides were induced in response to damage after UV irradiation, which indicated an SOS-like response in this species. The RecA protein, however, seemed not to be significantly induced in Lactococcus lactis subsp. lactis IL1403. Some of the UV-induced polypeptides overlaped with stress proteins induced by the other treatments.     

Adaptive acid tolerance response in Lactobacillus acidophilus

Stationary phase cultures of Lactobacillus acidophilus CRL 639 are naturally acid resistant to pH 3.0 while exponential phase cells induce an acid tolerance response upon exposure to sublethal pH (3.8–6.0). Maximal response was achieved after adaptation at pH 4.2 and pH 5.0. Protein synthesis was required in the latter case. © Rapid Science Ltd. 1998     

Characterization of antagonistic and acid formation properties of Lactobacillus casei

Cultural, morphologic, and biochemical characteristics of Lactobacillus casei were studied as well as their acid-forming and antagonistic activity and resistance to antibiotics. 60 cultures identified as L. caseiwere isolated from 250 samples of sour-milk products and clinical specimens. All isolated strains had respectively high antagonistic activity regardless of their source. Dependence between antagonistic activity and acid formation was not detected. Conclusion about promise for using these lactobacilli for manufacturing of probiotics has been done.     

SigM,an extracytoplasmic function sigma factor of Bacillus subtilis,is activated in response to cell wall antibiotics,ethanol, heat,acid, and superoxide stress

The extracytoplasmic function sigma M of Bacillus subtilis is required for normal cell growth under salt stress. It is expressed maximally during exponential growth and is further induced by the addition of 0.7 M NaCl. The promoter region of the sigM operon contains two promoters; one (P(A)) is sigma A dependent, and the other (P(M)) is sigma M dependent. These have been placed separately at the amy locus, directing expression of a lacZ reporter gene. Only the P(M) fusion responded to salt induction. This promoter, which was responsive to the level of active sigma M in the cell, was also induced by 5% ethanol, by vancomycin, bacitracin, or phosphomycin (inhibitors of cell wall biosynthesis; 2 micro g per ml), and by heat shock of 50 degrees C for 10 min. It was very strongly induced by acid (pH 4.3) and 80 micro M paraquat, but after a 15- to 30-min delay. There was no induction by alkali (pH 9), 5 mM H(2)O(2), the detergents 0.1% Triton X-100 and 0.1% Tween 20, or 50 micro M monensin. In addition to their reduced tolerance to salt, null mutants of sigM were unable to grow at pH 4.3 and lysed after exposure to 5% ethanol. Genes regulated by SigM were also tested for their response to pH 4.3, 5% ethanol, and 2 micro g of vancomycin per ml. Expression of the genes may have been activated by increased levels of sigma M, but at least some were also subject to additional controls, as they responded to one type of stress but not another. Expression of yrhJ, which encodes a cytochrome P450/NADPH reductase, was induced in response to acid and vancomycin. yraA expression was acid, ethanol, and vancomycin induced, whereas yjbD showed only ethanol induction. YraA protein was extremely important to acid survival-a mutation in yraA, like a sigM mutation, resulted in the failure of B. subtilis to grow at pH 4.3. Sigma M is therefore involved in maintaining membrane and cell wall integrity in response to several different stresses in exponential growth phase and is activated by such stresses.     

温度和渗透应激对植物乳杆菌稳定性的影响

在植物乳杆菌对数期后期,高温（43—47℃）、低温（15—25℃）及10-30g/L NaCl等应激处理60min均可使植物乳杆菌耐热性和耐酸性得到较大的提高。其中,尤以高温应激45℃应激处理效果最好,细胞耐热残存率和耐酸残存率较对照分别提高124％和56．8％。