Decolorization and bioremediation of molasses wastewater by white-rot fungi in a semi-solid-state condition

Molasses wastewater (vinasse; the by-product of distillation of fermented sugar) was decolorized and its chemical oxygen demand (COD) was reduced in static cultivation using the fungi Coriolus versicolor, Funalia trogii, Phanerochaete chrysosporium and Pleurotus pulmonarius ('Pleurotus sajorcaju'). The effect of cotton stalk on decolorizing and COD removing capability of four fungi was determined. In the entire concentration range tested (10-30%), wastewater was effectively decolorized by C. versicolor and F. trogii. Cotton stalk addition stimulated the decolorization activity of all fungi. The utilization of cotton stalk represents several advantages due to its function as an attachment place and as a source of nutrients; its use also reduces process costs.     

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.     

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。     

植物乳杆菌RS2-2发酵法生产乳酸脱氢酶

筛选到一株产乳酸脱氢酶的植物乳杆菌RS2-2（Lactobacillus plantarum RS2-2）。产酶条件研究表明,以蔗糖为碳源,酵母膏为氮源,起始pH6．5左右,30℃发酵,接种量为10％,产酶量最高。在25L发酵罐放大实验,并通过流加NaOH控制发酵过程中的pH,所产乳酸脱氢酶比活力为18．73U／mg,菌重量为lg／L以上。粗酶液经分离纯化得到成品酶液,比酶活提高了51．7倍,达到1210、7U／mg,总收率为30、2％。用凝胶过滤检测其分子量分布,分子量约为85,000Da,酶纯度为88％。     

L-Glutamate-glyoxylate aminotransferase in Lactobacillus plantarum.

Glutamate-glyoxylate aminotransferase which mediates the reaction of glyoxylic acid with glutamic acid to yield glycine and alpha-oxoglutaric acid has been isolated and purified 84-fold from extracts of Lactobacillus plantarum. Purified enzyme requires the addition of pyridoxal phosphate and magnesium ions for its activity. The molecular weight of the enzyme estimated by Sepharose 4B gel filtration amounts to 37.000. Micaelis constants for glyoxylate and glutamate are corresponding to 6.25 X 10(-3) M and 2.75 X 10(-3) M, respectively. Optimal pH in phosphate and veronal buffers is 8.0 and optimal temperature 35--37 degrees C.     

Cloning and analysis of bile salt hydrolase genes from Lactobacillus plantarum CGMCC No. 8198

Genes coding for bile salt hydrolase of Lactobacillus plantarum CGMCC 8198, a novel probiotic strain isolated from silage, were identified, analyzed and cloned. L. plantarum strongly resisted the inhibitory effects of bile salts and also decreased serum cholesterol levels by 20 % in mice with hypercholesterolemia. Using RT-PCR analysis, bsh2, bsh3 and bsh4 were upregulated by bile salts in a dose-dependent manner. All three bsh genes had high similarity with those of other Lactobacillus strains. All three recombinant BSHs had high activities for the hydrolysis of glycodeoxycholic acids and taurodeoxycholic acids.     

Oxygen dependent lactate utilization by Lactobacillus plantarum

Lactobacillus plantarum P5 grew aerobically in rich media at the expense of lactate; no growth was observed in the absence of aeration. The oxygen-dependent growth was accompanied by the conversion of lactate to acetate which accumulated in the growth medium. Utilization of oxygen with lactate as substrate was observed in buffered suspensions of washed whole cells and in cell-free extracts. A pathway which accounts for the generation of adenosine triphosphate during aerobic metabolism of lactate to acetate via pyruvate and acetyl phosphate is proposed. Each of the enzyme activities involved, nicotinamide adenine dinucleotide independent lactic dehydrogenase, nicotinamide adenine dinucleotide dependent lactic dehydrogenase, pyruvate oxidase, acetate kinase and NADH oxidase were demonstrated in cell-free extracts. The production of pyruvate, acetyl phosphate and acetate was demonstrated using cell-free extracts and cofactors for the enzymes of the proposed pathway.Abbreviations MRS Man, Rogosa and Sharpe (1960) medium modified as in Materials and methods - TY Tryptone Yeast Extract broth - OUL Oxygen uptake with lactate as substrate - DCPIP 2,6-Dichlorophenolindophenol - LDH Lactic dehydrogenase     

Culture conditions of tannase production by Lactobacillus plantarum

Lactobacillus plantarum produced an extracellular tannase after 24 h growth on minimal medium of amino acids containing 2 g tannic acid l^–1. Enzyme production (6 U ml^–1) was optimal at 37 °C and pH 6 with 2 g glucose l^–1 and 7 g tannic acid l^–1 in absence of O₂.     

Melibiose transport system in Lactobacillus plantarum.

Lactobacillus plantarum ATCC 8014 grew on melibiose at 30 C, but not at 37 C, although it grew on galactose or lactose at either temperature. ATCC 8014 grown on lactose at 30 or 37 C accumulated melibiose slowly, suggesting that melibiose may partly be transported by a lactose transport system. A lactose-negative mutant, NTG 21, derived from ATCC 8014 was isolated. The mutant was totally deficient in lactose transport, but retained normal melibiose transport activity. In NTG 21, the melibiose transport activity was induced by melibiose at 30 C, but not at 37 C. The transport activity itself was found to be stable for at least 3 hr at 37 C, suggesting that the induction process in the cytoplasm rather than the inducer entrance is temperature-sensitive in the organism. The organism also failed to form alpha-galactosidase at 37 C when grown on melibiose. The enzyme synthesis, however, was induced by galactose in NTG 21 (and also by lactose in ATCC 8014) even at 37 C, indicating that the induction of the enzyme is essentially not temperature-sensitive. In NTG 21, melibiose transport system and alpha-galactosidase were induced by galactose, melibiose and o-nitrophenyl-alpha-D-galactopyranoside when the strain was grown at 30 C. Raffinose induced melibiose transport system only a little, while it was a good inducer for alpha-galactosidase. Inhibition studies revealed that galactose may be a weak substrate of the melibiose transport system; no inhibition was demonstrated with lactose and raffinose.     

Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of l-malate to l-lactate and CO₂ concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C₄-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.     

Characterization of undecaprenol kinase from Lactobacillus plantarum.

A membrane-bound undecaprenol kinase from Lactobacillus has been identified by observing the ATP-dependent phosphorylation of [14C]undercaprenol. The product of this reaction was shown to be [14C]undecaprenyl monophosphate by comparison of its chromatographic mobilities with authentic undecaprenyl monophosphate. It was shown that 32P from [gamma-32P]ATP was incorporated into undecaprenyl monophosphate. The kinase was partially solubilized by a variety of methods utilizing Triton X-100. Both the membrane-associated and solubilized enzymes required Mg2+, Triton X-100 and dimethylsulfoxide for activity. The enzyme preferentially phosphorylated the C34, C50 AND C 55 polyprenols. Geranylgeraniol (C20) and dolichol (C100), however, were utilized only 6% and 13% as well as undecaprenol, respectively. Despite the 8-fold difference in apparent V values, the apparent Km values for dolichol and undecaprenol were both 14 microM. The apparent Km for the nucleotide cosubstrate, ATP, was 2 mM. No other nucleoside triphosphate could substitute for ATP.     

Enzymatic Decolorization of Melanoidin by Coriolus sp. No. 20

Coriolus sp. No. 20 decolorized a melanoidin solution, a decrease of about 80% in darkness under the optimal conditions. This decolorization occurred with an intracellular enzyme which was prepared from an extract of integrated mycelia, and required aeration and some kinds of sugars, particularly glucose and sorbose. The fraction with melanoidin-decolorizing activity was collected and purified by DEAE-cellulose and Sephadex G-200 column chromatographies. The optimal pH and temperature were pH 4.5 and 35°C, respectively. The molecular weight was found to be about 200,000 by SDS-gel electrophoresis. The purified enzyme was identified as sorbose oxidase; decolorization proceeded in the presence of oxygen and sugars such as maltose, sucrose, lactose, galactose and xylose, besides glucose and sorbose. Glucose in the reaction mixture was converted to gluconic acid. Melanoidin was suggested to be decolorized by the active oxygen formed.     

Plasmid-associated Bacteriocin Production by a Lactobacillus plantarum Strain

A Lactobacillus plantarum strain, LTF154, isolated from a fermented sausage, produces a bacteriocin, designated plantacin 154. Plantacin 154 was stable to heat treatment, and its activity was sensitive to proteolytic enzymes. The molecular mass, as indicated by activity detection after SDS-PAGE, was estimated to be 3.0 kDa or less. A plasmid-curing experiment and transformation analysis indicated that a 9.5-MDa plasmid, pLP1542, may be involved in the production of plantacin 154.     

Exploring Lactobacillus plantarum genome diversity by using microarrays

Lactobacillus plantarum is a versatile and flexible species that is encountered in a variety of niches and can utilize a broad range of fermentable carbon sources. To assess if this versatility is linked to a variable gene pool, microarrays containing a subset of small genomic fragments of L. plantarum strain WCFS1 were used to perform stringent genotyping of 20 strains of L. plantarum from various sources. The gene categories with the most genes conserved in all strains were those involved in biosynthesis or degradation of structural compounds like proteins, lipids, and DNA. Conversely, genes involved in sugar transport and catabolism were highly variable between strains. Moreover, besides the obvious regions of variance, like prophages, other regions varied between the strains, including regions encoding plantaricin biosynthesis, nonribosomal peptide biosynthesis, and exopolysaccharide biosynthesis. In many cases, these variable regions colocalized with regions of unusual base composition. Two large regions of flexibility were identified between 2.70 and 2.85 and 3.10 and 3.29 Mb of the WCFS1 chromosome, the latter being close to the origin of replication. The majority of genes encoded in these variable regions are involved in sugar metabolism. This functional overrepresentation and the unusual base composition of these regions led to the hypothesis that they represented lifestyle adaptation regions in L. plantarum. The present study consolidates this hypothesis by showing that there is a high degree of gene content variation among L. plantarum strains in genes located in these regions of the WCFS1 genome. Interestingly, based on our genotyping data L. plantarum strains clustered into two clearly distinguishable groups, which coincided with an earlier proposed subdivision of this species based on conventional methods.     

New types of antimicrobial compounds produced by Lactobacillus plantarum

New types of antimicrobial compounds were identified in the culture filtrate of Lactobacillus plantarum VTT E-78076. Activity was detected in the low molecular mass fraction separated by gel chromatography. This fraction totally inhibited the growth of the Gram-negative test organism, Pantoea agglomerans (Enterobacter agglomerans) VTT E-90396. Characteristic compounds from this fraction were identified by GC/MS-analysis and the identification was confirmed using pure commercial reference compounds in identical chromatographs and in antimicrobial tests. The active fraction included benzoic acid (CAS 65-85-0), 5-methyl-2,4-imidazolidinedione (CAS 616-03-5, methylhydantoin), tetrahydro-4-hydroxy-4-methyl-2H- pyran-2-one (CAS 674-26-0, mevalonolactone) and 3-(2-methylpropyl)-2,5-piperazinedione (CAS 5845-67-0, cyclo(glycyl-L-leucyl)). These compounds in concentrations of 10 ppm inhibited growth of the test organism by 10-15% when acting separately, but 100% when all were applied together with 1% lactic acid. The inhibition was 40% by 1% lactic acid alone. The compounds were also active against Fusarium avenaceum (Gibberella avenacea) VTT-D-80147. The inhibition was 10-15% by separate compounds in concentrations of 10 ppm and maximally 20% in combinations. Fungal growth was not inhibited by lactic acid. Inhibition by unfractionated Lact. plantarum culture filtrate was 37% and by the low molecular mass fraction, 27%.