Bacteria associated with orchid roots and microbial production of auxin

Associative bacteria of terrestrial (Paphiopedilum appletonianum) and epiphytic (Pholidota articulata) tropical orchids were investigated. Microbial community of epiphytic plant differed from that of the terrestrial one. Streptomyces, Bacillus, Pseudomonas, Burkholderia, Erwinia and Nocardia strains populated Paphiopedilum roots, whereas Pseudomonas, Flavobacterium, Stenotrophomonas, Pantoea, Chryseobacterium, Bacillus, Agrobacterium, Erwinia, Burkholderia and Paracoccus strains colonized Pholidota roots. Endophytic bacteria populations were represented with less diversity: Streptomyces, Bacillus, Erwinia and Pseudomonas genera were isolated from P. appletonianum, and Pseudomonas, Bacillus, and Flavobacterium genera were isolated from Ph. articulata. Microorganisms produced indole-3-acetic acid (IAA). Variations in its biosynthesis among the strains of the same genus were also observed. The highest auxin level was detected during the stationary growth phase. Biological activity of microbial IAA was proved by treatment of kidney bean cuttings with bacterial supernatants, revealing considerable stimulation of root formation and growth.     

Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004

An extracellular protease was produced under stress conditions of high temperature and high salinity by a newly isolated moderate halophile, Salinivibrio sp. strain AF-2004 in a basal medium containing peptone, beef extract, glucose and NaCl. A modification of Kunitz method was used for protease assay. The isolate was capable of producing protease in the presence of sodium chloride, sodium sulfate, sodium nitrate, sodium nitrite, potassium chloride, sodium acetate and sodium citrate. The maximum protease was secreted in the presence of 7.5 to 10% (w/v) sodium sulfate or 3% (w/v) sodium acetate (4.6 U ml⁻¹). Various carbon sources including glucose, lactose, casein and peptone were capable of inducing enzyme production. The optimum pH, temperature and aeration for enzyme production were 9.0, 32 °C and 220 rpm, respectively. The enzyme production corresponded with growth and reached a maximum level during the mid-stationary phase. Maximum protease activity was exhibited in the medium containing 1% (w/v) NaCl at 60 °C, with 18% and 41% activity reductions at temperature 50 and 70 °C, respectively. The optimum pH for enzyme activity was 8.5, with 86% and 75% residual activities at pH 10 and 6, respectively. The activity of enzyme was inhibited by EDTA. These results suggest that the protease secreted by Salinivibrio sp. strain AF-2004 is industrially important from the perspectives of its activity at a broad pH ranges (5.0–10.0), its moderate thermoactivity in addition to its high tolerance to a wide range of salt concentration (0–10% NaCl).     

Antifungal lactic acid bacteria with potential to prolong shelf-life of fresh vegetables

AIM: The aim of this study was to isolate and identify antifungal lactic acid bacteria from fresh vegetables, and evaluate their potential in preventing fungal spoilage of vegetables. METHODS AND RESULTS: Lactic acid bacteria from fresh vegetables were enriched in MRS (de Man Rogosa Sharpe) broth and isolated by plating on MRS agar. All the isolates (359) were screened for activity against Aspergillus flavus of which 10% showed antifungal activity. Potent antifungal isolates were identified by phenotypic characters and confirmed by partial 16S rRNA gene sequencing. These were screened against additional spoilage fungi viz. Fusarium graminearum, Rhizopus stolonifer, Sclerotium oryzae, Rhizoctonia solani, Botrytis cinerea and Sclerotinia minor by overlay method. Most of the isolates inhibited wide range of spoilage fungi. When fresh vegetables were inoculated with either cell suspension (10(4) cells ml(-1)) or cell-free supernatant of Lact. plantarum, followed by application of vegetable spoilage fungi (A. flavus and F. graminearum, R. stolonifer, B. cinerea each with 10(4) conidia ml(-1)) the vegetable spoilage was significantly delayed than control. CONCLUSIONS: Fresh vegetables constitute a good source of lactic acid bacteria with ability to inhibit wide range of spoilage fungi. Such bacteria can be applied to enhance shelf-life of vegetables. In the present study, we report for the first time the antifungal activity of Weissella paramessenteroides and Lact. paracollinoides isolated from fresh vegetables, against wide range of food spoilage fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Fresh vegetables can be used as a source of antifungal lactic acid bacteria. Their exploitation as biopreservative will help in prolonging shelf-life of fresh vegetables.     

Identification of the agent from Lactobacillus plantarum KFRI464 that enhances bacteriocin production by Leuconostoc citreum GJ7

AIM: To provide evidence that the production of bacteriocin by lactic acid bacteria can be enhanced by the presence of a bacteriocin-sensitive strain and identify the agent that is responsible for enhancing bacteriocin production. METHODS AND RESULTS: One bacteriocin-producing lactic acid bacterium was isolated from kimchi. The strain GJ7 was designated as Leuconostoc citreum GJ7 based on Gram staining, biochemical properties, and 16S rRNA gene sequencing. The isolate produced a heat- and pH-stable bacteriocin (kimchicin GJ7), which has antagonistic activity against a broad spectrum of micro-organisms. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified kimchicin GJ7 showed a single band of molecular weight c. 3500 Da. Cultures of Leuc. citreum GJ7 in the presence of thermally inactivated kimchicin GJ7-sensitive strains, Lactobacillus plantarum KFRI 464, Lactobacillus delbrueckii KFRI 347, or Leuconostoc mesenteroides KCTC 1628, increased bacteriocin production. This inducing factor was characterized and purified from Lact. plantarum KFRI 464, which showed the greatest enhancement of kimchicin GJ7 activity. The inducing factor was purified using a DEAE (diethyl aminoethyl)-Sephacel column and high-performance liquid chromatography, and yielded a single band of c. 6500 Da. N-terminal sequencing of the inducing factor identified 16 amino acids. The N-terminal sequence of the inducing factor was synthesized and examined for the induction of kimchicin GJ7 activity, and was found to induce activity, but at a level about 10% lower than that of the entire molecule. CONCLUSIONS: The presence of a bacteriocin-sensitive strain, Lact. plantarum KFRI 464, acts as an environmental stimulus to activate the production of kimchicin GJ7 by Leuc. citreum GJ7. The inducing factor from Lact. plantarum KFRI 464 is highly homologous to the 30S ribosomal protein S16 from various micro-organisms. The N-terminal sequence of the inducing factor examined in this study is a very important sequence related to the inducing activity. Nevertheless, the inducing factor may not be part of the ribosomal protein S16 itself. SIGNIFICANCE AND IMPACT OF THE STUDY: We believe that the present study is the first to identify an agent that is produced by one micro-organism and influences bacteriocin production in another. The bacteriocin-enhancing system described in this study could be effectively used to control the growth of other micro-organisms (sensitive cells) in food systems. Moreover, this enhancement of bacteriocin production can be applied usefully in industrial production of natural food preservatives.     

Screening and selection of lactic acid bacteria strains suitable for ensiling grass

AIM: Lactic acid bacteria (LAB) strains shown to have broad-spectrum antimicrobial activity were screened for potential as grass silage inoculants. The strains capable of rapidly lowering the pH of the grass matrix and with low proteolytic activity were assessed in laboratory-scale silos in a grass matrix containing natural microbial flora. METHODS AND RESULTS: Screening of nine candidate strains was performed first in a grass extract medium. The four most promising strains were selected on the basis of growth rate in the medium, capacity to reduce pH and ability to limit the formation of ammonia-N. The efficiency of the selected strains was further assessed in a laboratory-scale ensiling experiment. Untreated (no additive) and formic acid served as controls. All tested inoculants improved silage quality compared with untreated. With one exception (Pediococcus parvulus E315) the fermentation losses in the inoculated silages were even lower than in the acid-treated control silage. Pure lactic acid fermentation was obtained in the timothy-meadow fescue silage with all inoculants. The results obtained in the ensiling experiments were consistent with those of the screening procedure, which appeared to predict correctly the potential of LAB as silage inoculants. The strains with a low ammonia production rate in the grass extract medium behaved similarly in the silage. Especially in this respect the strain Lactobacillus plantarum E76 was superior to the other candidates. CONCLUSIONS: The screening method using grass extract proved to be useful in strain selection. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid screening method developed for the LAB strains provides a useful tool for more systematic product development of commercial inoculant preparations. Time consuming and laborious ensiling experiments can be limited only to the most promising strains.     

Development of antioxidant activity in milk whey during fermentation with lactic acid bacteria

AIMS: To investigate the production of antioxidant activity during fermentation with commonly used dairy starter cultures. Moreover, to study the development of antioxidant activity during fermentation, and the connection to proteolysis and bacterial growth. METHODS AND RESULTS: Antioxidant activity was measured by analysing the radical scavenging activity using a spectrophotometric decolorization assay and lipid peroxidation inhibition was assayed using liposomal model system with a fluorescence method. Milk was fermented with 25 lactic acid bacterial (LAB) strains, and from these six strains, exhibiting the highest radical scavenging activity was selected for further investigation. Leuconostoc mesenteroides ssp. cremoris strains, Lactobacillus jensenii (ATCC 25258) and Lactobacillus acidophilus (ATCC 4356) showed the highest activity with both the methods used. However, the radical scavenging activity was stronger than lipid peroxidation inhibition activity. The development of radical scavenging activity was connected to proteolysis with four strains. Molecular distribution profiles showed that fermentates with high scavenging activity also possessed a higher proportion of peptides in the molecular mass range of 4-20 kDa, while others had mostly large polypeptides and compounds below 4 kDa. In addition, the amount of hydrophobic amino acids was higher in these fermentates. CONCLUSIONS: The development of antioxidant activity was strain-specific characteristic. The development of radical scavengers was more connected to the simultaneous development of proteolysis whereas, lipid peroxidation inhibitory activity was related to bacterial growth. However, high radical scavenging activity was not directly connected to the high degree of proteolysis. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this seems to be the first report, which screens possible antioxidant activity among most common dairy LAB strains. Use of such strains improve nutritional value of fermented dairy products.     

Dynamics of competitive population abundance of Lactobacillus plantarum ivi gene mutants in faecal samples after passage through the gastrointestinal tract of mice

AIM: This study aims to evaluate the impact of mutation of previously identified in vivo-induced (ivi) genes on the persistence and survival of Lactobacillus plantarum WCFS1 in the gastrointestinal (GI) tract of mice. METHODS AND RESULTS: Nine Lact. plantarum ivi gene replacement mutants were constructed, focussing on ivi genes that encode proteins with a predicted role in cell envelope functionality, stress response and regulation. The in vitro growth characteristics of the mutants appeared identical to those observed for the wild-type strain, which agrees with the recombination-based in vivo expression technology suggestion that these genes are not transcribed in the laboratory. Quantitative PCR experiments demonstrated differences in the relative population dynamics of the Lact. plantarum ivi mutants in faecal samples after passage through the GI tract of mice. CONCLUSIONS: The in situ competition experiments revealed a 100- to 1000-fold reduction of the relative abundance of three of the ivi gene mutants, harbouring deletions of genes predicted to encode a copper transporter, an orphan IIC cellobiose PTS and a cell wall anchored extracellular protein. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments clearly establish that the proteins encoded by these three genes play a key role in Lact. plantarum performance during passage of the GI tract.     

Effects of substratum on the diversity and stability of ammonia-oxidizing communities in a constructed wetland used for wastewater treatment

AIM: To study the relationship between the nature of the substratum and the diversity and stability of the ammonia-oxidizing microbial community in a constructed wetland for the treatment of wastewaters. METHODS AND RESULTS: Samples have been taken the year around from sections of the wetland filled with different substrata. When present, the root zones of the helophyte Phragmites australis were also sampled. The diversity of the ammonia-oxidizing community was established by a coupled PCR-DGGE method based on the 16s rRNA gene. Averaged over the seasons, no large differences in community composition were observed between the different substrata, although the section with zeolite always showed the highest frequencies of bands belonging to ammonia-oxidizing bacteria of the beta-subclass of the Proteobacteria. Only sequences related to the Nitrosospira lineage were detected. Averaged again over the seasons, the section with zeolite was also most constant with respect to the potential ammonia-oxidizing activity. CONCLUSIONS: Although the ammonia-oxidizing communities did not differ significantly between the different sections of the constructed wetland, the characteristics of zeolite were most appropriate to accommodate a stable and active community of ammonia-oxidizing bacteria. The presence of the helophyte had no effect on the diversity and stability of the ammonia-oxidizing community. SIGNIFICANCE AND IMPACT OF THE STUDY: It has been shown that substrata used in constructed wetlands made no distinction between ammonia-oxidizing strains in relation to attachment. However, zeolite had the best performance with respect to activity over the seasons.     

Relationship between sampling duration and concentration of culturable airborne mould and bacteria on selected culture media

AIMS: This study was designed to evaluate potential effects of sampling duration on observed concentrations of airborne culturable mould and bacteria on selected media. METHODS AND RESULTS: Airborne culturable mould and bacteria from lightly to moderately contaminated environments were collected on selected culture media using two co-located, concurrently operated, Andersen N-6 samplers for five sampling durations in the range of 1-10 min. Differences in mean concentrations, as well as linear relationships between sampling duration and both concentration and variability, were evaluated using nonparametric procedures. For the five sampling durations, there were no significant differences in mean concentrations of mould; for bacteria, there were significant differences, with a trend of decreasing concentrations as sampling duration increased. Data variability decreased with increasing sampling duration for both mould and bacteria. CONCLUSIONS: Airborne culturable mould concentrations were similar for sampling durations in the range of 1-10 min. Airborne bacteria concentrations tended to trend downwards with sampling durations exceeding 3 min. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that sampling durations of 1-10 min are appropriate for collection of airborne culturable mould on malt extract agar (MEA) and dichloran glycerol agar (DG-18); based on the apparent trend of decreasing bacterial sample concentrations associated with increasing sampling duration, sampling durations of 相似文献   

Microbiological quality of water and dialysate in a haemodialysis unit in Ponta Grossa-PR, Brazil

AIMS: The objective of the study was to determine the microbiological quality of samples of water and dialysate in a haemodialysis unit. METHODS AND RESULTS: Seventy-two samples each of water and dialysate were collected during November 2003 to April 2004. The following microbiological analyses were performed: test for total and faecal coliforms, which produced negative results for all the samples; counts of total heterotrophic bacteria, where three samples of water and two of dialysate showed levels higher than those permitted by national standards; and endotoxin assay, which revealed high quantities only in samples of water that preceded reverse osmosis. Nonfermenting Gram-negative bacteria were identified in 54 samples of dialysate and in 26 samples of water. The test for adhesion to an inert surface showed that various bacteria were capable of forming biofilms. Twenty-seven per cent of the bacteria were resistant to sodium hypochlorite at 500 ppm for 10-min contact time. Sixty per cent of the isolates were resistant to three or more antibiotics. CONCLUSIONS: Water and dialysate can be a source of infection for patients who need haemodialysis. SIGNIFICANCE AND IMPACT OF THE STUDY: An adequate system for water treatment, disinfection of the haemodialysis system and microbiological monitoring of the water and dialysate are necessary to reduce bacteraemia and pyrogenia outbreaks.