In vitro fermentation studies for selection and evaluation of Bacillus strains as starter cultures for the production of okpehe, a traditional African fermented condiment

Selected Bacillus and Enterococcus strains, isolated from traditional okpehe fermentations, were studied for their suitability as starter cultures in laboratory-scale fermentations of Prosopis africana seeds for the production of okpehe, a traditional fermented vegetable product of Nigeria. The strains were selected on the basis of highest proteolytic activity, as determined with the APIZYM (BioMerieux) test. The choice of starter strains was narrowed to Bacillus subtilis strains BFE 5301 and BFE 5372. These were determined as the best starter combination because of rapid growth, high amylolytic and proteolytic activities, high levels of polyglutamic acid production by strain BFE 5372, as well as bacteriocin production by strain BFE 5301. Other mixed culture fermentations did not yield sensorically acceptable products. Although a monoculture fermentation, using only B. subtilis strain BFE 5372, produced okpehe with very good sensory characteristics, the growth of B. cereus could be detected after 48 h fermentation, indicating that this starter did not sufficiently contribute to product safety. Mixed culture fermentation with the combination of bacteriocin-producing starter B. subtilis BFE 5301 and the non-bacteriocin-producing B. subtilis BFE 5372, produced a product with good sensory characteristics, in which growth of B. cereus was delayed. The bacteriocin produced by B. subtilis strain BFE 5301 was identified as subtilisin, using subtilisin-specific primers and PCR amplification of the subtilisin gene. The bacteriocin was heat-stable at 100 degrees C for 10 min and exhibited highest activity at pH values lower or equal to pH 6.0. The bacteriocin was sensitive to the proteolytic enzymes trypsin and alpha-chymotrypsin at concentrations of 10 mg/ml.     

Comparison of the most odour-active volatiles in different hop varieties by application of a comparative aroma extract dilution analysis

Application of a comparative aroma extract dilution analysis on the volatiles isolated from five different hops (Hallertau Perle, Hallertau Hersbrucker Spät, Slowenian Golding, Hallertau Smaragd, US Cascade) revealed linalool and myrcene with the highest Flavour Dilution (FD)-factors in all varieties, followed by 2-isopropyl-3-methoxypyrazine, 3-methylbutanoic acid and geraniol. Some odourants, however, showed high FD-factors only in certain varieties, for example, (5Z)-octa-1,5-dien-3-one and germacrene B in Hersbrucker Spät, (3E,5Z)-undeca-1,3,5-triene in Hersbrucker Spät and Cascade and nonanal in Cascade. The overall odour profile of the Cascade sample clearly differed from the other varietes, and was dominated by a black currant like odour note. The identification experiments revealed 4-methyl-4-sulfanylpentan-2-one, so far unknown as hop constituent, as key contributor to this odour. In addition, an odour-active undecatetraene was present, in particular, in Perle and Cascade. Synthesis and structural assignment of the four stereoisomers of (3E)-undeca-1,3,5,9-tetraene allowed the identification of the fresh, pineapple-like smelling compound as (3E,5Z,9E)-undeca-1,3,5,9-tetraene. Among the four isomers synthesised, this compound showed by far the lowest odour threshold of 0.01 ng/L in air.     

Cloning of the bile salt hydrolase (bsh) gene from Enterococcus faecium FAIR-E 345 and chromosomal location of bsh genes in food enterococci

Enterococcus faecium strain FAIR-E 345 isolated from food was shown to possess bile salt hydrolase (Bsh) activity in a plate screening assay and by high-performance liquid chromatography analysis. The bsh gene was cloned and sequenced. DNA sequence analysis revealed that it encoded a protein of 324 amino acids, with pI 4.877. A bsh gene probe was prepared from the cloned bsh gene and was used for probing plasmid and total genomic DNA of Bsh-positive enterococci isolated from food to determine the genomic location of their bsh genes. This probe was able to detect the bsh gene among total genomic DNA preparations but not from plasmid preparations of 10 plasmid-bearing Enterococcus strains. However, the probe could detect the bsh gene from total genomic DNA preparations of 12 Enterococcus strains that did not contain detectable plasmid DNA. In no cases did the probe hybridize with plasmid DNA preparations, suggesting that the bsh gene among enterococci is probably generally chromosomally encoded. This presumptive chromosomal location of bsh genes among food enterococci suggests that transfer of this trait by conjugative plasmids is unlikely.     

Concentration and distribution of platinum group elements (Pt, Pd, Rh) in airborne particulate matter in Frankfurt am Main, Germany

The concentrations and distribution of platinum group elements (Pt, Pd, Rh) in airborne particulate matter were studied in a period of one year from August 2001 to July 2002 in urban and in nonurban areas. Airborne dust samples were collected as a total amount (particles with an aerodynamic diameter <22 microm) and classified using an eight-stage Andersen impactor (<10 microm) at three locations with different traffic density roads in the Frankfurt am Main and nonurban areas. Sampling at the three locations was performed simultaneously for total airborne dust and fractionated airborne dust. Pd was determined by total reflection X-ray fluorescence after Hg coprecipitation. Pt and Rh were analyzed by adsorptive striping voltammetry after HPA digestion. The results show that the PGE concentrations in airborne samples depend on the traffic density. The highest PGE concentrations in air were found in the vicinity of major roads with heavy traffic, and the lowest ones were found in the nonurban area. The presence of PGE at the sampling station relatively free of traffic in a nonurban area hints to a transport of some of the emitted PGE from the city to this station by wind. At all three sampling locations, a heterogeneous distribution of the Pd, Pt, and Rh concentrations during the sampling year can be observed. The sum of PGE concentrations in total airborne dust is comparable with the sum of impactor samples. However, the concentration of Pt and Rh in total airborne dust (<22 microm) is on average higher than in impactor samples (<10 microm). On the contrary, Pd concentration is higher in impactor samples in most cases. The airborne PGE distribution is dominated by Pt, followed by Pd and Rh. The impactor samples are dominated by Pd, followed by Pt and Rh. This fact indicates that palladium occurs mainly in relatively fine airborne particles. The main fraction of PGE is found on average in particle sizes between 1.1 and 4.7 microm. Knowledge of the size distribution of particles containing PGE is important with respect to risk assessment of human inhalation.     

Effect of acid deposition on quantity and quality of dissolved organic matter in soil-water

The aim of this study was to explore how acid deposition may affect the concentration and quality of dissolved organic matter (DOM) in soil-water. This was done by a small-scale acidification experiment during two years where 0.5 × 0.5 m(2) plots were artificially irrigated with water with different sulfuric acid content, and soil-water was sampled using zero-tension lysimeters under the O-horizon. The DOM was characterized using absorbance, fluorescence, and size exclusion chromatography analyses. Our results showed lower mobility of DOM in the high acid treatment. At the same time, there was a significant change in the DOM quality. Soil-water in the high acid treatment exhibited DOM that was less colored, less hydrophobic, less aromatic, and of lower molecular weight, compared to the low acid treatment. This supports the hypothesis that reduction in sulfur deposition is an important driver behind the ongoing brownification of surface waters in many regions.     

Functional properties of Lactobacillus strains isolated from kimchi

The objective of this study was to evaluate the functional properties of lactic acid bacteria (LAB) from kimchi, a traditional Korean fermented vegetable product generally consumed raw as a side-dish with practically every meal.Twelve mild acid producing facultatively heterofermentative Lactobacillus strains were selected for their potential as starter cultures for fermentation of kimchi, and evaluated for their functional properties. Eleven strains were identified as Lactobacillus sakei and one as Lactobacillus plantarum. The strains identified as L. sakei differed in some physiological features; of particular interest was the fact that 9 of these strains produced L(+) lactic acid from glucose in presence of acetate.All strains were able to survive gastrointestinal conditions simulating stomach and duodenum passage. In addition, they showed higher adherence to HT-29 cells than Lactobacillus rhamnosus GG, a commercial probiotic strain used worldwide. These strains also showed antimicrobial activity against a number of food-borne pathogens. Their ability to lower cholesterol was demonstrated by BSH (bile salt hydrolytic) activity, and cholesterol assimilation tests in vitro. The results suggest the probiotic potential of these strains for use in kimchi fermentation.     

Enterococci as probiotics and their implications in food safety

Enterococci belong to the lactic acid bacteria (LAB) and they are of importance in foods due to their involvement in food spoilage and fermentations, as well as their utilisation as probiotics in humans and slaughter animals. However, they are also important nosocomial pathogens that cause bacteraemia, endocarditis and other infections. Some strains are resistant to many antibiotics and possess virulence factors such as adhesins, invasins, pili and haemolysin. The role of enterococci in disease has raised questions on their safety for use in foods or as probiotics. Studies on the incidence of virulence traits among enterococcal strains isolated from food showed that some can harbour virulence traits, but it is also thought that virulence is not the result of the presence of specific virulence determinants alone, but is rather a more intricate process. Specific genetic lineages of hospital-adapted strains have emerged, such as E. faecium clonal complex (CC) 17 and E. faecalis CC2, CC9, CC28 and CC40, which are high risk enterococcal clonal complexes. These are characterised by the presence of antibiotic resistance determinants and/or virulence factors, often located on pathogenicity islands or plasmids. Mobile genetic elements thus are considered to play a major role in the establishment of problematic lineages. Although enterococci occur in high numbers in certain types of fermented cheeses and sausages, they are not deliberately added as starter cultures. Some E. faecium and E. faecalis strains are used as probiotics and are ingested in high numbers, generally in the form of pharmaceutical preparations. Such probiotics are administered to treat diarrhoea, antibiotic-associated diarrhoea or irritable bowel syndrome, to lower cholesterol levels or to improve host immunity. In animals, enterococcal probiotics are mainly used to treat or prevent diarrhoea, for immune stimulation or to improve growth. From a food microbiological point of view, the safety of the bacteria used as probiotics must be assured, and data on the major strains in use so far indicate that they are safe. The advantage of use of probiotics in slaughter animals, from a food microbiological point of view, lies in the reduction of zoonotic pathogens in the gastrointestinal tract of animals which prevents the transmission of these pathogens via food. The use of enterococcal probiotics should, in view of the development of problematic lineages and the potential for gene transfer in the gastrointestinal tract of both humans and animals, be carefully monitored, and the advantages of using these and new strains should be considered in a well contemplated risk/benefit analysis.     

Enterococci in foods--a conundrum for food safety

Enterococci form part of the lactic acid bacteria (LAB) of importance in foods. They can spoil processed meats but they are on the other hand important for ripening and aroma development of certain traditional cheeses and sausages, especially those produced in the Mediterranean area. Enterococci are also used as human probiotics. However, they are important nosocomial pathogens that cause bacteraemia, endocarditis and other infections. Some strains are resistant to many antibiotics, but antibiotic resistance alone cannot explain the virulence of some of these bacteria. Virulence factors such as adhesins, invasins and haemolysin have been described. The role of enterococci in disease has raised questions on their safety for use in foods or as probiotics. Studies on the incidence of virulence traits among enterococcal strains isolated from food showed that some harbour virulence traits and generally, Enterococcus faecalis harbours more of them than Enterococcus faecium. Regulations in Europe stipulate that safety of probiotic or starter strains is the responsibility of the producer; therefore, each strain intended for such use should be carefully evaluated. For numerous questions, immediate answers are not fully available. It is therefore suggested that when considering an Enterococcus strain for use as a starter or probiotic culture, it is imperative that each particular strain should be carefully evaluated for the presence of all known virulence factors. Ideally, such strains should harbour no virulence determinants and should be sensitive to clinically relevant antibiotics. In general, E. faecium appears to pose a lower risk for use in foods, because these strains generally harbour fewer recognised virulence determinants than E. faecalis. Generally, the incidence of such virulence determinants among E. faecium strains is low, as compared to E. faecalis strains, probably as a result of the presence of pheromone-responsive plasmids.     

Concentration and distribution of heavy metals in urban airborne particulate matter in Frankfurt am Main, Germany

Heavy metal concentrations were measured in airborne dust collected at three sites with different traffic densities from August 2001 to July 2002 in the Frankfurt am Main area. Bulk samples of particulate matter (PM) with an aerodynamic equivalent diameter of <22 microm were collected on cellulose nitrate filters using air filtration devices. Fractionated samples of PM with an aerodynamic equivalent diameter of <10 microm were collected using an eight-stage Andersen impactor. Pb, Cd, Mn, Ni, Zn, V, As, Sb, Cu, Cr, Co, and Ce were determined by inductively coupled plasma sector field mass spectrometry, Pt and Rh were determined by adsorptive voltammetry, and Pd was determined by total reflection X-ray fluorescence analysis. The results show that the highest airborne heavy metal concentrations occurred at the main street with a large volume of traffic. With the exception of Co, V, Ce, and Mn, the heavy metals had an elevated enrichment factor compared to their concentrations in the continental crust. The main street site was especially contaminated with Sb, Zn, Cu, V, and Ni. Motor vehicles are the likely source of emissions. With the exception of Cr, Cu, and Zn, most of the airborne heavy metal concentrations determined for impactor samples deviate slightly from the results for total airborne dust. Heavy metal particle size distributions can be divided into three groups. For metals such as As, Cd, Pb, and V, the main fraction can be found in fine particles with a diameter of <2.1 microm, whereas Ce, Cr, Co, and Ni occur mainly in coarse particles with a diameter of >2.1 microm. Cu, Mn, Sb, Zn, Pt, Pd, and Rh occur in high concentrations in the medium range of the impactor stages (particle diameters of 1.1-4.7 microm). Metal concentrations in fine dust particles are needed to assess the human health risks of their inhalation.     

Phenotypic and genotypic identification of lactic acid bacteria isolated from ethnic fermented bamboo tender shoots of North East India

Mesu, soidon, soibum and soijim are ethnic fermented bamboo tender shoot products prepared by the people in North East India. Microbiological analysis of mesu, soidon, soibum and soijim showed the population dominated by lactic acid bacteria (LAB) ranging up to 10(8) cfu g(-1). The phenotypic characterisation of predominant LAB isolated from the fermented bamboo shoot products was based on general morphology, physiological tests, API and Biolog systems. The genotypic characterisation of LAB was based on RAPD-PCR, rep PCR, species-specific PCR techniques, 16S rRNA gene sequencing and DNA-DNA hybridisation. Predominant functional LAB strains associated with the fermented bamboo shoot products were identified as Lactobacillus brevis, Lb. plantarum, Lb. curvatus, Pediococcus pentosaceus, Leuconostoc mesenteroides subsp. mesenteroides, Leuc. fallax, Leuc. lactis, Leuc. citreum and Enterococcus durans.