A review of the microtremor horizontal-to-vertical spectral ratio (MHVSR) method.

The single-station microtremor horizontal-to-vertical spectral ratio (MHVSR) method was initially proposed to retrieve the site amplification function and its resonance frequencies produced by unconsolidated sediments overlying high-velocity bedrock. Presently, MHVSR measurements are predominantly conducted to obtain an estimate of the fundamental site frequency at sites where a strong subsurface impedance contrast exists. Of the earthquake site characterization methods presented in this special issue, the MHVSR method is the furthest behind in terms of consensus towards standardized guidelines and commercial use. The greatest challenges to an international standardization of MHVSR acquisition and analysis are (1) the what - the underlying composition of the microtremor wavefield is site-dependent, and thus, the appropriate theoretical (forward) model for inversion is still debated; and (2) the how - many factors and options are involved in the data acquisition, processing, and interpretation stages. This paper reviews briefly a historical development of the MHVSR technique and the physical basis of an MHVSR (the what). We then summarize recommendations for MHVSR acquisition and analysis (the how). Specific sections address MHVSR interpretation and uncertainty assessment.

B-group vitamin production by lactic acid bacteria--current knowledge and potential applications.

Although most vitamins are present in a variety of foods, human vitamin deficiencies still occur in many countries, mainly because of malnutrition not only as a result of insufficient food intake but also because of unbalanced diets. Even though most lactic acid bacteria (LAB) are auxotrophic for several vitamins, it is now known that certain strains have the capability to synthesize water-soluble vitamins such as those included in the B-group (folates, riboflavin and vitamin B(12) amongst others). This review article will show the current knowledge of vitamin biosynthesis by LAB and show how the proper selection of starter cultures and probiotic strains could be useful in preventing clinical and subclinical vitamin deficiencies. Here, several examples will be presented where vitamin-producing LAB led to the elaboration of novel fermented foods with increased and bioavailable vitamins. In addition, the use of genetic engineering strategies to increase vitamin production or to create novel vitamin-producing strains will also be discussed. This review will show that the use of vitamin-producing LAB could be a cost-effective alternative to current vitamin fortification programmes and be useful in the elaboration of novel vitamin-enriched products.

Combined effect of synthetic enterocin CRL35 with cell wall, membrane-acting antibiotics and muranolytic enzymes against Listeria cells.

AIMS: To evaluate the inhibition effectiveness of enterocin CRL35 in combination with cell wall, membrane-acting antibiotics and muranolytic enzymes against the foodborne pathogen Listeria. METHODS AND RESULTS: Synthetic enterocin CRL35 alone and in combination with monensin, bacitracin, gramicidin, mutanolysin and lysozyme were used in this study. Minimal inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) index assays were performed using Listeria innocua 7 and Listeria monocytogenes FBUNT as sensitive strains. Antibiotics showed positive interactions with the bacteriocin in both strains tested. On the other hand, when mutanolysin and enterocin CRL35 were added to resting cells in a buffer system, the lytic effect of mutanolysin was enhanced. However, the addition of mutanolysin showed no effect on the growth of L. innocua 7 cells in a culture medium. Moreover, mutanolysin allowed the overgrowth of L. innocua 7 cells to an OD similar to control cells in the presence of inhibitory concentration of enterocin CRL35. In contrast, the combination of lysozyme and enterocin CRL35 resulted in a 50% inhibition of the L. innocua 7 growth. CONCLUSIONS: Based on our results, we conclude that the combination of synthetic enterocin CRL35 with some antibiotics is effective against L. innocua 7 and L. monocytogenes FBUNT cells, and more importantly the amount of these agents to be used was considerably reduced. The effectiveness of the combination of synthetic enterocin CRL35 with muramidases seems to depend on complex environments, and more detailed studies need to be performed to elucidate this issue. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterocin CRL35 represents a promising agent that not only can ensure the quality and safety of food but it can also be combined with several antimicrobial agents important in the medical field.

Two perspectives on equipartition in diffuse elastic fields in three dimensions.

The elastodynamic Green function can be retrieved from the cross correlations of the motions of a diffuse field. To extract the exact Green function, perfect diffuseness of the illuminating field is required. However, the diffuseness of a field relies on the equipartition of energy, which is usually described in terms of the distribution of wave intensity in direction and polarization. In a full three dimensional (3D) elastic space, the transverse and longitudinal waves have energy densities in fixed proportions. On the other hand, there is an alternative point of view that associates equal energies with the independent modes of vibration. These two approaches are equivalent and describe at least two ways in which equipartition occurs. The authors gather theoretical results for diffuse elastic fields in a 3D full-space and extend them to the half-space problem. In that case, the energies undergo conspicuous fluctuations as a function of depth within about one Rayleigh wavelength. The authors derive diffuse energy densities from both approaches and find they are equal. The results derived here are benchmarks, where perfect diffuseness of the illuminating field was assumed. Some practical implications for the normalization of correlations for Green function retrieval arise and they have some bearing for medium imaging.

A thermostable alpha-galactosidase from Lactobacillus fermentum CRL722: genetic characterization and main properties.

Alpha-galactosidase (alpha-Gal) enzyme, which is encoded by the melA gene hydrolyzes alpha-1,6 galactoside linkages found in sugars, such as raffinose and stachyose. These alpha-galacto-oligosaccharides (alpha-GOS), which are found in large quantities in vegetables, such as soy, can cause gastrointestinal disorders in sensitive individuals because monogastric animals (including humans) do not posses alpha-Gal in the gut. The use of microbial alpha-Gal is a promising alternative to eliminate alpha-GOS in soy-derived products. Using degenerate primers, the melA gene from Lactobacillus (L.) fermentum CRL722 was identified. The complete genomic sequence of melA (2223 bp), and of the genes flanking melA, were obtained using a combination of polymerase chain reaction-based techniques, and showed strong similarities with the alpha-Gal gene of thermophilic microorganisms. The alpha-Gal gene from L. fermentum CRL722 was cloned and the protein purified from cell-free extracts of the native and recombinant strains using various techniques (ion exchange chromatography, salt precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ultra-filtration); Its main biochemical properties were determined. The enzyme was active at moderately high temperatures (55 degrees C) and stable at wide ranges of temperatures and pH. The thermostable alpha-Gal from L. fermentum CRL722 could thus be used for technological applications, such as the removal of alpha-GOS found in soy products. The complete melA gene could also be inserted in other micro-organisms, that can survive in the harsh conditions of the gut to degrade alpha-GOS in situ. Both strategies would improve the overall acceptability of soy-derived products by improving their nutritional value.

Ingestion of milk fermented by genetically modified Lactococcus lactis improves the riboflavin status of deficient rats.

Riboflavin deficiency is common in many parts of the world, particularly in developing countries. The use of riboflavin-producing strains in the production of dairy products such as fermented milks, yogurts, and cheeses is feasible and economically attractive because it would decrease the costs involved during conventional vitamin fortification and satisfy consumer demands for healthier foods. The present study was conducted to assess in a rat bioassay the response of administration of milk fermented by modified Lactococcus lactis on the riboflavin status of deficient rats. Rats were fed a riboflavin-deficient diet during 21 d after which this same diet was supplemented with milk fermented by Lactoccus lactis pNZGBAH, a strain that overproduces riboflavin during fermentation. The novel fermented product, with increased levels of riboflavin, was able to eliminate most physiological manifestations of ariboflavinosis, such as stunted growth, elevated erythrocyte glutathione reductase activation coefficient values and hepatomegaly, that were observed using a riboflavin depletion-repletion model, whereas a product fermented with a nonriboflavin-producing strain did not show similar results. A safety assessment of this modified strain was performed by feeding rodents with the modified strain daily for 4 wk. This strain caused no detectable secondary effects. These results pave the way for analyzing the effect of similar riboflavin-overproducing lactic acid bacteria in human trials. The regular consumption of products with increased levels of riboflavin could help prevent deficiencies of this essential vitamin.

Reduction of alpha-galactooligosaccharides in soyamilk by Lactobacillus fermentum CRL 722: in vitro and in vivo evaluation of fermented soyamilk.

AIMS: Consumption of soya-derived products has been hampered by the presence of alpha-galactooligosaccharides (alpha-GOS) because mammals lack pancreatic alpha-galactosidase (alpha-Gal) which is necessary for their hydrolysis. These sugars thus reach the large intestine causing gastrointestinal disorders in sensitive individuals. The use of lactic acid bacteria (LAB) expressing alpha-Gal is a promising solution for the degradation of alpha-GOS in soyamilk. METHODS AND RESULTS: The capacity of the LAB Lactobacillus fermentum CRL 722 to properly degrade alpha-GOS was studied in vitro using controlled fermentation conditions and in vivo using a rat model. Lactobacillus fermentum CRL 722 was able to grow on commercial soyamilk and completely eliminated stachyose and raffinose during fermentation because of its high alpha-Gal activity. Rats fed soyamilk fermented by this LAB had smaller caecums compared with rats fed unfermented soyamilk. CONCLUSIONS: Soyamilk fermentation by Lact. fermentum CRL 722 results in the reduction of alpha-GOS concentrations in soyamilk, thus eliminating possible undesirable physiological effects normally associated with its consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: Fermentation with Lact. fermentum CRL 722 could prevent gastrointestinal disorders in sensitive individuals normally associated with the consumption of soya-based products. This LAB could thus be used in the elaboration of novel fermented vegetable products which better suit the digestive capacities of consumers.

Reduction of non-digestible oligosaccharides in soymilk: application of engineered lactic acid bacteria that produce alpha-galactosidase

Human consumption of soy-derived products has been limited by the presence of non-digestible oligosaccharides (NDO), such as the alpha-galactooligosaccharides raffinose and stachyose. Most mammals, including man, lack pancreatic alpha-galactosidase (alpha-Gal), which is necessary for the hydrolysis of these sugars. However, such NDO can be fermented by gas-producing microorganisms present in the cecum and large intestine, which in turn can induce flatulence and other gastrointestinal disorders in sensitive individuals.The use of microorganisms expressing alpha-Gal is a promising solution to the elimination of NDO before they reach the large intestine. In the present study, lactic acid bacteria engineered to degrade NDO have been constructed and are being used as a tool to evaluate this solution. The alpha-Gal structural genes from Lactobacillus plantarum ATCC8014 (previously characterized in our laboratory) and from guar have been cloned and expressed in Lactococcus lactis. The gene products were directed to different bacterial compartments to optimize their possible applications. The alpha-Gal-producing strains are being evaluated for their efficiency in degrading raffinose and stachyose: i) in soymilk fermentation when used as starters and ii) in situ in the upper gastrointestinal tract when administered to animals orally, as probiotic preparations. The expected outcomes and possible complications of this project are discussed.

Examination of adhesive determinants in three species of Lactobacillus isolated from chicken.

The microbial adhesion process includes passive forces; electrostatic interactions; hydrophobic, steric forces; lipoteichoic acids; and specific structures, such as external appendages (lectins) and (or) extracellular polymers. In a previous work, we showed that Lactobacillus animalis, L. fermentum, and L. fermentum ssp. cellobiosus had lectinlike proteic structures on their surfaces and high hydrophobicity values on the cell surface of L. fermentum ssp. cellobiosus. Here, we examined the presence of the bacterial forces or structures that could be involved in the interaction between bacteria and epithelial cells. Lactobacillus animalis and L. fermentum possessed a net negative surface charge, whereas L. fermentum ssp. cellobiosus showed similar affinity to both cationic and anionic exchange resins, aggregated in the presence of ammonium sulfate, and had high affinity (75.4%) to a hydrophobic matrix. Only L. animalis was shown to have ribitol teichoic acids in the cell wall. The amount of polysaccharides from cell walls varied between different strains, with L. fermentum ssp. cellobiosus having the highest concentration. Lectin extracts obtained from lactobacilli did not possess sugar residues, thereby demonstrating the proteic nature of the superficial surface structures of three strains. The lactic acid bacteria studied here showed different surface determinants, which could be involved in the interactions between these lactobacilli and intestinal epithelial cells.

Selection of bacteriocin producer strains of lactic acid bacteria from a dairy environment.

Two strains showing bacteriocin production were selected from a total of 206 lactic acid bacteria isolated from samples of milk, milk serum, whey and homemade cheeses in Southern Cordoba, Argentina. This property was detected by means of well diffusion assays. The strains were identified as Enterococcus hirae and Enterococcus durans. The protein nature of those substances was proved by showing their sensitivity to type IV and XXV proteases, papaine, trypsin, pepsin and K proteinase. The bacteriocins inhibited the growth of Listeria monocytogenes, Bacillus cereus, Clostridium perfringes and two strains of Staphylococcus aureus, an A-enterotoxin and a B-enterotoxin producers. All of these bacteria are common pathogens usually associated with food borne diseases (ETA). These lactic acid bacteria or their bacteriocins could be suitable candidates for food preservation and specially useful in the our regional dairy industry.

Heterofermentative pattern and exopolysaccharide production by Lactobacillus helveticus ATCC 15807 in response to environmental pH.

AIMS: The objective of this work was to evaluate the fermentation pattern of and the exopolysaccharide (EPS) production by Lactobacillus helveticus ATCC 15807 in milk batch cultures under controlled pH (4.5, 5.0 and 6.2). METHODS AND RESULTS: EPS concentration was estimated by the phenol/sulphuric acid method and the chemical composition of purified EPS by HPLC. Fermentation products and residual sugars were determined by HPLC and enzymatic methods. The micro-organism shifted from a homofermentative to a heterofermentative pattern, producing acetate (9.5 and 5.8 mmol l(-1)) at pH 5.0 and 6.2, respectively, and acetate (7.1 mmol l(-1)) plus succinate (1.2 mmol l(-1)) at pH 4.5. At pH 5.0 and 6.2, acetate derived from citrate while at pH 4.5 it came from both citrate and pyruvate splitting. The EPS has a MW of 10(5)-10(6) and contains phosphate (81% in average), rhamnose (traces), and glucose and galactose in a ratio of 1 : 1 (pH 6.2) and 2 : 1 (pH 4.5 and 5.0). The highest production (549 mg l(-1)) corresponded to pH 5.0 and the lowest (49 mg l(-1)) to pH 6.2. CONCLUSIONS: The heterofermentative pattern of Lact. helveticus ATCC 15807 was linked to alternative pyruvate pathways and/or citrate metabolism according to the environmental pH. The EPS production was improved under low environmental pH conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides relevant information of the effect of pH on the metabolism of citrate and EPS production by Lact. helveticus. It may contribute to improve technological aspects of ropy and citrate-utilizing lactic acid bacteria.

Combined effect of bacteriocins on the survival of various Listeria species in broth and meat system.

The antilisterial efficiency of three bacteriocins from lactic acid bacteria, lactocin 705 (produced by L. casei CRL705, 17000 AU/ml), enterocin CRL35 (produced by E. faecium CRL35, 17000 AU/ml), and nisin (2000 IU/ml), was tested in broth, individually and in combination against Listeria monocytogenes and Listeria innocua. Both Listeria species showed an initial decrease in viable counts followed by the regrowth of the survivors after 1 h in the presence of each bacteriocin. A greater antilisterial effect was observed when the bacteriocins were combined in pairs, maximal inhibition being reached when nisin was involved. When a mix of the three bacteriocins was used, no survivors were observed after 24 h of incubation. Similar results were obtained when the bacteriocin combinations were tested in a meat system, indicating that the use of more than one LAB bacteriocin in combination may be effective in preventing the spontaneous emergence of a bacteriocin-resistant Listeria population.

Effect of enterocin CRL35 on Listeria monocytogenes cell membrane.

The antimicrobial peptide Enterocin CRL35, a class II bacteriocin, produces at high concentrations (8 microg ml(-1)) localized holes in the wall and cellular membrane of Listeria monocytogenes, reflected in the efflux of macromolecules such as proteins and other ultraviolet-absorbing materials. At lower concentrations (0.5 microg ml(-1)), neither ultra structural changes nor macromolecules efflux were observed, however potassium and phosphate ions were released, dissipating the proton motive force. As a result the bacteria were killed.

Identification and nucleotide sequence of genes involved in the synthesis of lactocin 705, a two-peptide bacteriocin from Lactobacillus casei CRL 705.

The structural gene determinants of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705, have been amplified from a plasmid of approximately 35 kb and sequenced. Lactocin 705 is a class IIb bacteriocin, whose activity depends upon the complementation of two peptides (705alpha and 705beta) of 33 amino acid residues each. These peptides are synthesized as precursors with signal sequences of the double-glycine type, which exhibited high identities with the leader peptides of plantaricin S and J from Lactobacillus plantarum, brochocin C from Brochotrix campestris, sakacin P from Lactobacillus sake, and the competence stimulating peptides from Streptococcus gordonii and Streptococcus mitis. However, the two mature bacteriocins 705alpha and 705beta do not show significant similarity to other sequences in the databases.

Citrate utilization by homo- and heterofermentative lactobacilli.

Citrate utilization by several homo- and heterofermentative lactobacilli was determined in Kempler and McKay and in calcium citrate media. The last medium with glucose permitted best to distinguish citrate-fermenting lactobacilli. Lactobacillus rhamnosus ATCC 11443, Lactobacillus zeae ATCC 15820 and Lactobacillus plantarum ATCC 8014 used citrate as sole energy source, whereas in the other strains, glucose and citrate were cometabolized. Some lactobacilli strains produced aroma compounds from citrate. Citrate transport experiments suggested that all strains studied presented a citrate transport system inducible by citrate. The levels of induction were variable between several strains. Dot blot experiment showed that lactobacilli do not present an equivalent plasmid coding for citrate permease.

Purification and amino acid sequence of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705.

Lactobacillus casei CRL 705, isolated from a dry fermented sausage, produces an antibacterial peptide which is active against Listeria monocytogenes. Previous studies have shown that this compound is potentially useful to control food-borne pathogens in ground meat. In view of the potential application of this antimicrobial substance in food fermentation, a detailed biochemical analysis of this peptide is required. In this work, the purification and amino acid sequence of this bacteriocin is presented. The adsorption-desorption pH-dependent property of lactocin 705 was exploited for purification. The active extract was further subjected to RP-HPLC and SDS-PAGE. The active antimicrobial band was electroeluted from an SDS-PAGE gel and its amino acid sequence determined. Lactocin 705 had an estimated molecular weight of 3357.80 and an isoelectric point of 10.03. The peptide contains a high ratio of glycine residues and does not show any modified amino acids, like lanthionine or beta-methyllanthionine. The sequence was unique when compared to several databases.

Antiviral activity of enterocin CRL35 against herpesviruses.

Enterocin CRL35 is an antibacterial polypeptide of 3.5 x 10(3) Da produced by Enterococcus faecium CRL35. A series of experiments are described that show the enterocin also had antiviral activity against thymidine-kinase positive (tk+) and deficient (tk-) strains of herpes simplex (HSV) type 1 and 2 in Vero and BHK-21 cells. This activity was observed at 100 microg/ml, 15-fold lower than the cytotoxic concentration. In both cell lines there was a 2 log inhibition of infectivity. The compound inhibited viral multiplication in a dose-dependent manner and had no virucidal effect. Enterocin CRL35 also inhibited the virion-associated host shutoff in infected Vero cells showing that intracellular viral multiplication was affected.

[Enterocin-35, a bacteriocin with activity against Listeria monocytogenes. Possible use in the food industry]. / Enterocina-35, una bacteriocina con actividad sobre Listeria monocytogenes. Posible uso en la industria de alimentos.

The in vitro inhibitory activity of enterocin-35 produced by Enterococcus faecium CRL 35, was studied against Listeria monocytogenes, isolated from seafoods. Optimal growth conditions of the enterocin-35 producing strain, for higher bacteriocin production and improve the extraction and purification of these peptides, were applied. A crude extract of enterocin-35 was assayed in a frozen seafood artificially contaminated with Listeria monocytogenes isolate, simulating at laboratory scale an eventual application of this biopreservant in a routine production process at factory level. The feasibility of biopreservation of seafoods by means of bacteriocins is proposed and discussed.

Properties of beta-lactamase from Neisseria gonorrhoeae.

Beta-lactamase activity was studied in Neisseria gonorrhoeae strains. Optimum temperature was found to be 37 degrees C. The enzyme was inactivated at temperatures higher than 60 degrees C, but remained active during storage at low temperatures (4 degrees C, -30 degrees C and -70 degrees C) for two months. Enzyme activity was observed within a pH range of 5.8-8.0, while the optimum pH was 7.0-7.2. Addition of Ni2+, Fe2+, Mn2+ and p-chloromercurybenzoate to the reaction buffer exerted a negative effect upon the activity, whereas Hg2+ and ethylene diamine tetra-acetic acid produced complete inhibition. These results would indicate the presence of -SH groups at the catalytic site of the enzyme.

Association between hemolytic uremic syndrome and verotoxin-producing strains of E. coli.

One of the most frequent etiologic agents of Hemolytic Uremic Syndrome (HUS) is Escherichia coli O157H7, a microorganism that possesses virulence factors (Shiga-like Toxins I and II and adhesion fimbriae). The present study was set up to determine the relationship between HUS and the presence of Verotoxin in patients of "Niño Jesús" Children's Hospital. Tucumán, Argentina. 19 Children between 0 and 4 years old suffering from HUS (typical and atypical symptoms) and 15 control children of similar sex and age were selected. Presence of enterohemorrhagic E. coli was studied in both groups using molecular hybridization techniques. Free Verotoxin and Verotoxin-producing E. coli were analyzed in Vero cells. The following results were obtained: 1) The cytotoxic effect on Vero cells from fecal filtrates was observed in all children suffering from HUS 2) Verotoxin-producing E. coli was detected in only 12 of them 3) None of the filtrates of feces from control children presented a cytotoxic effect on Vero cells 4) In 8 of the patients suffering from HUS serotype O157H7 was isolated, in one O55K59 and in 3 typification of E. coli was not possible with the serums assayed 5) 77.5% of the strains isolated from HUS patients gave a positive molecular hybridization reaction, showing the following: Adhesion Fimbriae (AF) (25%); AF + Shiga-like Toxin I (13.75%); AF + Shiga-like Toxin II (20%); AF + Shiga-like Toxins I and II (41.25%). In patients suffering from atypical HUS a combination of AF + Shiga-like Toxins I and II was found. The 15 control children did not hybridize to the probes assayed. From the results obtained we may conclude that there exists a relationship between HUS and the presence of Verotoxin in the children suffering from HUS studied. The predominant serotype in our cases was O157H7 and Shiga-like Toxin II was found with highest frequency.