Repositório RCAAP

One strain (S32) of Clostridium perfringens type A was isolated from a case of catarrhal enteritis of piglets. This strain was able to adhere to HeLa cells showing an adherence index (AI) of 25.15 ± 1.26 (mean ± 1 standard error of the mean). Treatment of the bacterial cells with trypsin (0.25mg/ml) decreased in 70%-80% the AI and metaperiodate (10mg/ml) abolished completely the adherence, suggesting that the structure responsible for this phenomenon was probably a glycoprotein. Heating of bacterial suspensions (100ºC/5 min) before carrying out the adhesion test decreased the AI rendering it equal to the negative controls. Rabbit homologous S32 antiserum inhibited the adherence up to dilutions of 1: 640, at least. The piglet ileal loop assay, carried out with strains S32 and Jab-1 (negative control) demonstrated that the strain S32 was able to adhere to the intestinal epithelial cells when examined after Gram staining. Transmission electron microcopy (TEM) demonstrated that S32 strain displayed a loose fibrillar material not seen with Jab-1. Stabilization of the bacterial cells with homologous antiserum of strain S32, followed by staining with rhuteniun red, revealed loose long fibrillar material on the outer surface of the cells, that sometimes could be seen spreading out from the cells and linking bacterial cells. The question whether this structure might be an adhesin for this strain of Cl. perfringes type A, perhaps playing a role in the pathogenesis of the catarrhal enteritis of piglets, is dependent on further studies.

The strain Saccharomyces cerevisiae Y500-4L, previously selected from the must of alcohol producing plants and showing high fermentative and killer capacities, was characterized according to the interactions between the yeasts and examined for curing and detection of dsRNA plasmids, which code for the killer character. The killer yeast S. cerevisiae Y500-4L showed considerable killer activity against the Fleischmann and Itaiquara commercial brands of yeast and also against the standard killer yeasts K2 (S. diastaticus NCYC 713), K4 (Candida glabrata NCYC 388) and K11 (Torulopsis glabrata ATCC 15126). However S. cerevisiae Y500-4L showed sensitivity to the killer toxin produced by the standard killer yeasts K8 (Hansenula anomala NCYC 435), K9 (Hansenula mrakii NCYC 500), K10 (Kluyveromyces drosophilarum NCYC 575) and K11 (Torulopsis glabrata ATCC 15126). No M-dsRNA plasmid was detected in the S. cerevisiae Y500-4L strain and these results suggest that the genetic basis for toxin production is encoded by chromosomal DNA. The strain S. cerevisiae Y500-4L was more resistant to the loss of the phenotype killer with cycloheximide and incubation at elevated temperatures (40oC) than the standard killer yeast S. cerevisiae K1.

Investigations were carried out with the aim of producing L-glutamic acid from Brevibacterium sp. by utilizing a locally available starchy substrate, cassava (Manihot esculenta Crantz). Initial studies were carried out in shake flasks, which showed that even though the yield was high with 85-90 DE (Dextrose Equivalent value), the maximum conversion yield (~34%) was obtained by using only partially digested starch hydrolysate, i.e. 45-50 DE. Fermentations were carried out in batch mode in a 5 L fermenter, using suitably diluted cassava starch hydrolysate, using a 85-90 DE value hydrolysate. Media supplemented with nutrients resulted in an accumulation of 21 g/L glutamic acid with a fairly high (66.3%) conversation yield of glucose to glutamic acid (based on glucose consumed and on 81.74% theoretical conversion rate). The bioreactor conditions most conducive for maximum production were pH 7.5, temperature 30°C and an agitation of 180 rpm. When fermentation was conducted in fed-batch mode by keeping the residual reducing sugar concentration at 5% w/v, 25.0 g/L of glutamate was obtained after 40 h fermentation (16% more the batch mode). Chromatographic separation by ion-exchange resin was used for the recovery and purification of glutamic acid. It was further crystallized and separated by making use of its low solubility at the isoelectric point (pH 3.2).

<FONT FACE="Symbol">b</font>-Galactosidase or <FONT FACE="Symbol">b</font>-D-galactoside-galactohydrolase (EC. 3.2.1.23) is an important enzyme industrially used for the hydrolysis of lactose from milk and milk whey for several applications. Lately, the importance of this enzyme was enhanced by its galactosyltransferase activity, which is responsible for the synthesis of transgalactosylated oligosaccharides (TOS) that act as functional foods, with several beneficial effects on consumers. Penicillium simplicissimum, a strain isolated from soil, when grown in semi-solid medium showed good productivity of <FONT FACE="Symbol">b</font>-galactosidase with galactosyltransferase activity. The optimum pH for hydrolysis was in the 4.04.6 range and the optimum pH for galactosyltransferase activity was in the 6.07.0 range. The optimum temperature for hydrolysis and transferase activity was 55-60°C and 50°C, respectively, and the enzyme showed high thermostability for the hydrolytic activity. The enzyme showed a potential for several industrial applications such as removal of 67% of the lactose from milk and 84% of the lactose from milk whey when incubated at their original pH (4.5 and 6.34, respectively) under optimum temperature conditions. When incubated with a 40% lactose solution in 150 mM McIlvaine buffer, pH 4.5, at 55°C the enzyme converted 86.5% of the lactose to its component monosaccharides. When incubated with a 60% lactose solution in the same buffer but at pH 6.5 and 50°C, the enzyme can synthetize up to 30.5% TOS, with 39.5% lactose and 30% monosaccharides remaining in the preparation.

Escherichia coli O157:H7 is a foodborne pathogen of increasing importance. It has been involved in several threatening outbreaks, most of them associated with meat products. In this study, the influence of some bacteria from the natural background flora of raw meat over E.coli O157:H7 in ground beef stored under refrigeration and at room temperature was evaluated. Different levels of E.coli O157:H7 (101-102, 103-104 and 106-107 CFU/g), inoculated in ground beef samples, were challenged with strains of non-pathogenic E.coli, Pseudomonas putida or Leuconostoc sp. Growth of the pathogen was monitored using standard cultural methods and an ELISA-type rapid method. Non-pathogenic E.coli, Pseudomonas putida and Leuconostoc sp. did not affect growth of E.coli O157:H7 in ground beef, both under refrigeration and at room temperature. Based on these findings, the low occurrence of E.coli O157:H7 in raw meat may not be attributed to antagonistic effects of bacteria from the natural background flora.

Cyanobacteria (Microcystis aeruginosa), which produce powerful hepatotoxic cyclopeptides, were collected and submitted to the determination of toxicity through intraperitoneal injections made in 30 and 90 days-old Swiss albino mice. The liver and the spleen were histopathologically analyzed and the weight and vital signs development were monitored. Test of toxicity resulted in a LD50 of 154.28 mg.Kg-1. M. aeruginosa represented 95% of the analyzed biomass. The ratios between liver weight and body weight in the animal inoculated with a single dose were 6.0% and 7.2%, with multi doses 7.0% and 7.5% and in the control animals 4.0% and 5.0%, for adult and young animals, respectively. There was an accentuated increase in the volume and weight of the spleen, and the animals inoculated with a single dose showed a ratio between spleen weight and body weight of 0.67% and 0.37%, with multidoses 1.22% and 1.05% and the control animals the ratio was 0.12% and 0.15%, for adult and young animals, respectively. The young animals inoculated with single and multi doses had an increase of 150% and 407% in the spleen size while the adults increased, 607% and 845%, respectively, in relation to the control. The histopathological analysis showed strong differences in the structure of the hepatic parenchyme in control animals and in those exposed to the M. aeruginosa extract. The main alterations were the congestive aspect, including the sinusoid, and intrahepatic haemorrhagia. The histopathological analysis showed considerable increase in the number of multinuclear giant cells in the spleen of the animals intoxicated by M. aeruginosa.

Thermophilic and hyperthermophilic microorganisms are found as normal inhabitants of continental and submarine volcanic areas, geothermally heated sea-sediments and hydrothermal vents and thus are considered extremophiles. Several present or potential applications of extremophilic enzymes are reviewed, especially polymer-hydrolysing enzymes, such as amylolytic and hemicellulolytic enzymes. The purpose of this review is to present the range of morphological and metabolic features among those microorganisms growing from 70oC to 100°C and to indicate potential opportunities for useful applications derived from these features.

One hundred sixty eight bacterial strains, isolated from soil and samples of vegetable in decomposition, were screened for the use of citrus pectin as the sole carbon source. 102 were positive for pectinase depolymerization in assay plates as evidenced by clear hydrolization halos. Among them, 30% presented considerable pectinolytic activity. The cultivation of these strains by submerged and semi-solid fermentation for polygalacturonase production indicated that five strains of Bacillus sp produced high quantities of the enzyme. The physico-chemical characteristics, such as optimum pH of 6.0 - 7.0, optimum temperatures between 45oC and 55oC, stability at temperatures above 40oC and in neutral and alkaline pH, were determined.

A Brazilian strain of Fusarium solani was tested for extracellular lipase production in peptone-olive oil medium. The fungus produced 10,500 U.l-1 of lipase after 72 hours of cultivation at 25oC in shake-flask at 120 rpm in a medium containing 3% (w/v) peptone plus 0.5% (v/v) olive oil. Glucose (1% w/v) was found to inhibit the inductive effect of olive oil. Peptone concentrations below 3% (w/v) resulted in a reduced lipase production while increased olive oil concentration (above 0.5%) did not further stimulate lipase production. The optimum lipase activity was achieved at pH 8.6 and 30oC and a good enzyme stability (80% activity retention) was observed at pH ranging from 7.6 to 8.6, and the activity rapidly dropped at temperatures above 50oC. Lipase activity was stimulated by the addition of n-hexane to the culture medium supernatants, in contrast to incubation with water-soluble solvents.

The growth kinetics of Acetivibrio cellulolyticus grown in medium containing different carbon sources (cellobiose, amorphous or crystalline cellulose) was investigated. The specific growth rate was higher in cellobiose fed cultures than in the presence of the other two substrates. Endoglucanase production was greater in cultures grown on amorphous cellulose; enzyme activity increased during the stationary phase in cultures grown on crystalline cellulose.

In this study we determined some properties of the cholesterol oxidase from a Brevibacterium strain isolated from buffalo's milk and identified the cholesterol degradation products by the bacterial cell. A small fraction of the enzyme synthesized by cells cultured in liquid medium for 7days was released into the medium whereas a larger fraction remained bound to the cell membrane. The extraction of this fraction was efficiently accomplished in 1 mM phosphate buffer, pH 7.0, containing 0.7% Triton X-100. The enzyme stability under freezing and at 45oC was improved by addition of 20% glycerol. The optimum temperature and pH for the enzyme activity were 53°C and 7.5, respectively. The only steroidal product from cholesterol oxidation by the microbial cell and by the crude extract of the membrane-bound enzyme was 4-colesten-3-one. Chromatographic analysis showed that minor no steroidal compounds as well as 4-colesten-3-one found in the reaction media arose during fermentation process and were extracted together with the enzyme in the buffer solution. Cholesterol oxidation by the membrane-bound enzyme was a first order reaction type.

This work investigated the partitioning of b-galactosidase from Kluyveromyces fragilis in aqueous two-phase systems (ATPS) by bioaffinity. PEG 4000 was chemically activated with thresyl chloride, and the biospecific ligand p-aminophenyl 1-thio-b-D-galactopyranoside (APGP) was attached to the activated PEG 4000. A new two-step method for extraction and purification of the enzyme b-galactosidase from Kluyveromyces fragilis was developed. In the first step, a system composed of 6% PEG 4000-APGP and 8% dextran 505 was used, where b-galactosidase was strongly partitioned to the top phase (K = 2,330). In the second step, a system formed of 13% PEG-APGP and 9% phosphate salt was used to revert the value of the partition coefficient of b-galactosidase (K = 2 x 10-5) in order to provide the purification and recovery of 39% of the enzyme in the bottom salt-rich phase.

Fifty individuals of both sexes aged on average 45.2 years were evaluated at the Semiology Clinic of FORP-USP in order to isolate and identify yeasts from the oral cavity, with and without lesions, and to determine the maximal inhibitory dilution (MID) of the commercial products Propolis (Apis-Flora) and Periogard (Colgate) against the strains isolated. Yeasts of the genus Candida were detected in the saliva of 9/19 (47.4%) individuals with a clinically healthy mouth, 18/22 (81.8%) of individuals with oral lesions, and in 4/9 (44.4%) of patients with deviation from normality, and were detected in 19/22 (86.4%) of the lesions. In the group with oral candidiasis, we isolated in tongue and lesion, respectively for each specie: C.tropicalis (8% and 10.7%), C.glabrata (4% and 3.6%) and C.parapsilosis (2% and 3.6%), in addition to C.albicans (71.4% and 67.8%) as the only species and the prevalent. The total cfu counts/ml saliva showed a higher mean value in the group with oral candidiasis (171.5% x 10(3)) than in the control group (72.6 x 10(3)) or the group with abnormalities (8.3 x 10(3)). Most of the test strains 67/70 (95.71%) were sensitive to the antiseptics, with Propolis presenting a MID of 1:20 for 54/70/77.1%), and Periogard a MID of 1:160 for 42/70 (60%) strains from healthy sites, results similar to those obtained with strains from oral lesions. Different results were mainly observed among different species. The results indicate the possibility of using the antiseptics Propolis and Periogard (chlorhexidine) for the prevention and treatment of oral candidiasis.

Fusobacterium nucleatum is indigenous of the human oral cavity and has been involved in different infectious processes. The production of bacteriocin-like substances may be important in regulation of bacterial microbiota in oral cavity. The ability to produce bacteriocin-like substances by 80 oral F. nucleatum isolates obtained from periodontal patients, healthy individuals and Cebus apella monkeys, was examinated. 17.5% of all tested isolates showed auto-antagonism and 78.8% iso- or hetero-antagonism. No isolate from monkey was capable to produce auto-inhibition. In this study, the antagonistic substances production was variable in all tested isolates. Most of the F. nucleatum showed antagonistic activity against tested reference strains. These data suggest a possible participation of these substances on the oral microbial ecology in humans and animals. However, the role of bacteriocins in regulating dental plaque microbiota in vivo is discussed.

Hazards and critical control points (CCP) associated with meat balls and kibbe preparations in a hospital kitchen were determined using flow diagrams and microbiological testing of samples collected along the production line. Microbiological testing included counts of mesophilic and psicrothrophic microorganisms, yeasts and molds, total and fecal coliforms, C. perfringens, coagulase positive staphylococci, bacteria of the B. cereus group and detection of Salmonella. Time/temperature binomial was measured in all steps of preparation. A decision tree was used to help in the determination of CCPs. The detected hazards were: contamination of raw meat and vegetables, multiplication of the microorganisms during meat manipulation, poor hygiene of utensils and equipment, and survival of microorganisms to the cooking process. Cooking and hot-holding were considered CCPs. The results stress the importance of the implementation of a training program for nutricionists and foodhandlers and the monitoring of CCPs and other measures to prevent foodborne diseases.

A total of 30 strains of Listeria monocytogenes isolated from different foods (16 of differents kinds of sausage, 14 cheese,) purchased at groceries of São Paulo City were ribotyped and analysed for the presence and expression of hemolysin gene and production of phosphatidylinositol-specific phosphalipase C - PI-PLC enzyme. The L. monocytogenes strains were differentiated into six ribotype classes. A total of 13 (43.3%) from these strains belong to the same ribotype (ribotype I), and was coincident to the ribotype of the standard L. monocytogenes prototype strain (ATCC-15313). The hemolytic activity was observed in 29 (96.7%) strains when incubated at 37oC, but not at 4oC. The direct colony hybridization method for hemolysin gene detection showed a positive reaction whit all the 30 L. monocytogenes strains, while showed negative reaction with other Listeria spp. The PI-PLC was produced by 27 (90%) of the strains analysed. There was no correlation between the six identified ribotypes and the virulence factors (hemolysin and PI-PLC) studied.

The genetic diversity of 41 typical and atypical enteropathogenic Escherichia coli (EPEC) strains of the serogroup O55 was analyzed by using the random amplified polymorphic DNA (RAPD) method. All typical EPEC O55 strains were grouped in two clusters (A and C) and belonged to the serotype O55:H6, while cluster B included all atypical strains, which were of the serotype O55:H7. The three groups also included non-motile strains. RAPD may be a useful method for epidemiological studies on E. coli O55 infection.

The susceptibility of the five cell lines - IB-RS-2, RK-13, Vero, BHK-21, CER - to reovirus S1133 and infectious bursal disease virus (IBDV vaccine GBV-8 strain) was studied to better define satisfactory and sensitive cell culture systems. Cultures were compared for presence of CPE, virus titers and detection of viral RNA. CPE and viral RNA were detected in CER and BHK-21 cells after reovirus inoculation and in RK-13 cell line after IBDV inoculation and with high virus titers. Virus replication by production of low virus titers occurred in IB-RS-2 and Vero cells with reovirus and in BHK-21 cell line with IBDV.

Supramolecular chemistry deals with the association of several chemical species, in an organized way and according to well defined purposes. Based on a molecular engineering approach, supramolecular structures can be designed from pre-formed building blocks, providing a promising route from chemistry to molecular nanotechnology. New supramolecular systems have been assembled in our laboratory with the use of bridging unities such as tetrapyridylporphyrins, porphyrazines and polypyrazines, connecting transition metal complexes and clusters. These systems display a very exciting electrochemical and catalytic behavior, and interact with DNA, generating ¹O2 and leading to efficient oxidative clivage for photodynamic terapy applications. Molecular interfaces have been developed, exhibiting photocurrent response in the presence of visible-UV light, and rectifying properties in the presence of electroactive species. Successful applications of the supramolecular species in chemical and bio-sensors have been developed.

A relevant series of symmetric supramolecular porphyrins has been obtained by attaching four [Ru II(bipy)2Cl] groups to the pyridyl substituents of meso-tetra(4-pyridyl)porphyrin and its metallated derivatives. These compounds display a rich electrochemistry and versatile catalytic, electrocatalytic and photochemical properties, associated with the ruthenium-bipyridine and the porphyrin complexes. These properties can be transferred to the electrodes by attaching thin molecular films of the compounds, by dip-coating, electrostatic assembly or electropolymerization. In this way, the interesting properties of those supermolecules and supramolecular assemblies can be used to prepare molecular devices and sensors.