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PubMed1975

Catalysis of phosphoryl group transfer. The role of divalent metal ions in the hydrolysis of lactic acid O-phenyl phosphate and salicylic acid O-aryl phosphates.

The spontaneous hydrolyses of lactic acid O-phenyl phosphate (I) and, to a lesser extent, 3-hydroxybutyric acid O-phenyl phosphate (II) have been investigated and compared with similar intramolecular and bimolecular reactions. Compared to bimolecular nucleophilic reactions, the reactivity of II is similar to other systems involving the formation of a six-membered ring intermediate, which suggests that the electrostatic barrier to attack of an anionic nucleophile on a phosphate diester anion is fully present in II. The reactivity of I, as compared to that of II, would suggest that at least a partial overcoming of the electrostatic barrier takes place upon closer approimation of the two reacting centers. The Mn-2+-catalyzed hydrolysis of I exhibits saturation kinetics, consistent with the enhanced reactivity of the metal ion-substrate complex. The binding constant for this complex, determined from kinetics, is in good agreement with that obtained by electron spin resonance (ESR) titration. It is argued that the complex of Mn-2+ with II, as observed by pulsed Fourier transform nuclear magnetic resonance (NMR) techniques, is a precursor to the complex of catalytic significance. The hydrolysis of I as catalyzed by a variety of divalent metal ions suggests an optimal metal ion size. The spontaneous and metal ion catalyzed hydrolyses of salicyclic acid O-aryl phosphates (IIIa-d) proceed through cyclic acyl phosphate intermediates after expulsion of phenol. Product studies on the parent compound have failed to detect phenyl phosphate as a product in either the spontaneous or metal ion catalyzed process. The dependence of the second-order rate constant for the metal-catalyzed hydrolysis on leaving group pKa, beta-1-g, decreases significantly relative to beta-1-g for the spontaneous hydrolysis. From the collective data a specific interation of the metal ion with a pentacovalent intermediate is inferred in the rate-determining step for esters I and III. The probable consequences of these mechanistic postulates for phosphoryl transfer reactions in biological systems are discussed.

Cations, Divalent↗
PubMed2003

The survival of silage inoculant lactic acid bacteria in rumen fluid.

AIMS: To determine whether lactic acid bacteria (LAB) used in inoculants for silage can survive in rumen fluid (RF), and to identify those that survive best. METHODS AND RESULTS: Twelve commercial silage inoculants were added at 107 CFU ml-1 to strained RF (SRF) taken from dairy cows, with and without 5 g l-1 glucose and incubated in vitro at 39 degrees C. Changes in pH, LAB numbers and fermentation products were monitored for 72 h. In the inoculated RF with glucose, the pH decreased and numbers of LAB increased. The inoculants varied with regard to their effect on pH change and growth. In the SRF, both with and without glucose, the pH values of the inoculated samples were generally higher than those of the uninoculated controls throughout most of the incubation period. This may suggest a positive effect on the rumen environment. CONCLUSIONS: LAB used in silage inoculants can survive in RF in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first step in studying the probiotic potential of silage LAB inoculants for dairy cattle. The survival of these LAB in RF may enable them to interact with rumen microorganisms and to affect rumen functionality.

Animals↗
PubMed2004

Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: their characteristics and potential applications in biotechnology.

Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. The use of genetic engineering techniques is an effective means of enhancing the industrial applicability of LAB. However, when using genetic engineering technology, safety becomes an essential factor for the application of improved LAB to the food industry. Cloning and expression systems should be derived preferably from LAB cryptic plasmids that generally encode genes for which functions can be proposed, but no phenotypes can be observed. However, some plasmid-encoded functions have been discovered in cryptic plasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can be used as selective marker systems in vector construction. This article presents information concerning LAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmids collated are discussed.

Biotechnology↗
PubMed1998

Poly-beta-hydroxybutyrate production by lactic acid bacteria.

Poly-beta-hydroxybutyrate was determined in lactic acid bacteria belonging to the genera Lactobacillus, Lactococcus, Pediococcus and Streptococcus. Lactobacilli were grown in MRS broth, the others were grown in Elliker broth medium. Cell biomass was obtained by centrifugation. The cell walls were lysed with sodium hypochlorite. Poly-beta-hydroxybutyrate was extracted using chloroform in a Soxhlet system. Then it was converted to crotonic acid using sulfuric acid and the amount of crotonic acid was measured spectrophotometrically. The yield of poly-beta-hydroxybutyrate (% of cell dry weight) of Lactobacillus species was 6.6-35.8%. The values for Lactococcus, Pediococcus and Streptococcus species were 9.0-20.9, 1.1-8.0 and 6.8-17.2, respectively. It was observed that one of the Lactobacillus species did not produce poly-beta-hydroxybutyrate. Generally, Lactobacillus species produced more poly-beta-hydroxybutyrate than the other tested bacteria and no significant correlation was observed between poly-beta-hydroxybutyrate production and cell density of the cultures.

Hydroxybutyrates↗
PubMed1999

Phenotypic and genotypic characterization of non-starter lactic acid bacteria in mature cheddar cheese.

Non-starter lactic acid bacteria were isolated from 14 premium-quality and 3 sensorially defective mature Irish Cheddar cheeses, obtained from six manufacturers. From countable plates of Lactobacillus-selective agar, 20 single isolated colonies were randomly picked per cheese. All 331 viable isolates were biochemically characterized as mesophilic (i.e., group II) Lactobacillus spp. Phenotypically, the isolates comprised 96.4% L. paracasei, 2.1% L. plantarum, 0.3% L. curvatus, 0.3% L. brevis, and 0.9% unidentified species. Randomly amplified polymorphic DNA (RAPD) analysis was used to rapidly identify the dominant strain groups in nine cheeses from three of the factories, and through clustering by the unweighted pair group method with arithmetic averages, an average of seven strains were found per cheese. In general, strains isolated from cheese produced at the same factory clustered together. The majority of isolates associated with premium-quality cheese grouped together and apart from clusters of strains from defective-quality cheese. No correlation was found between the isomer of lactate produced and RAPD profiles, although isolates which did not ferment ribose clustered together. The phenotypic and genotypic methods employed were validated with a selection of 31 type and reference strains of mesophilic Lactobacillus spp. commonly found in Cheddar cheese. RAPD analysis was found to be a useful and rapid method for identifying isolates to the species level. The low homology exhibited between RAPD banding profiles for cheese isolates and collection strains demonstrated the heterogeneity of the L. paracasei complex.

Bacterial Typing Techniques↗
PubMed1966

Effect of lactic acid bacteria on growth of Staphylococcus aureus.

Cultures of lactic acid bacteria, mostly from foods, were tested for their effect on the growth of Staphylococcus aureus in Trypticase Soy Broth (BBL). Some of the effectors, e.g., Streptococcus faecalis, S. faecium, Lactobacillus lactis, L. brevis, and Leuconostoc mesenteroides, stimulated growth of S. aureus during early hours of growth, especially at higher temperatures of incubation, but most cultures were inhibitory, and some (S. faecium and L. mesenteroides) were even killing by the time of attainment of the maximal phase of growth of the Staphylococcus. Low-temperature meat lactobacilli and Leuconostoc dextranicum inhibited S. aureus at 10, 15, 20, and 25 C throughout its growth. Streptococcus faecalis var. liquefaciens inhibited at these temperatures and at 30 and 37 C, as well. When the ratio of effectors to staphylococci in the inoculum was 100:1, the three enterococci, the meat Lactobacillus, and L. dextranicum prevented the attainment of 5 x 10(6) staphylococci per milliliter at 15 C, and all but the meat Lactobacillus did so at 22 C. A ratio of 1:1 accomplished similar results at 15 C, except that S. aureus was only delayed for 12 hr by S. faecalis. A ratio of 1:100 usually was ineffective. In general, the more effector bacteria there were in the inoculum, the greater was the overall inhibition (or stimulation) of S. aureus. Inhibition was most effective at 10 or 15 C, less so at 20 or 25 C, and least at 30 or 37 C, whereas stimulation during early growth was greater at the higher temperatures. Results with different strains of the effectors and with two strains of S. aureus were similar, for the most part.

Enterococcus faecalis↗
PubMed1987

Regulation of beta-galactoside transport and accumulation in heterofermentative lactic acid bacteria.

Galactose-grown cells of the heterofermentative lactic acid bacteria Lactobacillus brevis and Lactobacillus buchneri transported methyl-beta-D-thiogalactopyranoside (TMG) by an active transport mechanism and accumulated intracellular free TMG when provided with an exogenous source of energy, such as arginine. The intracellular concentration of TMG resultant under these conditions was approximately 20-fold higher than that in the medium. In contrast, the provision of energy by metabolism of glucose, gluconate, or glucosamine promoted a rapid but transient uptake of TMG followed by efflux that established a low cellular concentration of the galactoside, i.e., only two- to fourfold higher than that in the medium. Furthermore, the addition of glucose to cells preloaded with TMG in the presence of arginine elicited a rapid efflux of the intracellular galactoside. The extent of cellular TMG displacement and the duration of the transient effect of glucose on TMG transport were related to the initial concentration of glucose in the medium. Exhaustion of glucose from the medium restored uptake and accumulation of TMG, providing arginine was available for ATP generation. The nonmetabolizable sugar 2-deoxyglucose elicited efflux of TMG from preloaded cells of L. buchneri but not from those of L. brevis. Phosphorylation of this glucose analog was catalyzed by cell extracts of L. buchneri but not by those of L. brevis. Iodoacetate, at a concentration that inhibits growth and ATP production from glucose, did not prevent efflux of cellular TMG elicited by glucose. The results suggested that a phosphorylated metabolite(s) at or above the level of glyceraldehyde-3-phosphate was required to evoke displacement of intracellular TMG from the cells. Counterflow experiments suggested that glucose converted the active uptake of TMG in L. brevis to a facilitated diffusion mechanism that allowed equilibrium of TMG between the extra- and intracellular milieux. The means by which glucose metabolites elicited this vectorial regulation is not known, but similarities to the inducer expulsion that has been described for homofermentative Streptococcus and Lactobacillus species suggested the involvement of HPr, a protein that functions as a phosphocarrier protein in the phosphotransferase system, as well as a presumptive regulator of sugar transport. Indeed, complementation assays wit extracts of Staphylococcus aureus ptsH mutant revealed the presence of HPr in L. brevis, although this lactobacillus lacked a functional phaosphoenolpyruvate-dependent phosphortransferase system for glucose, 2-deoxyglucose, or TMG.

Arginine↗
PubMed2003

Antagonism of Helicobacter pylori by bacteriocins of lactic acid bacteria.

Antimicrobial activity of seven bacteriocins produced by lactic acid bacteria against Helicobacter pylori strains (ATCC 43504, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH [DSM] 4867, DSM 9691, and DSM 10242) was investigated in vitro using a broth microdilution assay. The bacteriocins chosen for the study were nisin A; lacticins A164, BH5, JW3, and NK24; pediocin PO2; and leucocin K. Antimicrobial activity of the bacteriocins varied among the H. pylori strains tested, of which strain ATCC 43504 was the most tolerant. Among the bacteriocins tested, lacticins A164 and BH5 produced by Lactococcus lactis subsp. lactis A164 and L. lactis BH5, respectively, showed the strongest antibacterial activity against H. pylori strains. MICs of the lacticins against H. pylori strains, when assessed by the critical dilution micromethod, ranged from 0.097 to 0.390 mg/liter (DSM strains) or from 12.5 to 25 mg/liter (ATCC 43504), supporting the strain-dependent sensitivity of the pathogen. Pediocin PO2 was less active than the lacticins against four strains of H. pylori, and leucocin K was the least active peptide, with no inhibition toward H. pylori ATCC 43504. Anti-Helicobacter activity of lacticin A164 was dependent on initial inoculum size as well as concentration of the bacteriocin added.

Antibiosis↗
PubMed2001

Lactic acid bacteria and their effect on the immune system.

Lactic acid bacteria (LAB) are present in the intestine of most animals. The beneficial role played by these microorganisms in the humans and other animals, including the effect on the immune system, has been extensively reported. They are present in many foods and are frequently used as probiotics to improve some biological functions in the host. The activation of the systemic and secretory immune response by LAB requires many complex interactions among the different constituents of the intestinal ecosystem (microflora, epithelial cells and immune cells). Through different mechanisms they send signals to activate immune cells. Thus the knowledge of the normal intestinal microflora, the contribution of LAB and their role in the numerous functions in the digestive tract as well as the functioning of the mucosal immune system form the basis for the study and selection of a probiotic strain with immunostimulatory properties. In the selection of LAB by their immunostimulatory capacity it helps to know not only the effect which they have on the mucosal immune system, but the specific use to which these oral vaccine vectors are being put. Although there are reports of the protection of animals and humans against diseases such as microbial infections and cancer, more work remains to be done on the factors affecting the design of oral vaccine vectors and the use of LAB for therapeutic purposes. The basic knowledge of LAB immunostimulation and the criteria for selection of LAB by their immunostimulatory capacity, will be extensively discussed and appraised in this review.

Animals↗
PubMed1979

[Transplacental blastomogenic effect of p-hydroxyphenyl lactic acid].

Transplacental administration of tyrosine metabolite p-hydroxyphenyl lactic acid having blastomogenic activity induced development of malignant and benign neoplasms (leukemias and lymphosarcomas, adenomas and pulmonary carcinoma, hepatomas, papillomas, and cancer of the urinary bladder and other tumours) in progeny of CC57BR and C57BL mouse strains, in 88 and 78% of cases, respectively. The number of neoplasms was significantly higher, than in the control, they appeared earlier and were of more malignant character. The role of endogenous blastomogenic substances in the development of leukemias in the newborn and yound children is discussed.

Animals↗
PubMed2003

Thermal and mechanical characteristics of poly(L-lactic acid) nanocomposite scaffold.

Inorganic nanosized silicate nanoplatelets were incorporated into biodegradable poly(L-lactic acid) (PLLA) for the purpose of tailoring mechanical stiffness of PLLA porous scaffold systems. Increasing the nucleation density around the foreign body surfaces, the montmorillonite (MMT) nanoplatelets modified with dimethyl dihydrogenated tallow ammonium cations decreased the glass transition temperature and the degree of PLLA crystallinity, which seemingly caused the accelerated biodegradation rate of PLLA nanocomposites due to the enhanced segmental mobility of backbone chains and the expanded amorphous region of PLLA matrix. The tensile modulus was increased from 121.2MPa of pristine polymer scaffold to 170.1MPa of MMT/PLLA nanocomposite scaffold (ca. 40% increment) by the addition of small amount of MMT platelets (5.79 vol%) acting as a mechanical reinforcement of polymer chains in the nanoscale molecular level. Overall, the nanotechnology used in this study may be applied to various scaffold systems of biodegradable polymers and hard/soft scaffold structures requiring critical control and design characteristics of mechanical stiffness and biodegradation rate.

Culture Techniques↗
PubMed2005

Homofermentative lactic acid bacteria of a traditional cheese, Comlek peyniri from Cappadocia region.

Comlek peyniri is a typical artisanal cheese in Central Anatolia. This type of cheese was made by using the indigenous lactic acid bacteria (LAB) flora of cow or ewes' milk. Majority of the samples were taken from fresh cheese because the aim was to isolate homofermentative LAB. Initially 661 microbial isolates were obtained from 17 cheese samples. Only 107 were found to be homofermentative LAB. These isolates were selected and identified by using both phenotypic and molecular methods. Phenotypic identification included curd formation from skim milk, catalase test, Gram staining and light microscopy, growth at different temperatures and salt concentrations, arginine hydrolysis, gas production from glucose, and carbohydrate fermentation. Molecular identification was based on the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 16S rRNA gene-ITS (internally transcribed spacer) region. By combining the phenotypic and molecular identification results, isolates belonging to each of the following genera were determined at species or subspecies level: 54 Lactococcus lactis subsp. lactis, 21 Enterococcus faecium, 3 Ec. faecalis, 2 Ec. durans, 10 Ec. sp., 15 Lactobacillus paracasei subsp. paracasei, and 2 Lb. casei strains. Technological characterisation was also performed by culturing each of the strains in UHT skim milk, and by monitoring pH change and lactic acid production at certain time intervals through the 24 h incubation. Results of the technological characterisation indicated that 33% of the isolates (35 strains) were capable of lowering the pH of UHT milk below 5.3 after 6 h incubation at 30 degrees C. Thirty four of these strains were Lc. lactis subsp. lactis, and only one was an Ec. faecium strain.

Animals↗
PubMedDate not supplied

Microencapsulation using poly(DL-lactic acid). II: Effect of polymer molecular weight on the microcapsule properties.

Poly(DL-lactic acid) (DL-DPA) of three different molecular weights, 20,500; 13,300 and 5200, was used to prepare microcapsules containing differing contents of phenobarbitone (PB), as a reference core. A water/oil (W/O) emulsion evaporation method was used. The effect of polymer molecular weight on the particle size, 'encapsulation efficiency', morphology, density, thermal behaviour and swelling property has been reported. A general trend towards lowering the mean microcapsule size, both by volume and population, was observed with respect to lower polymer molecular weight. The gross morphology of the microsapsule surface, encapsulation efficiency and density were unaffected by variations in polymer molecular weight. Differential scanning calorimetric analysis of the microcapsules showed a lowering of glass transition temperature after microencapsulation. The melting endotherm for phenobarbitone also indicated the presence of crystalline drug in the microcapsule matrix. These microcapsules were found to swell in the aqueous environment and the mean size increased linearly with time. However, the rate of swelling was higher with low molecular weight polymer and also depended on core loading.

Chemical Phenomena↗
PubMed2004

Plasticization and spraying of poly (DL-lactic acid) using supercritical carbon dioxide: control of particle size.

Exposure of poly(DL-lactic acid) (PDLLA), and related polymers, to supercritical CO2 (scCO2) at or below, physiological temperatures leads to very effective plasticization and liquefying of the polymers. The phenomenon arises from the high solubility and interaction of the scCO2 in the polymer. Under these unique conditions, temperature and solvent labile molecules can be mixed efficiently into the liquefied polymer. This liquefied polymer/drug/CO2 mixture can then be sprayed into a collecting chamber, and during this process particles of drug-loaded polymer are formed. This process is very different from rapid expansion and antisolvent based techniques that have been previously reported. In this article, we describe a method of controlling particle size during the spray process by introducing a backpressure of N2 in the collecting chamber. This backpressure dynamically regulates the loss of CO2 from the issuing polymer/CO2 mixture, leading to control over sprayed particle size. In situ observation of the viscosity of the plasticized polymer indicates that a backpressure of 68 bar or greater is necessary to ensure the production of fine particles. The influences of backpressure and saturation temperature on particle size for the sprayed products are discussed in terms of observed PDLLA/CO2 mixture viscosities.

Air Pressure↗
PubMedDate not supplied

Microencapsulation using poly (L-lactic acid) III: Effect of polymer molecular weight on the microcapsule properties.

Poly (L-lactic acid) microcapsules were prepared using an emulsification and organic solvent evaporation technique (w/o system) with phenobarbitone as a reference core. Three polymers of different molecular weight (61,300; 43,200, 2400), were used to prepare different core loaded microcapsules. Microcapsule size increased with increase in polymer molecular weight. Microcapsule size was also found to increase with increase in core loading with the two high molecular weight polymers, whilst the low molecular weight polymer tended to aggregate to form larger microcapsules than expected. The calculated microcapsule density was found to decrease with an increase in the polymer molecular weight and core loading. 'Encapsulation efficiency' was reduced with the decrease in initial theoretical core loading. However, the phenobarbitone content of the microcapsules was not affected by the difference in polymer molecular weight. Significant morphological differences were observed due to variations in the polymer molecular weight. The two high molecular weight polymers were found to produce non-uniform, porous microcapsules, whilst low molecular weight polymer formed a uniform non-porous surface when core loading was low. With increasing core loading, an increasing number of phenobarbitone crystals were observed on the surface and microcapsules became increasingly porous. This was more evident after release of the drug. Differential scanning calorimetry of the microcapsules showed thermal events for both the polymer and phenobarbitone.

Chemical Phenomena↗
PubMed2001

Evaluation of poly-L-lactic acid and polyglycolic acid resorbable stents for repair of tracheomalacia in a porcine model.

Poly-L-lactic acid and polyglycolic acid (PLPG) resorbable stents may offer a potential solution to the problem of tracheomalacia. Advantages of this material include its strength, its versatile shaping characteristics, and its resorbability, which would preclude surgical removal and allow for airway growth. The purpose of this pilot study was to examine the usefulness of PLPG stents for temporary external airway stenting of tracheomalacia in a porcine model. A severe tracheomalacia was created in 6 pigs by submucosal resection of segments of tracheal cartilage from 6 consecutive rings. The PLPG stent was then shaped to recreate the tracheal contour and sutured to the underlying airway. Endoscopic photodocumentation during spontaneous ventilation was obtained before and after reconstruction. After creation of the malacic tracheal segment, all animals developed stridor, retractions, and cyanosis during spontaneous ventilation. After repair, all animals were extubated without complication. All animals survived the follow-up period of 9 to 12 weeks without evidence of respiratory distress and with rapid weight gain. Repeat bronchoscopy showed no evidence of airway collapse during spontaneous ventilation. Tracheal measurements revealed growth of the stented segment with a mild narrowing within the repaired region. Histologic examination showed preservation of respiratory epithelium. These preliminary findings suggest that PLPG stents may serve a useful role in the surgical management of tracheomalacia.

Absorbable Implants↗
PubMed2005

Use of absorbable poly (d,l) lactic acid plates in cranial-vault remodeling: presentation of the first case and lessons learned about its use.

OBJECTIVE: To present the first clinical use of a new bioabsorbable material, poly (D,L) lactic acid (PDLLA), in pediatric cranial-vault remodeling procedures. This discussion will highlight the benefits and detriments of PDLLA in comparison with currently used absorbable plating systems. DESIGN: This was a case study documenting the first North American case in which PDLLA was used to treat craniosynostosis. MATERIALS AND METHODS: Evaluation of pure PDLLA, a copolymer product of the mixture of poly L-lactic acid and its D-isomer, was used in an 8-month-old boy with a severe phenotypic expression of sagittal craniosynostosis. No signs of elevated intracranial pressure were present, and the neurological examination did not show impairments. Total cranial-vault remodeling with the "hung-span" technique was performed. The Resorb X system, containing 2.2-mm screws and 0.6- to 1-mm-thick plates, was used to stabilize the reconstructed cranial vault. RESULTS: No surgical complications occurred. The preoperative cranial index measured 62. The scaphocephalic appearance of the skull was eliminated, and the cranial index was normalized to 77. The screws and plates were less palpable than other plating systems. Twelve months postoperatively, none of the plates and screws were identifiable by external palpation. CONCLUSION: Resorb X has been successfully used in the treatment of sagittal craniosynostosis. Its rapid rate of resorption and lower profile make it an advantageous system for pediatric skull reconstruction. This represents the first use of this product in the United States for any pathology.

Absorbable Implants↗