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Analysis of blood of severely intoxicated patients always requires prompt investigation. Diagnosis of intoxication with ethylene glycol, gamma-hydroxybutyric acid or D-lactic acid takes hours, since several different procedures are required. Rapid derivatization of the common hydroxyl function may resolve this analytical problem. Here we describe a fast method for the simultaneous measurement of ethylene glycol, glycolic acid, gamma-hydroxybutyric acid and racemic lactic acid. Only 20 microl of serum, plasma or urine are required for immediate derivatization at 70 degrees C with 750 microl of bis-N,O-trimethylsilyl trifluoroacetamide after adding 20 microl of internal standard solution (1,3-propylene glycol) and 20 microl of the catalyst dimethylformamide. After centrifugation an aliquot is transferred to a gas chromatographic system and analyzed with electron-impact mass spectrometry in selective ion monitoring mode. The derivatized acids and ethylene glycol are well separated and detected with a limit of detection ranging from 0.12 mg/l for ethylene glycol to 0.95 mg/l for gamma-hydroxybutyric acid, while the limit of quantification ranged from 0.4 mg/l for ethylene glycol to 3.15 mg/l for gamma-hydroxybutyric acid. The method is linear from 0.5 to 1800 mg/l blood for ethylene glycol, from 0.7 to 1200 mg/l for lactic acid, from 1.2 to 1800 mg/l for glycolic acid, and from 3.2 to 200 mg/l for gamma-hydroxybutyric acid, with analytical recoveries, accuracy, day-to-day and within-day precision well within the required limits. Total analysis time with one calibrator was 30 min, derivatization time included. This method is very suitable for emergency toxicology, since several toxic substances can be quantified simultaneously in a fast and sensitive manner.
Repeated batch electrodialysis for lactic acid recovery was investigated using lactic acid solution and fermentation broth. In both cases, lactate fluxes averaged more than 7.0 moles/m2.h, lactate recovery reached more than 99% for all the batch runs, and specific energy consumption per unit lactate transported was lower than 0.25 kWh/kg-lactate. When electrodialysis wastewater was used as a fermentation medium, supplemented with 100 g/l glucose, up to 92.4 g/l lactic acid was produced with a productivity of 0.67 g/l.h. In addition, when electrodialysis wastewater was supplemented with 150 g/l whole-corn flour hydrolyzate and 5 g/l corn steep liquor, 2.5-fold and 1.8-fold increases in lactic acid productivity and maximum cell growth, respectively, were achieved, as compared with lactic acid fermentation using electrodialysis wastewater supplemented with glucose only.
A simple, efficient procedure for removing lactic acid and for reducing nonprotein nitrogen and ash in lactic acid whey has been developed. The procedure consists of culturing Candida ingens on the whey. This organism could assimilate >98% of the lactic acid and approximately 40% of the nonprotein nitrogen. Ash reduction of up to 45% resulted from precipitation of calcium apatite due to the increase in pH from 4.4 to approximately 8.0 which occurred during growth of C. ingens. Improved fluxes during laboratory-scale ultrafiltration were obtained for the treated lactic acid whey. C. ingens treatment of lactic acid whey appears to facilitate processing of this material to a more useful product.
We have combined automated fluorescence microscopy with a combinatorial approach for creating polymer blend gradients to yield a rapid screening method for characterizing cell proliferation on polymer blends. A gradient in polymer blend composition of poly(L-lactic acid) (PLLA) and poly(D,L-lactic acid) (PDLLA) was created in the form of a strip-shaped film and was annealed to allow PLLA to crystallize. Fourier transform infrared (FTIR) microspectroscopy was used to determine the composition in the gradients and atomic force microscopy was used to characterize surface topography. Osteoblasts were cultured on the gradients and proliferation was assessed by automated counting of cells using fluorescence microscopy. Surface roughness varied with composition, was smooth on PDLLA-rich regions and was rough on the PLLA-rich regions. Cell adhesion was similar on all regions of the gradients while proliferation was faster on the smooth, PDLLA-rich end of the gradients than on the rough, PLLA-rich end of the gradients. These results demonstrate the feasibility of a new, combinatorial approach for evaluating cell proliferation on polymer blends.
One hundred and twelve different strains of lactic acid bacteria, belonging to the genera Leuconostoc, Streptococcus, and Lactobacillus, were examined for the ability to degrade 10 organic acids by detecting gas production, using the conventional Durham tube method. All the strains did not break down succinate, glutarate, 2-oxo-glutarate, and mucate. Malate, citrate, pyruvate, fumarate, tartrate, and gluconate were variably attacked. Streptococcus cremoiris AM2, ML8, and SK11 required glucose to produce gas from citrate, whereas Leuconostoc citrovorum and Streptococcus faecalis did not. Streptococcus cremoris differed from the other streptococci in not producing gas from gluconate. From all lactic acid bacteria examined, only Lactobacillus plantarum formed gas from tartarate. Determination of acetoin and diacetyl proved to be a more reliable evidence for assessing the degradation of pyruvate, compared with detection of gas production. Homofermentative lactobacilli and Leuconostoc citrovorum produced acetoin and diacetyl from pyruvate, whereas beta-bacteria did not, a character that would be of taxonomic value. Streptobacteria degraded pyruvate in the presence of glucose with lactate as the major product together with a mean acetate of 4.1%, ethanol 7.9%, acetoin 1.7%, and diacetyl 2.6% yield on a molar basis after 60 days at 30 degrees C. L. brevis produced acetate and lactate. Formation of diacetyl from pyruvate by lactic acid bacteria may play an important role in flavour development in fermenting dairy products, especially in cheese, where lactic acid bacteria usually predominate, and pyruvate is probably excreted in the breaking down of lactose and in the oxidative deamination of alanine by the accompanying microflora.
1. The effects in the cow of intraruminal infusions of acetic acid, propionic acid or butyric acid on the secretion of the component fatty acids of the milk fat, and of these acids and of lactic acid on the composition of the blood plasma of the jugular vein, have been studied. 2. The infusion of acetic acid or butyric acid increased the yield of the C(4)-C(16) acids of milk fat but decreased the yield of C(18) acids. The infusion of propionic acid decreased the yields of all major component acids except palmitic acid and possibly lauric acid. 3. The changes in the concentrations in blood plasma of glucose and of ketone bodies were consistent with the glucogenic effect of propionic acid and the ketogenic effects of butyric acid and acetic acid. The effects of lactic acid were not consistent from cow to cow. Only with the infusion of acetic acid was a significant increase in the concentration of total volatile fatty acids in blood plasma found. Infusions of butyric acid and of propionic acid tended to depress the concentration of citric acid in the blood plasma and infusion of acetic acid increased it. No consistent effects of the infused acids on the concentration in blood plasma of esterified cholesterol, free cholesterol, triglyceride or phospholipid were observed. 4. The possibility is discussed that the effects of the infused acids on milk-fat secretion are caused through an alteration of the concentrations of precursors of milk fat in mammary arterial blood.
Microspheres of a new kind of copolymer, poly(lactic acid)-poly(ethylene glycol)-poly(lactic acid) (PLA-PEG-PLA), are proposed in the present work for clinical administration of an antineoplastic drug paclitaxel with hypothesis that incorporation of a hydrophilic PEG segment within the hydrophobic PLA might facilitate the paclitaxel release. Paclitaxel-loaded PLA-PEG-PLA microspheres of various compositions were prepared by the solvent extraction/evaporation method. Characterization of the microspheres was then followed to examine the particle size and size distribution, the drug encapsulation efficiency, the colloidal stability, the surface chemistry, the surface and internal morphology, the drug physical state and its in vitro release behavior. The effects of polymer types, solvents and drug loading were investigated. It was found that in the microspheres the PEG segment was homogeneously distributed and caused porosity. Significantly faster release from PLA-PEG-PLA microspheres resulted in comparison with the PLGA counterpart. Incorporation of water-soluble solvent acetone in the organic solvent phase further increased the porosity of the PLA-PEG-PLA microspheres and facilitated the drug release. A total of 49.6% sustained release of paclitaxel within 1 month was achieved. Potentially, the presence of PEG on the surface of PLA-PEG-PLA microspheres could improve their biocompatibility. PLA-PEG-PLA microspheres could thus be promising for the clinical administration of highly hydrophobic antineoplastic drugs such as paclitaxel.
Lipases from Rhizomucor miehei (Lipozyme IM20) and porcine pancreas (PPL) were employed as catalysts for the esterification reaction between the hydroxyl group of lactic acid and the carboxyl group of organic acids. Reactions were carried out at both shake-flask and bench-scale levels. Various parameters, such as solvent, temperature, substrate and enzyme concentrations, effect of buffer volume, buffer pH and water volume, were investigated for optimization of yields. While ethylmethyl ketone (EMK) was found to be the best solvent for shake-flask reactions, chloroform gave higher yields at bench-scale level. Detailed studies were carried out with respect to the synthesis of palmitoyl and stearoyl lactic acids. At shake-flask level, maximum yields of 37.5 and 40% were observed in case of palmitoyl and stearoyl lactic acids, respectively, with Lipozyme IM20; at bench-scale level, the maximum yields were 85.1 and 99% respectively, when PPL was employed. Of all the organic acids employed (C(2)--C(18)), only lauric, palmitic and stearic acids gave yields above 50%. At bench-scale level, PPL could be reused for up to three cycles with yields above 40%. Esters prepared were found to conform to Food Chemical Codex (FCC) specifications in terms of acid value, ester value, sodium and lactic acid contents.
Stereoisomers of lactic acid produced by lactic acid bacteria were determined by HPLC by using an enantiomeric resolution column. In general, the ratios of L-form to D-form (the type of stereoisomer) obtained were compared with those in references. Values of the type of stereoisomer of lactic acid were discussed from the viewpoint of identification of lactic acid bacteria.
When surfactant-stabilized biodegradable poly(lactic acid) (PLA) particles are injected into rats, the rate of clearance from blood is fast. The rate can be strongly reduced by using particles made from diblock copolymers of PLA and poly(ethylene oxide) (PLA-PEO), resulting in an increased duration of contact with the components of the coagulation system. Thus, possible adverse effects such as activation of the coagulation cascade could occur. In this paper, the interactions of surfactant-stabilized PLA and PLA-PEO nanoparticle suspensions with the plasma factors of the coagulation system are presented. PLA suspensions stabilized by sodium cholate (PLA-Ch) interact with thrombin, factor V and calcium ions. Formation of complexes and aggregates is induced by addition of calcium ions to PLA-Ch suspensions in the presence or in the absence of plasma. On the contrary, PLA-PEO suspensions are remarkably inert towards the coagulation factors and calcium ions, even when cholate is present. Steric repulsion owing to the high surface density of PEO is sufficient to avoid strong interations with the proteins and formation of aggregates between particles.
The amino acid composition of summary proteins in the strains 17, 33, 35 of the lactic acid bacteria cultures was studied in a nutrient medium with an automatic amino acids analyzer manufactured by the firm "Hitachi". The qualitative amino acids composition of various strains in the lactic acid bacteria cultures is identical with a total of 18 amino acids definable therein. As concerns the content of leucine, treonine, phenylalanine, isoleucine, methionine, tryptophan, arginine, glutamic and asparagic acids, as well as proline--the strains 17, 33 and 35 of the lactic acid bacteria cultures practically do not differ from one another.
Seven methods commonly used for fatty acid analysis of microorganisms and foods were compared to establish the best for the analysis of lyophilized lactic acid bacteria. One of these methods involves fat extraction followed by methylation of fatty acids, while the other methods use a direct methylation of the samples, under different operating conditions (e.g., reaction temperature and time, reagents, and pH). Fatty acid methyl esters were identified by gas chromatography-mass spectrometry and quantified by on-column capillary gas chromatography. Two reliable methods for the analysis of fatty acids in bacteria were selected and further improved. They guarantee high recovery of classes of fragile fatty acids, such as cyclopropane and conjugated acids, and a high degree of methylation for all types of fatty acid esters. These two direct methylation methods have already been successfully applied to the analysis of fatty acids in foods. They represent a rapid and highly reliable alternative to classical time- and solvent-consuming methods and they give the fatty acid profile and the amount of each fatty acid. Using these methods, conjugated linoleic acids were identified and quantified in lactic acid bacteria.
Lactic acid was added to batch very high gravity (VHG) fermentations and to continuous VHG fermentations equilibrated to steady state with Saccharomyces cerevisiae. A 53% reduction in colony-forming units (CFU) ml(-1) of S. cerevisiae was observed in continuous fermentation at an undissociated lactic acid concentration of 3.44% w/v; and greater than 99.9% reduction was evident at 5.35% w/v lactic acid. The differences in yeast cell number in these fermentations were not due to pH, since batch fermentations over a pH range of 2.5-5.0 did not lead to changes in growth rate. Similar fermentations performed in batch showed that growth inhibition with added lactic acid was nearly identical. This indicates that the apparent high resistance of S. cerevisiae to lactic acid in continuous VHG fermentations is not a function of culture mode. Although the total amount of ethanol decreased from 48.7 g l(-1) to 14.5 g l(-1) when 4.74% w/v undissociated lactic acid was added, the specific ethanol productivity increased ca. 3.2-fold (from 7.42 x 10(-7) g to 24.0 x 10(-7) g ethanol CFU(-1) h(-1)), which indicated that lactic acid stress improved the ethanol production of each surviving cell. In multistage continuous fermentations, lactic acid was not responsible for the 83% (CFU ml(-1)) reduction in viable S. cerevisiae yeasts when Lactobacillus paracasei was introduced to the system at a controlled pH of 6.0. The competition for trace nutrients in those fermentations and not lactic acid produced by L. paracasei likely caused the yeast inhibition.
The conversion of peptides to free amino acids and their subsequent utilization is a central metabolic activity in prokaryotes. At least 16 peptidases from lactic acid bacteria (LAB) have been characterized biochemically and/or genetically. Among LAB, the peptidase systems of Lactobacillus helveticus and Lactococcus lactis have been examined in greatest detail. While there are homologous enzymes common to both systems, significant differences exist in the peptidase complement of these organisms. The characterization of single and multiple peptidase mutants indicate that these strains generally exhibit reduced specific growth rates in milk compared to the parental strains. LAB can also catabolize amino acids produced by peptide hydrolysis. While the catabolism of amino acids such as Arg, Thr, and His is well understood, few other amino acid catabolic pathways from lactic acid bacteria have been characterized in significant detail. Increasing research attention is being directed toward elucidating these pathways as well as characterizing their physiological and industrial significance.
In order to realize resource recycling of kitchen waste, the enhancement of fermentative production of lactic acid (LA) by a strain of amylolytic lactic acid bacterium (ALAB) isolated from kitchen waste was investigated. A total of 6 strains of ALAB were isolated from anaerobically fermented kitchen waste. Among these isolates, the strain FH164 exhibited the highest starch-hydrolyzing rate and LA production. 32.67 g x L(-1) of LA was produced from 40.50 g x L(-1) of soluble starch by strain FH164 at pH 5.5-6.0 in 48 h of fermentation. On the basis of its morphological and physio-biochemical characteristics as well as 16S rDNA sequence, the strain FH164 was tentatively identified as Streptococcus sp.. 28.23 g x L(-1) of LA was obtained from kitchen waste by the strain FHl64 employing an open fermentation mode (substrate non-autoclaved), which was 19.2% higher than that of the fermentation without inoculum (control).
Pyroglutamic acid is present in high amounts (0.5g/ 100g) in many cheese varieties-and particularly in extensively ripened Italian cheeses such as Grana Padano and Parmigiano Reggiano. An in vivo model system for cooked mini-cheese production and ripening acceleration was set up to demonstrate the ability of thermophilic lactic acid bacteria, used as a starter, to produce pyroglutamic acid (pGlu). In mini-cheeses stored at 38 and 30 degrees C for up to 45 d, all starters tested produced different amounts of pGlu. In descending order of pGlu production, the bacteria analyzed were: Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactobacillus delbrueckii subsp. lactis. Evidence for the presence of glutamine to pGlu cyclase activity in lactic acid bacteria was provided. Cell lysates obtained from cultures of L. helveticus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, and S. thermophilus showed the ability to cyclize glutamine to pGlu, resulting in processing yields from 1.4 to 30.3%, depending on the subspecies. Formation of pGlu from free glutamine appeared to be similar to that observed using a glutamine-glutamine dipeptide substrate. Under the experimental conditions applied, pGlu aminopeptidase activity was only detected in L. helveticus. Thus, pGlu formation in long-ripened cooked cheese may depend on the activity of thermophilic lactic acid bacteria.
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