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Inhibition of reticulo-ruminal motility by volatile fatty acids and lactic acid in sheep.

1. A study was made of the influence on reticulo-ruminal motility, recorded by electromyography, of ruminal infusions of volatile fatty acids (VFAs) and lactic acid in twenty-four sheep maintained by intragastric infusion of a complete liquid diet, in three sheep fed grass pellets, and in nine chronically vagotomized sheep; abomasal and duodenal infusions of VFA and lactic acid were tested in five sheep fed grass pellets. 2. Ruminal infusions of VFAs and lactic acid progressively inhibited the amplitude of the reticulo-ruminal contractions. In many experiments there was no effect on contraction frequency until the cessation of all reticulo-ruminal contractions at which point the maximal concentration of VFA recorded in the abomasum was 28 mM, and that of lactic acid was 20 mM. 3. The concentrations of undissociated VFAs causing cessation of reticulo-ruminal contractions in the vagus-intact sheep were very similar to the concentrations causing abolition of the organized intrinsic motility of the chronically vagotomized sheep. 4. The inhibition of reticulo-ruminal motility with ruminal infusions of mixtures of VFAs and of lactic acid together with VFAs could largely be explained by the sum of the effects of the individual acids present. 5. Abomasal infusion of VFA or lactic acid inhibited the amplitude of ruminal, especially primary ruminal, contractions at concentrations of undissociated acid of 60 mM and above and increased the frequency of reticulum and primary ruminal contractions at about 80 mM. 6. Duodenal infusion of VFAs and lactic acid (100 mM, 5 ml/min) strongly inhibited abomasal motility without affecting reticulo-ruminal motility, and at a higher rate (100 mM, 10 ml/min) abolished motility and inhibited both the amplitude and frequency of reticulo-ruminal contractions. 7. It is concluded that the initial inhibition of reticulo-ruminal motility in ruminal acidosis is unlikely to involve any significant influence from duodenal, or abomasal receptors. The final cessation of reticulo-ruminal motility with ruminal acidosis could involve local effects of VFAs in the reticulo-rumen as well as through excitation of acid-sensitive reticulo-ruminal receptors.

Action Potentials↗

Characterization of yeasts with high L[+]-lactic acid production: lactic acid specific soft-agar overlay (LASSO) and TAFE-patterns.

Only few yeast strains are known for the high level production of L[+]-lactate. We report indications for the conspecifity of Kluyveromyces thermotolerans (formerly Saccharomyces veronae) strain CBS 4728 with Stamm 42 (formerly Saccharomyces pretoriensis, RADLER 1984). We suggest that Stamm 42 has little, if any relationship to Saccharomyces cerevisiae. Furthermore, we have optimized the method of Subden et al. (1982) for the detection of lactate producing microorganisms. Using this method in a screening with 100 yeast strains of our institute collection, we could not find additional strains with high L[+]-lactate production. This method may provide a useful tool for the molecular cloning of the unique yeast L[+]-LDH1) gene (s).

Blotting, Southern↗

Production of conjugated fatty acids by lactic acid bacteria.

Conjugated fatty acids have attracted much attention as a novel type of biologically beneficial functional lipid. Some isomers of conjugated linoleic acid (CLA) reduce carcinogenesis, atherosclerosis, and body fat. Considering the use of CLA for medicinal and nutraceutical purposes, a safe isomer-selective process is required. The introduction of biological reactions for CLA production could be an answer. We screened microbial reactions useful for CLA production, and found several unique reactions in lactic acid bacteria. Lactic acid bacteria produced CLA from linoleic acid. The produced CLA comprised a mixture of cis-9,trans-11-octadecadienoic acid (18:2) and trans-9,trans-11-18:2. Lactobacillus plantarum AKU 1009a was selected as a potential CLA producer. Using washed cells of L. plantarum AKU 1009a as a catalyst, CLA production from linoleic acid reached 40 mg/ml under the optimized conditions. The CLA-producing reaction was found to consist of two successive reactions, i.e., hydration of linoleic acid to 10-hydroxy-12-octadecenoic acid and dehydrating isomerization of the hydroxy fatty acid to CLA. On the basis of these results, the transformation of hydroxy fatty acids by lactic acid bacteria was investigated. Lactic acid bacteria transformed ricinoleic acid (12-hydroxy-cis-9-octadecenoic acid) to CLA (a mixture of cis-9,trans-11-18:2 and trans-9,trans-11-18:2). Castor oil, which is rich in the triacylglycerol form of ricinoleic acid, was also found to act as a substrate for CLA production by lactic acid bacteria with the aid of lipase-catalyzed triacylglycerol hydrolysis. L. plantarum AKU 1009a produced conjugated trienoic fatty acids from alpha- and gamma-linolenic acid. The trienoic fatty acids produced from alpha-linolenic acid were identified as cis-9,trans-11,cis-15-octadecatrienoic acid (18:3) and trans-9,trans-11,cis-15-18:3. Those produced from gamma-linolenic were cis-6,cis-9,trans-11-18:3 and cis-6,trans-9,trans-11-18:3. The conjugated trienoic fatty acids produced from alpha- and gamma-linolenic acid were further saturated by L. plantarum AKU 1009a to trans-10,cis-15-18:2 and cis-6,trans-10-18:2, respectively.

Fatty Acids↗

Comparative studies of lactic acid dehydrogenases in lactic acid bacteria. I. Purification and kinetics of the allosteric L-lactic acid dehydrogenase from Lactobacillus casei ssp. casei and Lactobacillus curvatus.

The stability, pH-dependence and kinetic properties of the Mn2+ and FDP-activated NAD-dependent lactic acid dehydrogenases from Lactobacillus casei ssp. casei (ATCC 393) and L. curvatus (DSM 20010) were studied after the enzymes were purified to homogeneity by affinity chromatography. Both enzymes are virtually unidirectional, catalysing efficiency only the reduction of pyruvate. They are similar with respect to the effector requirement and pH-optimum. They differ, however, in their electrophoretic mobility, heat stability, pH-dependence of the Mn2+ requirement and several kinetic properties. It is suggested that most of these differences are caused by differences of the negative charges in the vicinity of the FDP-binding site or the site responsible for the interaction of the subunits of the enzymatically active oligomeres.

Allosteric Regulation↗

The influence of a single acetic acid and lactic acid injection on rat normal thyroid tissue.

Until now, the use of acetic and lactic acid for the treatment of thyroid nodular lesions, as well as histopathologic changes in the thyroid tissue after their intrathyroidal injections has not been described. The aim of this study is to evaluate macro- and microscopic changes in rat normal thyroid tissue induced by single intrathyroidal injection of acetic acid (AA) and lactic acid (LA) in different concentrations, as well as the influence of intrathyroidal acid injection on function of the recurrent laryngeal nerves. Male Wistar rats were divided into two groups of 50 (Group A and B). Rats of each group were divided into the subgroups of five. In each subgroup of Group A and B, AA and LA water solutions of different concentrations (0%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 80%), respectively, were injected into the right thyroid lobe. From Group A and B, 7 and 8 rats died, respectively (after injections of 50-80% AA and LA solutions). After 4 weeks post AA or LA injection the thyroid along with the surrounding tissues was excised and assessed macro- and microscopically in all rats that survived. Before the injection and just before the second operation, the vocal cord function was evaluated. Our results show that both, acetic and lactic acid of different concentrations damage rat normal thyroid tissue after a single intrathyroidal injection. The minimal concentrations of acetic and lactic acid that cause lethal changes in rat normal thyroid tissue are 20% and 10%, respectively. Acetic and lactic acid in the concentrations of 40% and 50%, respectively, and higher can cause injury of the tissues adjacent to the thyroid.

Acetic Acid↗

[Experimental study of the viability of warm ischemic kidney. Relationship between ATP value in renal tissue by isotachophoresis and serum pyruvic acid to lactic acid ratio (P/L ratio)].

The viability of preserved kidney is considered to be evaluated by mitochondrial function. We used the three experimental conditions of WIT 60 minutes (group I), 90 minutes (group II), and 120 minutes (group III) using rabbit. We measured the ATP value of renal tissue as a direct method before ischemia (control), immediately before recirculation, and at one hour, two hours, one day, two days, four days and seven days after recirculation, using isotachophoresis (Tachophor LKB 2127), and as an indirect method, serum pyruvic acid, lactic acid and LDH values were measured simultaneously. The ATP value in groups I, II, and III before recirculation were 1/10 lower than controls. However, as WIT became longer, the recovery speed was diminished. The serum P/L ratio reflected the mitochondrial function in group III. (p less than 0.05) The measurement of ATP value by this method, although it is somewhat invasive, is a useful method with a simple technique and a short analysis time. The serum P/L ratio is considered not to reflect the mitochondrial function directly, but can be measured adequately in WIT 120 minutes.

Adenosine Triphosphate↗

Metabolic acidosis: separation methods and biological relevance of organic acids and lactic acid enantiomers.

Metabolic acidosis can result from accumulation of organic acids in the blood due to anaerobic metabolism or intestinal bacterial fermentation of undigested substrate under certain conditions. These conditions include short-bowel syndrome, grain overfeeding of ruminants and, as recently reported, severe gastroenteritis. Measuring fermentation products such as short-chain fatty acids (SCFAs) and lactic acid in various biological samples is integral to the diagnosis of bacterial overgrowth. Stereospecific measurement of D- and L-lactic acid is necessary for confirmation of the origin and nature of metabolic acidosis. In this paper, methods for the separation of SCFAs and lactic acid are reviewed. Analysis of the organic acids involved in carbohydrate metabolism has been achieved by enzymatic methods, gas chromatography, high-performance liquid chromatography and capillary electrophoresis. Sample preparation techniques developed for these analytes are also discussed.

Acidosis, Lactic↗

Determination of lactic acid and poly(lactic acid)s in a dermatological formulation by capillary electrophoresis.

A stability-indicating capillary electrophoresis (CE) method was developed to determine lactic acid at a level of 0.9% m/m in a dermatological formulation. This level includes up to 34% present in the form of poly(lactic acid)s. Current methods which involve treatment of samples with sodium hydroxide to hydrolyse the various lactic acid oligomers to free lactic acid cannot strictly be regarded as true measures of stability. The harsh hydrolysis conditions used can also lead to the generation of many degradation peaks from excipients such as natural extracts which can interfere with the detection of lactic acid in HPLC and CE methods. Application of indirect UV detection using 4-methoxybenzoic acid (p-anisate) as the background electrolyte and negative voltage polarity (detector towards anode) allows the direct quantification of the total available lactic acid from the monomer and the predominant linear oligomers in the raw material, i.e., the dimer (lactoyllactate) and the trimer. Tetradecyltrimethylammonium bromide was used as the electroosmotic flow modifier, lactic acid, lithium salt, as a calibration standard and butyric acid as an internal standard. A typical RSD for standard response using migration time-corrected peak area ratios was 1.17% (n = 9). Duplicate analysis of total lactic acid from two similar product placebos spiked with raw material gave average recoveries of 101.0 and 99.6%. The method, when applied to fully and partially hydrolysed raw material, showed good agreement (99.6%) with a standard titration assay for raw materials and was successfully applied to 5 month storage samples of a product. Advantages of the method include speed, simplicity, low consumption of reagents and no organic solvents.

Dermatologic Agents↗

A PCR assay for detection of acetic acid-tolerant lactic acid bacteria in acidic food products.

A PCR assay for the detection of acetic acid-tolerant lactic acid bacteria in the genera of Lactobacillus and Pediococcus was developed in this study. Primers targeting the bacterial 16S rRNA gene were newly designed and used in this PCR assay. To determine the specificity of the assay, 56 different bacterial strains (of 33 genera), 2 fungi, 3 animals, and 4 plants were tested. Results were positive for most tested bacterial members of 16S rRNA gene-based phylogenetic groups (classified in the Lactobacillus casei and Pediococcus group), including Lactobacillus fructivorans, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus plantarum, and Lactobacillus paracasei. For all other bacterial strains and eukaryote tested, results were negative. Bacterial DNA for PCR was prepared with a simple procedure with the use of Chelex 100 resin from culture after growth in deMan Rogosa Sharpe broth (pH 6.0). To test this PCR assay for the monitoring of the acetic acid-tolerant lactic acid bacteria, L. fructivorans was inoculated into several acidic food as an indicator. Before the PCR, the inoculation of 10 to 50 CFU of bacteria per g of food was followed by a 28-h enrichment culture step, and the PCR assay allowed the detection of bacterial cells. Including the enrichment culture step, the entire PCR detection process can be completed within 30 h.

Acetic Acid↗

Evaluation of clastogenicity of formic acid, acetic acid and lactic acid on cultured mammalian cells.

Using Chinese hamster ovary K1 cells, chromosomal aberration tests were carried out with formic acid, acetic acid and lactic acid, and the relationship between the pH of the medium and the clastogenic activity was examined. The medium used was Ham's F12 supplemented with 17 mM NaHCO3 and 10% fetal calf serum. All of these acids induced chromosomal aberrations at the initial pH of ca. 6.0 or below (about 10-14 mM of each acid) both with and without S9 mix. Exposure of cells to about pH 5.7 or below (about 12-16 mM of each acid) was found to be toxic. When the culture medium was first acidified with each of these acids and then neutralized to pH 6.4 or pH 7.2 with NaOH, no clastogenic activity was observed. Using F12 medium supplemented with 34 mM NaHCO3 as a buffer, no clastogenic activity was observed at doses up to 25 mM of these acids (initial pH 5.8-6.0). However, it was found that about 10% of the cells had aberrations at pH 5.7 or below (27.5-32.5 mM of each acid). Furthermore, when 30 mM HEPES was used as a buffer, chromosomal aberrations were not induced at doses up to 20 mM formic acid and acetic acid (initial pH 7.0-7.1), and at doses up to 30 mM lactic acid (initial pH 6.6). In the initial pH range of 6.4-6.7 (25-32.5 mM of each acid), chromosomal aberrations were observed. The above results show that these acids themselves are non-clastogenic, and the pseudo-positive reactions attributable to non-physiological pH could be eliminated by either neutralization of the treatment medium or enhancement of the buffering ability.

Acetates↗

Degradation of polydispersed poly(L-lactic acid) to modulate lactic acid release.

Polydispersed poly(L-lactic acid) (PLLA) membranes comprised of blends of monodispersed PLLA of weight average molecular weight of 82,500 and 7600 were fabricated to investigate the effect of polydispersity on degradation characteristics. The PLLA blends exhibited large spherulites of high molecular weight chains embedded in a low molecular weight matrix. During degradation in phosphate buffer at pH 7.4 and 37 degrees C for 28 d, the release rate of lactic acid increased as the percentage of the low molecular weight component in the blend was increased. For low molecular weight compositions larger than 50%, voids were created in the degrading blends due to the degradation of low molecular weight chains and the concurrent dissolution of lactic acid, and also the release of undegraded particles of high molecular weight. These studies demonstrate the feasibility of modulating lactic acid release during in vivo degradation of PLLA implants by adjusting the polymer polydispersity.

Calorimetry, Differential Scanning↗

The effect of dietary fatty acids on lactic acid bacteria associated with the epithelial mucosa and from faecalia of Arctic charr, Salvelinus alpinus (L.).

Arctic charr, Salvelinus alpinus (L.), held in fresh water, were fed four experimental diets containing different polyunsaturated fatty acids (PUFA). In addition, one group fed a diet containing only coconut oil as sole lipid source served as control. The population of aerobic heterotrophic bacteria associated with the epithelial mucosa and the faecalia was estimated using the dilution plate technique. Generally, the population level of adherent bacteria increased along the digestive tract (stomach, small intestine and large intestine). Adherent Gram-negative and Gram-positive bacteria seemed to be present at equal levels in all parts of the alimentary tract. Lactic acid bacteria dominated among the Gram-positive bacteria, and they were detected in all regions of fish fed the PUFA supplemented diets. The frequency of lactic acid bacteria was highest in the digestive tract of fish fed diets with added 7.0% linolenic acid (18:3 n-3) or 4% of a PUFA mix. A lower frequency of lactic acid bacteria was found in fish fed dietary linoleic acid (18:2 n-6), and they were absent or present in low numbers in fish fed the coconut oil diet. It is suggested that dietary fatty acids affect the attachment sites for the gastrointestinal microbiota, possibly by modifying the fatty acid composition of the intestine wall. Numerical taxonomy procedures showed that the lactic acid bacteria Carnobacterium spp. and a Carnobacterium piscicola-like strain were predominant, with smaller numbers of Lactobacillus plantarum, Streptococcus spp. and Leuconostoc mesenteroides present. Seven strains of Carnobacterium spp. were further identified on the basis of 16S rDNA sequence analysis, and all these strains were identified as Carnobacterium piscicola.

Animal Feed↗

The effect of poly (aspartic acid-co-lactic acid) nanospheres on the lung metastasis of B16BL6 melanoma cells by intravenous administration.

Poly (aspartic acid-co-lactic acid) (PAL) has been investigated as a new biodegradable material for Drug Delivery Systems (DDS). Similar to the poly (lactic acid-co-glycolic acid) (PLGA) nanospheres, the PAL nanospheres can control-release encapsulated drugs by hydrolysis and adhere to the mucous membranes to improve the drug absorption. In this study, the vitamin encapsulated PAL nanospheres were applied on mice to examine their effect on tumor metastasis and safety as an injectable DDS material for anti-cancer and other drugs. In the experiment, 6 C57BL/6 mice per group were intravenously administered with B16BL6 melanoma cells (1 x 10(5) per mouse) and non-encapsulated PAL nanospheres or pro-vitamin encapsulated nanospheres respectively, while the control group was administered with B16BL6 cells alone. Two weeks later, the lungs of the mice were excised and metastatic foci on the lung surface were counted. The melanoma cell metastasis to lungs was prevented by intravenous co-injection of B16BL6 melanoma cells with 1.7 microg of pro-vitamin E encapsulated PAL nanospheres. Its metastatic foci count (mean +/- SD) was 127+/-80, which was better than the control (246+/-95, p<0.02). Also, applying the pro-vitamin C and pro-vitamin A encapsulated PAL nanospheres as well as the non-encapsulated PAL nanospheres slightly decreased the number of metastasis colonies in the lungs as compared to that of the control. These results suggested that PAL nanospheres did not promote the lung metastasis of B16BL6 melanoma cells. Thus, the PAL nanospheres are safe material for injection applications.

Animals↗

HPLC analysis of organic acids in lactic acid fermented vegetables.

An HPLC technique is described for the analysis of organic acids in lactic acid fermented vegetables. An Aminex HPX-87 column with 0.013 M-H2SO4 as mobile phase was used. Separation data are presented for oxalic, citric, tartaric, malic, succinic, lactic, formic, acetic, propionic, and butyric acids. Interference problems from sugars are also reported.

Bacteria↗

Osteoinductive potential of freeze-dried, biodegradable, poly (glycolic acid-co-lactic acid) disks incorporated with bone morphogenetic protein in skull defects of rats.

Biodegradable, porous, polymer implant disks with an osteoinductive potential were prepared by a freeze-drying technique by incorporating bovine bone morphogenetic protein (BMP) in poly (glycolic acid-co-lactic acid) (PGLA). The PGLA disks with and without BMP were implanted in rat skull defects, and the defect sites were studied radiographically and histologically for 2 and 4 weeks after implantation. A quantitative radiographic analysis showed significantly thicker radiopacity for the disks with BMP than those without BMP (P < 0.01). After 2 weeks, chondrogenesis and new bone formation were observed on the BMP-incorporated disks. After 4 weeks, PGLA was completely replaced by new bone in the defects with implantation of the BMP-incorporated disks, whereas the defects implanted with PGLA alone were filled with fibrous connective tissue. The results suggest that BMP-incorporated PGLA is an ideal bone substitute with osteoinductive potential.

Animals↗

The metabolism of several carboxylic acids by lactic acid bacteria.

The anaerobic metabolism of citrate, fumarate, gluconate, malate, 2-oxoglutarate and pyruvate by 137 strains of 23 species of lactic acid bacteria was investigated. The bacteria were from various sources (plant material, meat and dairy products, dough and wine) and belonged to the genera Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus. The ability of metabolize the acids was determined by thin layer chromatography or by enzymatic analysis after growth of the strains in a glucose-containing medium. All strains metabolized pyruvate and only 12 mainly heterofermentative strains were malate negative. These strains were also unable to decompose citrate. This acid was fermented by 23 strains, all of which metabolized malate. Many lactic acid bacteria reduced 2-oxoglutarate to hydroxyglutarate. The strains of Lactobacillus plantarum did not metabolize 2-oxoglutarate whereas all strains of Leuconostoc oenos decarboxylated this acid and formed 4-hydroxybutyrate and succinate. Gluconate was fermented by 52 mainly heterofermentative strains. No correlation was observed between the ability to ferment citrate, malate or gluconate.

Bacteria↗