Search

Goals The aims of this study were to isolate, to identify, and to characterize new potential probiotic strains from the feces of Chinese neonates. Background Probiotic strains approved in China for use in infants were declared to originate from the human gut of Western subjects. Diet is listed among the main factors affecting the composition of the human gut along with other factors such as genetics, lifestyle, and health status. On the basis of this, the lifestyle of mothers, including dietary habits, could have an impact on the bacterial strains that colonize the gut of their babies. Study Starting from fecal samples, plated onto selective media, of 26 babies, a total of 38 Lactobacillus and 45 Bifidobacterium colonies were isolated and subcultured, identified at the specie level with the partial sequencing of the 16S ribosomal RNA gene, and assessed for safety according to international guidelines for probiotics and European guidance. Only 6 Lactobacillus and 5 Bifidobacterium spp. were included for further analysis for the evaluation of survival rate within the gastrointestinal tract and for adhesive properties on the Caco-2 cell line. Some tests for prebiotic metabolism and growth on reconstituted skimmed milk were also performed. Results Three Lactobacillus strains and 1 Bifidobacterium strain showing interesting adhesive abilities were included in the in vitro immune-stimulatory test with dendritic cells. Among these isolates, the Bifidobacterium breve 2TA showed the most interesting probiotic properties. Conclusions The results obtained led to the identification of 4 new potential probiotic strains from Chinese babies to be submitted to further investigations about their metabolic and functional features.

Purpose Subclinical vitamin D (vitD) deficiency enhances the predisposition to a myriad of acute and chronic pathologies in many people worldwide. Due to the scarcity of vitD-rich foods, the consumption of supplements or fortified foods can be required to maintain healthy serum levels of 25-hydroxyvitamin D [25(OH)D], and the major circulating form of vitD that is commonly measured in serum to determine the vitD status. Since the vitD absorption seems to resemble that of lipids, improved emulsification in the gut could favor vitD permeation through the enterocyte membrane. Contextually, we hypothesized that a microorganism with cholecalciferol (vitD3)-solubilization properties may potentially result in enhanced serum vitD levels. Methods and results Six probiotic strains were screened for their ability to create a stable suspension of vitD3 in water: Lacticaseibacillus paracasei DG, L. paracasei LPC-S01, L. paracasei Shirota, L. rhamnosus GG, Limosilactobacillus reuteri DSM 17938, and Lactobacillus acidophilus LA5. The DG strain displayed the strongest vitD3 solubilization ability and, consequently, were used in an in vivo trial where a commercial preparation of vitD3 in refined olive oil was administered by gavage to CD-1 mice with or without the concurrent administration of L. paracasei DG. ELISA measurements showed that the DG strain significantly increased the serum levels of 25(OH) D when administered once a day for 1 week in association with the vitD3 supplement. Conclusion This preliminary pre-clinical study suggests that the combined administration of L. paracasei DG with an oil-based cholecalciferol supplement could contribute to the maintenance of the adequate 25(OH) D serum levels in people at risk of vitD deficiency.

The human intestinal tract is colonized by a resilient integrated ecosystem represented by a complex consortium of trillions of microbes [...]

The growing body of clinical and experimental data support the view that the efficacy of probiotics is strain-specific and disease-specific. Thus, newly isolated probiotic strains should be disease- targeted. Therefore, we firstly characterized a novel strain of Lactobacillus reuteri LMG P-27481 for safety (sensitivity to antibiotics and genome analysis) and putative efficacy (resistance to gut transit, adhesiveness, induction of cytokines, release of antimicrobial metabolites) by the conventional in-vitro experimental approach proposed to identify beneficial microbes by national and international guidelines. Afterward, LMG P-27481 was submitted to in-vitro assays targeted on diarrhea (lactose utilization, inhibition of pathogens such as bacteria and Rotavirus) revealing a stronger ability, compared to well-known reference strains, to antagonize Clostridium difficile (CD). Final confirmation of LMG P-27481 features in controlling CD colitis was achieved by an in-vivo trial on mice conditioned with large spectrum antibiotics before receiving CD challenge. Two out of the 3 antibiotic-treated groups received daily the probiotic strain LMG P-27481 with different duration in order to simulate a preventive approach (probiotic administered prior to CD challenge) or an antagonistic approach (probiotic administered after CD challenge). LMG P-27481 administration, with either protocols, significantly reduced CD DNA concentration in caecum and C. difficile toxins titer in the gut lumen, as compared to untreated controls. Moreover, LMG P-27481 supplementation significantly mitigated body weight loss, extent of inflammatory infiltrate and tissue damage. Despite the requirement for further in-vivo assessments on humans, the selection and characterization process targeted to diarrhea and CD infection, described in this study, identify the strain L. reuteri LMG P-27481 as a promising probiotic candidate in the course of CD infection.

Gut microorganisms (microbiota) live in symbiosis with the host and influence human nutrition, metabolism, physiology, and immune development and function. The microbiota prevents pathogen infection to the host, and in turn the host provides a niche for survival. The alteration of gut bacteria composition (dysbiosis) could contribute to the development of immune-mediated diseases by influencing the immune system activation and driving the pro- and anti-inflammatory responses in order to promote or counteract immune reactions. Probiotics are nonpathogenic microorganisms able to interact with the gut microbiota and provide health benefits; their use has recently been exploited to dampen immunological response in several experimental models of autoimmune diseases. Here, we focus on the relationships among commensal bacteria, probiotics, and the gut, describing the main interactions occurring with the immune system and recent data supporting the clinical efficacy of probiotic administration in rheumatoid arthritis, multiple sclerosis, and myasthenia gravis (MG) animal models. The encouraging results suggest that selected strains of probiotics should be evaluated in clinical trials as adjuvant therapy to restore the disrupted tolerance in myasthenia gravis.

The Lactobacillus vaginalis LMG S-26419 strain, also named CBA-L88 (BV2), was isolated at the AAT-Advanced Analytical Technologies laboratories from a vaginal swab obtained from a healthy woman. The total genome size is 1,806,242 bp with a G+C content of 40.6%.

Gut microorganisms (microbiota) live in symbiosis with the host and influence human nutrition, metabolism, physiology, and immune development and function. The microbiota prevents pathogen infection to the host, and in turn the host provides a niche for survival. The alteration of gut bacteria composition (dysbiosis) could contribute to the development of immune-mediated diseases by influencing the immune system activation and driving the pro- and anti-inflammatory responses in order to promote or counteract immune reactions. Probiotics are nonpathogenic microorganisms able to interact with the gut microbiota and provide health benefits; their use has recently been exploited to dampen immunological response in several experimental models of autoimmune diseases. Here, we focus on the relationships among commensal bacteria, probiotics, and the gut, describing the main interactions occurring with the immune system and recent data supporting the clinical efficacy of probiotic administration in rheumatoid arthritis, multiple sclerosis, and myasthenia gravis (MG) animal models. The encouraging results suggest that selected strains of probiotics should be evaluated in clinical trials as adjuvant therapy to restore the disrupted tolerance in myasthenia gravis.

Probiotics beneficial effects on the host are associated with regulation of the intestinal microbial homeostasis and with modulation of inflammatory immune responses in the gut and in periphery. In this study, we investigated the clinical efficacy of two lactobacillus and two bifidobacterium probiotic strains in experimental autoimmune myasthenia gravis (EAMG) and experimental autoimmune encephalomyelitis (EAE) models, induced in Lewis rats. Treatment with probiotics led to less severe disease manifestation in both models; ex vivo analyses showed preservation of neuromuscular junction in EAMG and myelin content in EAE spinal cord. Immunoregulatory transcripts were found differentially expressed in gut associated lymphoid tissue and in peripheral immunocompetent organs. Feeding EAMG animals with probiotics resulted in increased levels of Transforming Growth Factor-β (TGFβ) in serum, and increased percentages of regulatory T cells (Treg) in peripheral blood leukocyte. Exposure of immature dendritic cells to probiotics induced their maturation toward an immunomodulatory phenotype, and secretion of TGFβ. Our data showed that bifidobacteria and lactobacilli treatment effectively modulates disease symptoms in EAMG and EAE models, and support further investigations to evaluate their use in autoimmune diseases.

Background&aims The classical ketogenic diet (KD) is a high-fat, very low-carbohydrate normocaloric diet used for drug-resistant epilepsy and Glucose Transporter 1 Deficiency Syndrome (GLUT1 DS). In animal models, high fat diet induces large alterations in microbiota producing deleterious effects on gut health. We carried out a pilot study on patients treated with KD comparing their microbiota composition before and after three months on the diet. Methods Six patients affected by GLUT1 DS were asked to collect fecal samples before and after three months on the diet. RT – PCR analysis was performed in order to quantify Firmicutes, Bacteroidetes, Bifidobacterium spp. , Lactobacillus spp. , Clostridium perfringens, Enterobacteriaceae, Clostridium cluster XIV, Desulfovibrio spp. and Faecalibacterium prausnitzii. Results Compared with baseline, there were no statistically significant differences at 3 months in Firmicutes and Bacteroidetes. However fecal microbial profiles revealed a statistically significant increase in Desulfovibrio spp. (p = 0.025), a bacterial group supposed to be involved in the exacerbation of the inflammatory condition of the gut mucosa associated to the consumption of fats of animal origin. Conclusions A future prospective study on the changes in gut microbiota of all children with epilepsy started on a KD is warranted. In patients with dysbiosis demonstrated by fecal samples, it my be reasonable to consider an empiric trial of pre or probiotics to potentially restore the «ecological balance» of intestinal microbiota.

The probiotic product consumption has recently increased with the prevalent intent to promote human and animal wellbeing. The complex selection process dealing with new-isolated probiotic candidates is the first challenge that has to be faced. From the isolation to the launch on the market, information about safety, tolerance to host physiological conditions, adhesion properties, genetics and interaction with the host has to be collected. Probiotics must be safe, survive to the exposition to bile salts and to gut transit, adhere to intestinal cells lining and colonize the lumen of the tract. The evaluation process of the possible probiotic health benefits is widely supported by in-vitro assays simulating the in-vivo conditions. The aim of this work is to summarize the classical models usually employed for the probiotic screening by underlying strengths and weaknesses of all models and to present some more recent analysis tools used in the probiotic field. The long term goal in new probiotic candidate selection experiencing these combined essays together would lead to the hypothetical assignment acknowledged as one strain-one function.

Biocides are antimicrobial compounds that are widely used in the food industry and medical environments. In this study, we determined the Minimum Inhibitory Concentrations (MICs) by the microdilution method of four different biocides for a large collection of LAB of different origins. The tested isolates belong to 11 species of the Lactobacillus genus and to Streptococcus thermophilus, Streptococcus salivarius and Lactococcus garvieae. The results obtained in this study indicate that low susceptibilities to benzalkonium chloride (BC), triclosan (Tr), chlorhexidine (Ch), and sodium hypochlorite (SH) are not frequent among LAB. Moreover, no systematic co-tolerance between two or more tested biocides was found; that is, strains displaying high MIC values and thus low sensitivity to one of the biocides did not show higher MIC values for the other biocides.

Accumulating evidence indicates that the peroxisome proliferator activated receptor (PPAR)-gamma is a major player in maintaining intestinal mucosa homeostasis, but whether PPAR-gamma is directly involved in probiotic-mediated effects and the molecular events involved in its activation are not known.We investigated the role of PPAR-gamma in the immunomodulatory effects of Lactobacillus crispatus M247 on intestinal epithelial cells (IEC) and the role of probiotic-derived H(2)O(2) on PPAR-gamma activity.L crispatus M247 supplementation in mice significantly increased PPAR-gamma levels and transcriptional activity in the colonic mucosa. L crispatus M247 induced PPAR-gamma nuclear translocation and enhanced transcriptional activity in epithelial (CMT-93) cells, as demonstrated by the increased luciferase activity of a PPAR-gamma-responsive element, PPAR-gamma-responsive gene up-regulation, and reduced activity of an nuclear factor-kappaB-responsive element. Pharmacologic PPAR-gamma inhibition or silencing by small interfering RNA cancelled the L crispatus M247-mediated effects in CMT-93 cells. Because Lactobacillus strains producing little H(2)O(2) failed to activate PPAR-gamma, we investigated the role of L crispatus M247-derived H(2)O(2) in PPAR-gamma activation. L crispatus M247 induced a transient rise in intracellular H(2)O(2) and PPAR-gamma transcriptional activity was cancelled by antioxidant or H(2)O(2) scavenger. Toll-like receptor (TLR)-2 was not required for PPAR-gamma up-regulation mediated by L crispatus M247 in mice, although the protective effects of L crispatus M247 on dextran sodium sulfate-induced colitis were less pronounced in TLR-2(-/-) mice.L crispatus M247 uses H(2)O(2) as a signal transducing molecule to induce PPAR-gamma activation in IEC, directly modulating epithelial cell responsiveness to inflammatory stimuli.

Aggregation process affects the ability of Lactobacillus crispatus, a probiotic, to survive into the gastro-intestinal environment and to adhere to the intestinal mucosa. To elucidate mechanisms underlying this process, a comparative proteomic study was carried out on a wild type strain M247 and its spontaneous isogenic mutant Mu5, which had lost the aggregative phenotype. Results highlighted an overall lower amount of enzymes involved in carbohydrate transport and metabolism in strain M247 compared to strain Mu5, suggesting a reduction in the general growth rate, probably caused by nutrient limitation in cell aggregates, coherently with the phenotypic traits of the strains. Moreover, the up-regulation of a putative elongation factor Tu in the wild type M247 strain could suggest a role of this particular protein in the adhesion mechanism of L. crispatus.

Aggregation-promoting factor (APF) was originally described as a protein involved in the conjugation and autoaggregation of Lactobacillus gasseri 4B2, whose corresponding apf gene was cloned and sequenced. In this report, we identified and sequenced an additional apf gene located in the region upstream of the previously published one. Inactivation of both apf genes was unsuccessful, indicating that APF function may be essential for the cell. Overproduction of APF proteins caused drastic alteration in the cell shape of this strain. These cells were irregular, twisted, enlarged, and tightly bound in unbreakable clumps of chains. Down-regulation of APF synthesis was achieved by cloning of the apf2 promoter region on a high-copy-number plasmid, which recruited a putative apf activator. As a consequence, the shape of the corresponding recombinant cells was elongated (filamentous) and cell division sites were no longer visible. None of the induced changes in APF production levels was clearly correlated with modifications of the aggregation phenotype. This report shows, for the first time, that APF proteins are mainly critical for L . gasseri 4B2 cell shape maintenance.

A completely chemically-defined growth medium, containing guanine, thymine, cytidine, 2'-deoxyadenosine and 2'-deoxyuridine as DNA precursors, was developed for Lactobacillus johnsonii, on the basis of statistically designed techniques suitable for other lactobacilli. Particular focus was given to the nucleotide composition of different defined media, and to the specific nucleotide requirements of Lact. johnsonii. Most of the lactobacilli tested grew in a medium containing five free bases, four ribonucleosides or five deoxyribonucleosides. Adenine and guanine were replaceable by inosine. The requirement for thymine and cytosine was satisfied with uracil. The presence of inosine and uracil was identified as being essential for the growth of different Lactobacillus species, displaying their inability to synthesize purines and pyrimidines de novo. Defined recipes with different nucleotide composition were used to investigate iron requirements of lactobacilli. Only marginal differences in growth were observed in iron-depleted media supplemented with five free bases, four ribonucleosides or five deoxyribonucleosides; iron depletion had a greater effect on growth when inosine and uracil were supplied as the only nucleotide sources. The results suggest that iron plays a role in the pyrimidine and purine metabolism of lactobacilli. Lactobacillus spp., particularly Lact. johnsonii, require iron under particular environmental conditions with limited or specific nucleotide sources.

Afin de comprendre la croissance, la stabilite et le comportement de lactobacilles lactiques dans certaines conditions de culture, nous avons etudie l'impact de differents traitements stressants sur la survie des souches selectionnees. Le traitement par la chaleur ou par un choc osmotique sublethal augmente la resistance a une lyophilisation ou des conditions de stockage ulterieurs. L'operon codant pour le chaperon grosES/grosEL a ete mis en evidence dans ces souches. De plus un second operon (dnaK, dnaJ, grpE and hcrA) a aussi ete detecte et un modele de regulation adapte. Nous avons etudie l'activite de transcription des deux chaperons principaux en reponse a differents stress (Dot-Blot et Northern-Blot). L'induction maximale du chaperon n'est pas directement correlee a une resistance accrue a la lyophilisation et aux conditions de stockage. Les premiers resultats suggerant une viabilite accrue des souches investiguees sous certaines conditions de stress semblent tres prometteurs. Nous avons observe que l'induction de proteines de stress pourrait augmenter la survie de souches bacteriennes (surtout des lactobacilles) utilisees comme levains dans une grande variete de fermentations. Par consequent l'evaluation des bases genetiques de ce comportement physiologique necessite de plus amples recherches.

Chemically undefined substances, like yeast extract or peptones, are commonly used as milk supplements in industrial fermentations to allow the growth of bacterial strains unable to grow in pure milk. In order to look for substances to replace their positive effects on the bacterial growth, the requirements of the probiotic strain L. johnsonii NCC 533 (La1) in skim and whole fat UHT milk were investigated. A mixture of 20 amino acids was added to skim milk and the removal of single amino acids identified alanine, serine, isoleucine and cysteine as the most important amino acids for growing L. johnsonii in milk. The requirements of L. johnsonii NCC 533 (La1) for nucleotide precursors were also investigated. Supplementation of milk with the four identified amino acids, four ribonucleosides and one source of iron was able to support the growth of the tested strain in milk with cell counts and final pH as obtained by the addition of yeast extract. The impact of milk supplementation was also investigated for additional 10 lactobacilli and a positive effect on the growth could be demonstrated for other L. johnsonii strains, a probiotic L. acidophilus strain and the L. gallinarum type strain.

Disinfectants have been used in a variety of environmental applications, in products for personal care and in the food industry. The food industry has increased the use of biocides and chemical-based disinfectants to control microbial ecology at production sites in an effort to improve hygiene measures and food safety. However, the susceptibility profile of micro-organisms to disinfectants has been largely neglected. This study therefore aimed to provide this type of information by focusing on the four most commonly used biocides in the food industry, determining their minimum inhibitory concentrations (MICs) and analysing the distribution of MICs across a variety of micro-organisms. In total, 99 different strains of Bifidobacterium spp. were studied. Results showed a unimodal distribution of MICs for chlorhexidine, triclosan (Irgasan) and sodium hypochlorite with no apparent species-specific correlation. Conversely, part of the tested bifidobacteria population (20%) showed reduced susceptibility to benzalkonium chloride compared with the susceptibility exhibited by the majority of the tested bacterial community. The highest MICs were distributed among almost all of the considered Bifidobacterium spp. In generally, the sensitivity of the studied strains to the four tested biocides appeared to be a genus-related trait.

Goal: To assess the effects of the consumption of psyllium seed husk on fecal bifidobacteria in healthy women and the ability of fecal bifidobacteria to metabolize psyllium seed husk in vitro. Background: Poor microbiologic evidences are nowadays available concerning the ability of psyllium seed husk to promote the growth of bifidobacteria in human gut. Study: Eleven healthy women consumed 7.0 g/d of psyllium seed husk for 1 month. Viability of bifidobacteria in feces was assessed at different time points. Results: In vivo results showed that the average fecal content of viable bifidobacteria was not significantly affected even if fecal counts were found to increase significantly after treatment in 6 out of 11 women having low initial concentration. In vitro trials conducted on bifidobacteria strains isolated from treated women failed to confirm the prebiotic potential of undigested psyllium seed husk, whereas treatment with simulated gastric and pancreatic juices and mimicking physical and chemical alterations during human gut transit allowed fecal Bifdobacterium isolates to metabolize psyllium seed husk as carbon source in a growth medium deprived of sugar. Conclusions: Psyllium seed husk can be metabolized by bifidobacteria only after partial hydrolysis. Bifidogenic potential can be detected in healthy women only in case of low level of fecal bifidobacteria before treatment.

The colonic microbiota is a major modulator of the mucosal immune system; therefore, its manipulation through supplementation with probiotics may significantly affect the host's immune responses. Since different probiotics seem to exert various effects in vivo, we tested the relevance of the autoaggregation phenotype on the intestinal persistence of lactobacilli and their ability to modulate the host's innate immune responses. After 14 days of diet supplementation, the aggregating strain Lactobacillus crispatus M247 but not aggregation-deficient isogenic mutant MU5 was recovered from the feces and colonic mucosa of mice. This observation was confirmed by strain-specific PCR amplification and by Lactobacillus -specific denaturing gradient gel electrophoresis analysis. Indeed, L. crispatus M247 increased Toll-like receptor 2 (TLR2) mRNA levels, while it reduced TLR4 mRNA and protein levels in the colonic mucosa, whereas MU5 was ineffective. In colonic epithelial cells (CMT-93 cells) L. crispatus M247 but not MU5 induced time-dependent extracellular signal-regulated kinase-1 (ERK1) tyrosine phosphorylation and TLR modulation, which were abolished in the presence of PD98059 (an ERK1 inhibitor). To assess the functional relevance of probiotic-induced TLR modulation, we determined the consequences of L. crispatus preexposure on TLR4 (lipopolysaccharide [LPS]) and TLR2 [Pam 3 Cys-Ser-(Lys) 4 ] ligand-mediated effects in intestinal epithelial cells. Preexposure to L. crispatus M247 blunted LPS-induced interleukin-6 (IL-6) release and inhibition of CMT-93 migration over a wound edge, whereas it enhanced TLR2-mediated IL-10 up-regulation. In summary, the aggregation phenotype is required for L. crispatus persistence in the colon and for modulation of TLR2/TLR4 expression through an ERK-dependent pathway. We speculate that the aggregation phenotype in L. crispatus M247 is required to temper epithelial cell responsiveness to bacterial endotoxins, which thus affects the evolution of intestinal inflammatory processes.

Whether Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus can be recovered after passage through the human gut was tested by feeding 20 healthy volunteers commercial yogurt. Yogurt bacteria were found in human feces, suggesting that they can survive transit in the gastrointestinal tract.

We tested the therapeutic relevance of auto aggregation in lactobacilli by comparing the effect on DSS induced colitis of viable Lactobacillus crispatus M247, isolated from healthy humans, to L. crispatus MU5, an isogenic spontaneous mutants of M247, the latter lacking the auto aggregation phenotype which allows the adhesion to human mucus. Aggregating L. crispatus M247, but not the non-aggregating MU5, was retrievable from mice feces and adherent to the colonic mucosa. Daily administration of L. crispatus M247, but not heat killed L. crispatus M247 or aggregation deficient L. crispatus MU5, dose-dependently reduced the severity of DSS colitis. Indeed, L. crispatus MU5 administered in a 30% sucrose solution, known to restore the aggregation phenotype, had a protective effect comparable to mice receiving L. crispatus M247. These results indicate that a surface-mediated property such as aggregation may play a pivotal role in the protective effects obtained by dietary supplementation with L. crispatus M247 during colitis.

One hundred and six isolates of the genus Bifidobacterium, isolated from different environments (mainly gastrointestinal), were identified and classified taxonomically to species level by amplified ribosomal DNA restriction analysis. Two restriction endonucleases (Sau3AI and BamHI) were chosen for aligning the 16S rRNA sequences of 16 bifidobacterial species retrieved from various databases, to obtain species-specific restriction patterns. A rapid and accurate identification scheme was obtained by comparing the resulting 16S rDNA digestion profiles of 16 Bifidobacterium type-strains and 90 strains of various origins. All of the investigated strains were previously confirmed at the species level as belonging to the genus Bifidobacterium by fluorescence in-situ hybridisation and by polymerase chain reaction amplification with genus- and species-specific primers. The present work demonstrates that species-specific detection of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium coryneforme, Bifidobacterium cuniculi, Bifidobacterium dentium, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium suis, Bifidobacterium magnum, Bifidobacterium pseudolongum, Bifidobacterium pseudocatenulatum and Bifidobacterium pullorum present in different micro-ecological environments (e.g. gastrointestinal tract) can be accomplished in a reliable, rapid and accurate manner, circumventing the recognised deficiencies of traditional identification techniques.

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.