Cholesterol removal by some lactic acid bacteria that can be used as probiotic

In the present study, the relationship between exopolysaccharide production and cholesterol removal rates of five strains of Lactobacillus delbrueckii subsp. bulgaricus isolated from home‐made yoghurt was studied. Test strains were selected according to their exopolysaccharide production capacity. Influence of different bile concentrations on cholesterol removal was investigated. It was confirmed that B3, ATCC 11842 and G11 strains which produce high amounts of exopolysaccharide (211, 200 and 159 mg/l, respectively) were able to remove more cholesterol from the medium compared to those that produce low amounts of exopolysaccharide (B2, A13). The highest cholesterol removal (31%) was observed by strain L. delbrueckii subsp. bulgaricus B3, producing a high amount of exopolysaccharide, in 3 mg/ml bile concentration. Cholesterol removal by resting and dead cells was investigated and it was found to be 4%–14% and 3%–10%, respectively. Cholesterol removal by immobilized and free cells of the B3 strain was studied and it was determined that immobilized cells are more effective. Influence of cholesterol on exopolysaccharide production has also been tested and it was found that cholesterol increased the production of EPS. The results indicated that: (i) there is a correlation between cholesterol removal and EPS production; and (ii) L. delbrueckii subsp. bulgaricus B3 is regarded as a suitable candidate probiotic and adjunct culture.     

The cell membrane and the struggle for life of lactic acid bacteria

The major life-threatening event for lactic acid bacteria (LAB) in their natural environment is the depletion of their energy sources and LAB can survive such conditions only for a short period of time. During periods of starvation LAB can exploit optimally the potential energy sources in their environment usually by applying proton motive force generating membrane transport systems. These systems include in addition to the proton translocating F_oF₁-ATPase: a respiratory chain when hemin is present in the medium, electrogenic solute uptake and excretion systems, electrogenic lactate/proton symport and precursor/ product exchange systems. Most of these metabolic energy-generating systems offer as additional bonus the prevention of a lethal decrease of the internal and external pH. LAB have limited biosynthetic capacities and rely heavily on the presence of essential components such as sources of amino acids in their environment. The uptake of amino acids requires a major fraction of the available metabolic energy of LAB. The metabolic energy cost of amino acid uptake can be reduced drastically by accumulating oligopeptides instead of the individual amino acids and by proton motive force-generating efflux of excessively accumulated amino acids. Other life-threatening conditions that LAB encounter in their environment are rapid changes in the osmolality and the exposure to cytotoxic compounds, including antibiotics. LAB respond to osmotic upshock or downshock by accumulating or releasing rapidly osmolytes such as glycine-betaine. The life-threatening presence of cytotoxic compounds, including antibiotics, is effectively counteracted by powerful drug extruding multidrug resistance systems. The number and variety of defense mechanisms in LAB is surprisingly high. Most defense mechanisms operate in the cytoplasmic membrane to control the internal environment and the energetic status of LAB. Annotation of the functions of the genes in the genomes of LAB will undoubtely reveal additional defense mechanisms.     

Cholesterol assimilation by lactic acid bacteria and bifidobacteria isolated from the human gut

The objective of this study was to evaluate the effect of human gut-derived lactic acid bacteria and bifidobacteria on cholesterol levels in vitro. Continuous cultures inoculated with fecal material from healthy human volunteers with media supplemented with cholesterol and bile acids were used to enrich for potential cholesterol assimilators among the indigenous bacterial populations. Seven potential probiotics were found: Lactobacillus fermentum strains F53 and KC5b, Bifidobacterium infantis ATCC 15697, Streptococcus bovis ATCC 43143, Enterococcus durans DSM 20633, Enterococcus gallinarum, and Enterococcus faecalis. A comparative evaluation regarding the in vitro cholesterol reduction abilities of these strains along with commercial probiotics was undertaken. The degree of acid and bile tolerance of strains was also evaluated. The human isolate L. fermentum KC5b was able to maintain viability for 2 h at pH 2 and to grow in a medium with 4,000 mg of bile acids per liter. This strain was also able to remove a maximum of 14.8 mg of cholesterol per g (dry weight) of cells from the culture medium and therefore was regarded as a candidate probiotic.     

Myosin-cross-reactive antigens from four different lactic acid bacteria are fatty acid hydratases

The 67 kDa myosin-cross-reactive antigen (MCRA) is a member of the MCRA family of proteins present in a wide range of bacteria and was predicted to have fatty acid isomerase function. We have now characterised the catalytic activity of MCRAs from four LAB stains, including Lactobacillus rhamnosus LGG, L. plantarum ST-III, L. acidophilus NCFM and Bifidobacterium animalis subsp. lactis BB-12. MCRA genes from these strains were cloned and expressed in Escherichia coli, and the recombinant protein function was analysed with lipid profiles by GC–MS. The four MCRAs catalysed the conversion of linoleic acid and oleic acid to their respective 10-hydroxy derivatives, which suggests that MCRA proteins catalyse the first step in conjugated linoleic acid production. This is the first report of MCRA from L. rhamnosus with such catalytic function.     

Free fatty acid accumulation by mesophilic lactic acid bacteria in cold-stored milk

This study was aimed to determine the accumulation of free fatty acid by mesophilic lactic acid bacteria (Lactococcus lactis subsp. lactis 1471, Lactococcus lactis subsp. cremoris 1000 and Lactobacillus casei 111) in cold-stored milk. According to the results, all cold-stored milks had higher acid degree values than those of fresh milk. This phenomenon showed that a slight increase occurred in the accumulation of free fatty acids as a result of spontaneous lipolysis during cold storage. All lactic acid bacteria showed good performance in production of titratable acidity, which increased during fermentation of the milk (fresh and stored milks). Moreover, as the storage time was prolonged, more free fatty acid accumulation was obtained from the fermentation of the cold-stored milk by the investigated lactic acid bacteria. The control milk, which was without lactic acid bacteria, showed no change in the accumulation of free fatty acid during fermentation. From this result, it can be suggested that longer cold-storage time can induce higher free fatty acid accumulation in milk by lactic acid bacteria.     

Metabolic diversity in biohydrogenation of polyunsaturated fatty acids by lactic acid bacteria involving conjugated fatty acid production

Lactobacillus plantarum AKU 1009a effectively transforms linoleic acid to conjugated linoleic acids of cis-9,trans-11-octadecadienoic acid (18:2) and trans-9,trans-11–18:2. The transformation of various polyunsaturated fatty acids by washed cells of L. plantarum AKU 1009a was investigated. Besides linoleic acid, α-linolenic acid [cis-9,cis-12,cis-15-octadecatrienoic acid (18:3)], γ-linolenic acid (cis-6,cis-9,cis-12–18:3), columbinic acid (trans-5,cis-9,cis-12–18:3), and stearidonic acid [cis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (18:4)] were found to be transformed. The fatty acids transformed by the strain had the common structure of a C18 fatty acid with the cis-9,cis-12 diene system. Three major fatty acids were produced from α-linolenic acid, which were identified as cis-9,trans-11,cis-15–18:3, trans-9,trans-11,cis-15–18:3, and trans-10,cis-15–18:2. Four major fatty acids were produced from γ-linolenic acid, which were identified as cis-6,cis-9,trans-11–18:3, cis-6,trans-9,trans-11–18:3, cis-6,trans-10–18:2, and trans-10-octadecenoic acid. The strain transformed the cis-9,cis-12 diene system of C18 fatty acids into conjugated diene systems of cis-9,trans-11 and trans-9,trans-11. These conjugated dienes were further saturated into the trans-10 monoene system by the strain. The results provide valuable information for understanding the pathway of biohydrogenation by anaerobic bacteria and for establishing microbial processes for the practical production of conjugated fatty acids, especially those produced from α-linolenic acid and γ-linolenic acid. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Low pH-induced membrane fatty acid alterations in oral bacteria

Four oral bacterial strains, of which two are considered aciduric and two are considered acid-sensitive, were grown under glucose-limiting conditions in chemostats to determine whether their membrane fatty acid profiles were altered in response to environmental acidification. Streptococcus gordonii DL1, as well as the aciduric strains S. salivarius 57.I, and Lactobacillus casei 4646 increased the levels of mono-unsaturated membrane fatty acids. The non-aciduric strain S. sanguis 10904 did not alter its membrane composition in response to pH values examined here. Thus, in response to low pH, aciduric oral bacteria alter their membrane composition to contain increased levels of long-chained, mono-unsaturated fatty acids. This suggests that membrane fatty acid adaptation is a common mechanism utilized by bacteria to withstand environmental stress.     

Effects of pressure on cell morphology and cell division of lactic acid bacteria

The effect of pressure and temperature on the growth of the mesophilic lactic acid bacteria Lactococcus lactis and Lactobacillus sanfranciscensis was studied. Both strains were piezosensitive. Lb. sanfranciscensis failed to grow at 50 MPa and the growth rate of Lc. lactis at 50 MPa was less than 30% of that at atmospheric pressure. An increase of growth temperature did not improve the piezotolerance of either organism. During growth under high-pressure conditions, the cell morphology was changed, and the cells were elongated as cell division was inhibited. At atmospheric pressure, temperatures above the optimal temperature for growth caused a similar effect on cell morphology and cell division in both bacteria as that observed under high-pressure conditions. The segregation and condensation of chromosomal DNA were observed by DAPI staining and occurred normally at high-pressure conditions independent of changes in cell morphology. Immunofluorescence microscopy of Lc. lactis cells demonstrated an inhibitory effect of high pressure on the formation of the FtsZ ring and this inhibition of the FtsZ ring formation is suggested to contribute to the altered cell morphology and growth inhibition induced by high pressure.Communicated by K. Horikoshi     

乳酸菌的系统分类概况

乳酸菌是重要的益生菌资源,在食品、农业、化工业、医学等领域具有广阔的应用前景。目前,人们熟知的乳酸菌主要集中在乳杆菌属、双歧杆菌属、链球菌属、肠球菌属、乳球菌属、片球菌属和明串珠菌属等少数属种。为了拓宽人们对乳酸菌的认知,本文就乳酸菌的系统分类学进行阐述。在系统分类学上,乳酸菌分别隶属于厚壁菌门4纲7目18科39属653种和放线菌门2纲2目3科12属88种。最后,对乳酸菌的益生作用、安全性与有效来源、益生潜能的体外评价指标等进行简要的讨论。     

Evaluation of lactic acid bacteria autolysate for the supplementation of lactic acid bacteria fermentation

At the end of culture in a carbon-limited medium, i.e. the best conditions for subsequent autolysis, lactic acid bacteria were harvested and autolysed at 50 °C for 24 h. The resulting supernatant was then successfully tested as a substitute for industrial yeast extract for the supplementation of whey permeate and its conversion into lactic acid: for almost equivalent total nitrogen amounts of both supplements, the same growth and production rates were recorded.     

Comparative studies of lactic acid dehydrogenases in lactic acid bacteria

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 efficiently 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.Abbreviations l-LDH l-Lactic acid dehydrogenase - FDP Fructose-1,6-bisphosphate - DTE Dithioerythrol AddendumIn the case of the L. casei-LDH the shape of the NADH saturation curve is not changed by omitting the effectors FDP and Mn 2⁺. The K _M under these conditions is 3 fold higher (10.10 ^–5 M).     

Bacteriocins of lactic acid bacteria

Lactic acid bacteria produce a variety of antagonistic factors that include metabolic end products, antibiotic-like substances and bactericidal proteins, termed bacteriocins. The range of inhibitory activity by bacteriocins of lactic acid bacteria can be either narrow, inhibiting only those strains that are closely related to the producer organism, or wide, inhibiting a diverse group of Gram-positive microorganisms. The following review will discuss biochemical and genetic aspects of bacteriocins that have been identified and characterized from lactic acid bacteria.     

Genomics of lactic acid bacteria

Lactic acid bacteria (LAB) are found to occupy a variety of ecological niches including fermented foods as well as mucosal surfaces of humans and other vertebrates. This review is based on the genomic content of LAB that is responsible for the functional and ecological diversity of these bacteria. These genomes reveal an ongoing process of reductive evolution as the LAB have specialized to different nutritionally rich environments. Species-to-species variation in the number of pseudogenes as well as genes directing nutrient uptake and metabolism reflects the adaptation of LAB to food matrices and the gastrointestinal tract. Although a general trend of genome reduction was observed, certain niche-specific genes appear to be recently acquired and appear on plasmids or adjacent to prophages. Recent work has improved our understanding of the genomic content responsible for various phenotypes that continue to be discovered, as well as those that have been exploited by man for thousands of years.     

Cholesterol and the cell membrane

Recent studies concerning cholesterol, its behavior and its roles in cell growth provide important new clues to the role of this fascinating molecule in normal and pathological states.