Acid and bile tolerance and cholesterol removal ability of lactobacilli strains

Eleven strains of lactobacilli were studied for their acid and bile tolerance. Possible mechanisms of cholesterol removal by strains of lactobacilli were examined. Cholesterol assimilation as determined by the difference in cholesterol content in the medium before and after the incubation period showed that all lactobacilli strains were able to assimilate cholesterol at varying levels ranging from 12.03 to 32.25 microg/mL. Cholesterol removal was associated with growth of cultures. Binding of cholesterol to lactobacilli cells was determined using growing, heat-killed, and resting cells in phosphate buffer. Cholesterol removed by dead and resting cells ranged from 0.79 to 3.82 mg/g of dry weight compared with growing cells, which ranged from 4.53 to 16.03 mg/g of dry weight. Fatty acid methyl esters, as quantified using gas chromatography, showed changes in lipid profiles in cells grown in the presence of cholesterol compared with those grown without cholesterol. Fatty acid profiles, especially of hexadecanoic, octadecanoic, total saturated, and unsaturated acids suggested that cholesterol from the medium was incorporated into the cellular membrane. These findings suggest that strains of lactobacilli could remove cholesterol via various mechanisms and may be promising candidates for use as a dietary adjunct to lower serum cholesterol in vivo.     

Bile salt deconjugation ability,bile salt hydrolase activity and cholesterol co-precipitation ability of lactobacilli strains

Eleven strains of lactobacilli were screened for their bile salt deconjugation ability, bile salt hydrolase activity (BSH) and co-precipitation of cholesterol with deconjugated bile. Bile salt deconjugation as determined by the release of cholic acid showed that more cholic acid was liberated from the deconjugation of sodium glycocholate than sodium taurocholate, and Lactobacillus acidophilus strains had higher deconjugation ability than L. casei strains. BSH activity, as quantified by the amount of taurine or glycine liberated from conjugated bile salts, indicated that substrate specificity was more towards glycine-conjugated bile compared to taurine-conjugated bile. Co-precipitation of cholesterol with cholic acid was observed from deconjugation of both conjugated bile, with more cholesterol being precipitated upon deconjugation of sodium glycocholate than upon that of sodium taurocholate. Cholesterol co-precipitation with deconjugated bile increased with decreasing pH. L. acidophilus ATCC 33200, 4356 and 4962 and L. casei ASCC 1521 showed highest deconjugation ability and BSH activity towards bile mixtures that resemble the human bile, and may be promising candidates to exert beneficial bile deconjugation activity in vivo.     

Bile salt deconjugation and BSH activity of five bifidobacterial strains and their cholesterol co-precipitating properties

Five strains of bifidobacteria were screened for their bile salt deconjugation ability, bile salt hydrolase (BSH) activity and co-precipitation of cholesterol with deconjugated bile. Bile salt deconjugation was determined by the release of cholic acid. All strains exhibited deconjugation of both sodium glycocholate and sodium taurocholate. More cholic acid was liberated from the deconjugation of sodium glycocholate than sodium taurocholate. BSH activity was quantified by determining the amount of glycine or taurine liberated from conjugated bile salts by bifidobacteria strains. There was higher substrate specificity for glycine-conjugated bile compared to taurine-conjugated bile. Co-precipitation of cholesterol with cholic acid was observed from deconjugation of both sodium glycocholate and sodium taurocholate, and by all bifidobacteria strains studied. More cholesterol was precipitated with cholic acid when sodium glycocholate was used compared to sodium taurocholate. Increased cholesterol co-precipitation with deconjugated bile was observed with decreasing pH levels. Bifidobacterium infantis 17930 showed highest deconjugation ability and BSH activity towards bile mixtures that resemble the human bile, and may be a promising candidate to exert beneficial bile deconjugation activity in vivo.     

Bile salt deconjugation and cholesterol removal from media by Lactobacillus strains used as probiotics in chickens

BACKGROUND: Bile salt deconjugation by Lactobacillus strains is often closely linked to bile tolerance and survival of the strains in the gut and lowering of cholesterol in the host. The present study investigated the deconjugation of bile salts and removal of cholesterol by 12 Lactobacillus strains in vitro. The 12 strains were previously isolated from the gastrointestinal tract of chickens. RESULTS: The 12 Lactobacillus strains could deconjugate sodium glycocholate (GCA, 16.87–100%) and sodium taurocholate (TCA, 1.69–57.43%) bile salts to varying degrees, with all strains except L. salivarius I 24 having a higher affinity for GCA. The 12 Lactobacillus strains also showed significant (P < 0.05) differences in their ability to remove cholesterol from the growth medium (26.74–85.41%). Significant (P < 0.05) correlations were observed between cholesterol removal and deconjugation of TCA (r = 0.83) among the L. reuteri strains (C1, C10 and C16) and between cholesterol removal and deconjugation of TCA (r = 0.38) and GCA (r = 0.70) among the L. brevis strains (I 12, I 23, I 25, I 211 and I 218). In contrast, although L. gallinarum I 16 and I 26 and L. panis C 17 showed high deconjugating activity, there was no correlation between cholesterol removal and deconjugation of bile salts in these strains. CONCLUSION: The results showed that the 12 Lactobacillus strains were able to deconjugate bile salts and remove cholesterol in vitro, but not all strains with high deconjugating activity removed cholesterol effectively. Copyright © 2009 Society of Chemical Industry     

三株植物乳杆菌降胆固醇机理的研究

测定了已筛选出的具有较高胆固醇去除能力和胆盐水解酶活性的3株植物乳杆菌(KLDS6.0330,KLDS6.0333,KLDS6.0335)的胆固醇同化作用,胆汁盐的去结合作用和胆固醇共沉淀作用。这3株植物乳杆菌能够同化较多的胆固醇(﹥43μg/mL);所有的菌株都表现出了对甘氨胆酸钠和牛磺胆酸钠的去结合作用,与牛磺胆酸钠相比,甘氨胆酸钠的去结合能够释放较多的胆酸;所有菌株都表现出了胆固醇与甘氨胆酸钠和牛磺胆酸钠去结合释放胆酸的共沉淀,与牛磺胆酸钠相比,甘氨胆酸钠去结合产生的胆酸表现出了与较多的胆固醇共沉淀;随着pH值的下降,解聚态的胆汁酸与胆固醇的共沉淀量增加,结合态胆盐与胆固醇的共沉淀量较少。结果表明这3株植物乳杆菌在体外可以通过3种机制去除胆固醇。     

Homemade traditional cheeses for the isolation of probiotic Enterococcus faecium strains

One hundred twenty-two strains of Enterococcus faecium isolated from Tafí Cheese, a homemade traditional cheese of the highlands in the province of Tucumán, Argentina, were evaluated for their potential application as starter cultures in the manufacture of this traditional cheese. Eleven of the 122 strains showing limited delays in growth in oxgall were selected for the study of bile salts hydrolase activity (BSH), cholesterol reduction, antimicrobial activity, and virulence determinants. Nine strains were able to remove cholesterol in in vitro assays, a property that was closely related to the bile salt hydrolase activity. Only two strains produced active bacteriocins against Listeria strains although genetic evidence for the bacteriocin structural gene was found in six other enterococci strains. No virulence factors were detected in any of the 11 selected strains of enterococci.     

Screening of bile resistance and bile precipitation in lactic acid bacteria and bifidobacteria.

A modification of the ecometric method was developed for a rapid screening of bile resistance in lactic acid bacteria and bifidobacteria. Validation of the MEM bile assay (modified ecometric method) was performed comparing the bile resistance index (RIbile) and the bile resistance ratio (R%). Most Bifidobacterium strains assayed were bile sensitive (83.3%), while only 62.1% of lactobacilli showed that behavior. Some bifidobacterial strains (55.6%) showed a crystalline precipitate when grown on solid medium supplemented with 0.5% ox bile. The crystalline structures produced by B. pseudolongum CIDCA 531 were isolated and analyzed by optical and scanning electron microscopy, thin-layer chromatography, melting point, and specific cholesterol reactions. Those studies confirmed the presence of cholesterol in these crystalline structures. On the other hand, none of the lactobacilli and streptococci studied had the ability to produce crystalline precipitates.     

植物乳杆菌的降胆固研究

对植物乳杆菌MA2的体外和体内降胆固醇活性进行了研究,结果显示:培养基中添加的胆固醇的初始浓度对植物乳杆菌MA2的胆固醇去除率影响显著,当胆固醇的终质量浓度为30mg/dL,不添加胆盐的条件下,菌株MA2的胆固醇去除率达到(55±4.5)%。死菌体和休眠菌体也可去除培养基中的胆固醇,且菌体细胞浓度的影响显著。添加胆盐对菌株MA2的胆固醇去除率影响显著。体外实验表明植物乳杆菌MA2通过吸附胆固醇,进而将胆固醇吸入菌体细胞中来降低培养基中的胆固醇。MA2菌株可显著降低大鼠血清总胆固醇、甘油三酯的水平,表明其作为降血脂的益生菌的应用潜力。     

Screening of lactic acid bacteria for bile salt hydrolase activity

Bile salt hydrolysis is an important metabolic reaction in the bile salt metabolism of mammals. This reaction has a facilitating effect for bile salt excretion but can also be involved in various illnesses. In recent years interest has increased to use bile salt hydrolysis to influence the cholesterol metabolism of humans and farm animals. To understand the distribution and range of bile salt hydrolase activity in lactic acid bacteria, we screened more than 300 strains of the genera Bifidobacterium and Lactobacillus and the species Lactococcus lactis, Leuconostoc mesenteroides, and Streptococcus thermophilus. Results obtained for 273 strains showed that bile salt hydrolase activity is common in Bifidobacterium and Lactobacillus but absent in L. lactis, Leu. mesenteroides, and S. thermophilus. Nearly all bifidobacteria species and strains have bile salt hydrolase activity, whereas this activity can only be found in selected species of lactobacilli. A strong correlation can be observed between the habitat of a genus or species and the presence of bile salt hydrolase activity. Most often bile salt hydrolase activity is found in strains that have been isolated from the intestines or from feces from mammals--an environment rich in conjugated and unconjugated bile acids. Strains and species from other habitats like milk or vegetables--environments from which bile salts are absent--do normally not have bile salt hydrolase activity. In two independent assays, we established that bile salt hydrolase activity in bifidobacteria is, in general, much higher than in lactobacilli.     

Selection of Lactobacillus strains from fermented sausages for their potential use as probiotics

A rapid screening method was used to isolate potentially probiotic Lactobacillus strains from fermented sausages after enrichment in MRS broth at pH 2.5 followed by bile salt stressing (1% bile salts w/v). One hundred and fifty acid- and bile-resistant strains were selected, avoiding preliminary and time-consuming isolation steps. Strains were further characterized for survival at pH 2.5 for 3 h in phosphate-buffered saline and for growth in the presence of 0.3% bile salts with and without pre-exposure at low pH. Twenty-eight strains showed a survival >80% at pH 2.5 for 3 h; moreover, most of the strains were able to grow in the presence of 0.3% bile salts. Low pH and bile resistance was shown to be dependent on both the species, identified by phenotypic and molecular methods, and the strain tested. This is the first report on the direct selection of potentially probiotic lactobacilli from dry fermented sausages. Technologically interesting strains may be used in the future as probiotic starter cultures for novel fermented sausage manufacture.     

Characterization of lactic acid bacteria isolated from a Greek dry-fermented sausage in respect of their technological and probiotic properties

A total of 147 lactic acid bacteria was isolated from two types of naturally fermented dry sausages at four different stages of the ripening process studied in order to select the most suitable strains according to their technological characteristics including probiotic properties and antimicrobial activity against food-borne pathogens. Identification of the isolates revealed that 90% were lactobacilli, 4% enterococci, 3% Pediococcus sp. and sporadic isolates of Weissella viridescens, Leuconostoc pseudomesenteroides, and Leuconostoc sp. The isolated strains of Lactobacillus sakei (49 isolates), Lactobacillus curvatus (24 isolates) and Lactobacillus plantarum (7 isolates) were further characterized. All strains could grow at 15?°C, whereas the majority of the strains was able to grow in the presence of 6.5% NaCl and on acetate agar. The enzymatic potential of the strains was evaluated using the API ZYM system. During in vitro investigations all strains exhibited high leucine and valine aminopeptidase activities and moderate acid phosphatase and phosphohydrolase activities. Some strains showed very weak lipolytic activity. The enzyme profiling is an important factor for selection of strains as starter cultures. A large majority of the strains tolerated 0.1% bile salts whereas 58% of Lactobacillus curvatus strains and all Lactobacillus plantarum strains were resistant to 0.3% bile salts. All Lactobacillus sakei strains and the majority of Lactobacillus curvatus and Lactobacillus plantarum strains exhibited an anti-listerial activity against three Listeria monocytogenes strains. A percentage of 75, 50 and 29% of Lactobacillus sakei, L. curvatus and L. plantarum strains, respectively, could inhibit two Staphylococcus aureus strains. The contribution of the selected strains to a possible inhibition of Listeria monocytogenes and S. aureus in situ on fermented meats would be of considerable interest to enhance the hygienic quality of these products.     

Removal of cholesterol by lactobacilli via incorporation and conversion to coprostanol

Fifteen strains of Lactobacillus and Bifidobacterium were screened based on their ability to adhere to hydrocarbons via the determination of cellular hydrophobicity. Lactobacillus acidophilus ATCC 314, L. acidophilus FTCC 0291, Lactobacillus bulgaricus FTCC 0411, L. bulgaricus FTDC 1311, and L. casei ATCC 393 showed greater hydrophobicity and, thus, were selected for examination of cholesterol-removal properties. All selected strains showed changes in cellular fatty acid compositions, especially total fatty acids and saturated and unsaturated fatty acids in the presence of cholesterol compared with those grown in the absence of cholesterol. In addition, we found that cells grown in media containing cholesterol were more resistant to sonication and enzymatic lysis compared with those grown without cholesterol. We further evaluated the location of the incorporated cholesterol via the insertion of fluorescence probes into the cellular membrane. In general, enrichment of cholesterol was found in the regions of the phospholipid tails, upper phospholipids, and polar heads of the cellular membrane phospholipid bilayer. Our results also showed that lactobacilli were able to reduce cholesterol via conversion of cholesterol to coprostanol, aided by the ability of strains to produce cholesterol reductase. Our results provided experimental evidence to strengthen the hypothesis that probiotics could remove cholesterol via the incorporation of cholesterol into the cellular membrane and conversion of cholesterol to coprostanol. The strains studied may be potential health adjunct cultures in fermented dairy products with possible in vivo hypocholesterolemic effects.     

Nonstarter lactobacilli isolated from soft and semihard Argentinean cheeses: genetic characterization and resistance to biological barriers

Nonstarter lactic acid bacteria isolated from Argentinean cheeses were identified and characterized by focusing on their resistance to biological barriers, along with other physiological features of potential interest, in the search for future probiotic organisms. Lactobacilli were enumerated and isolated from semihard and soft cheeses made with multistrain Streptococcus thermophilus starters. Lactobacilli counts in 1-week-old cheeses were between 10(5) and 10(7) CFU/g and then reached 10(7) CFU/ g in all 1-month samples, while streptococci were always above 10(9) CFU/g. A total number of 22 lactobacilli isolates were retained, identified, and characterized by in vitro tests. Species identity was determined by carbohydrate metabolism and species-specific PCR assays. Genetic diversity was explored by random amplified polymorphic DNA (RAPD) PCR analysis. The Lactobacillus strains were assigned to the species L. casei, L. plantarum, L. rhamnosus, L. curvatus, L. fermentum, and L. perolens. All the strains studied tolerated 25 ppm of lysozyme, and most of them showed resistance to 0.3% bile. After incubation in gastric solution (pH 2.0), counts decreased by several log units, ranging from 3.2 to 7.0. The strains were able to grow in the presence of bile salts, but only three isolates were capable of deconjugation. The nonstarter lactobacilli that were assayed fermented the prebiotic substrates (especially lactulose and inulin). Some strains showed high cell hydrophobicity and beta-galactosidase activity, as well as inhibitory activity against pathogenic bacteria. It was concluded that most of the lactobacilli isolated in this study demonstrated resistance to biological barriers and physiological characteristics compatible with probiotic properties, which make them suitable for further research in in vivo studies aimed at identifying new probiotic organisms.     

自然发酵肉制品中乳酸菌的体外降胆固醇特性

为筛选出具有高效降胆固醇作用的乳酸菌,以自然发酵肉制品中分离出的13株乳酸菌为实验对象,采用邻苯二甲醛法对其体外降胆固醇能力进行测定,并对其中2株菌（M12、M23）的作用机理进行初步探讨。结果表明,2株菌均通过同化吸收和共沉淀作用协同降低培养基中的胆固醇;添加0.3 g/100 mL胆盐可极显著增加菌体对胆固醇的去除作用（P＜0.01）,去除率分别为56.05%和58.49%;添加胆固醇和胆盐均可显著提高菌株经超声波处理后的存活率（P＜0.05）,存活率分别为32.09%和37.93%;2株菌的热灭活菌体和休眠菌体也具有一定的胆固醇去除能力,且休眠菌体的去除率高于热灭活菌体。经16S rRNA序列分析,菌株M12为弯曲乳杆菌（Lactobacillus curvatus）,菌株M23为戊糖片球菌（Pediococcus pentosaceus）。     

Survival of lactic acid bacteria from fermented milks in an in vitro digestion model exploiting sequential incubation in human gastric and duodenum juice

In the present study, the survival of 9 lactic acid bacteria (5 Lactococcus strains, 3 Lactobacillus strains, and 1 strain of Enterococcus hirae), was investigated in vitro under conditions similar to human digestion using human gastric and duodenal juices. The tolerance of the bacteria was also tested with traditional methods using acidic conditions and bile salts. The strains were subjected to a model digestive system comprising sequential incubation in human gastric and duodenal juices, in a 2-step digestion assay at 37°C, simulating the human upper gastrointestinal tract with human gastric juices at pH 2.5 and human duodenal juices at pH 7. The bacterial strains were tested either as washed cells from culture media or in fermented milk. The initial in vitro testing in acid and bile salts showed that Lactobacillus strains and the E. hirae strain displayed a significantly higher acid tolerance than the lactococci. The lactobacilli and the Enterococcus numbers increased, whereas the lactococci decreased at least 1 log during the bile salt treatment. The Lactobacillus strains showed the highest survival rate in the model digestive system when washed bacterial cultures were used with a minor log reduction, whereas the lactococci numbers were reduced by at least log 4. However, when using fermented milks in the model digestion system it was demonstrated that the Enterococcus strain and 2 strains of Lactococcus lactis ssp. cremoris benefited significantly from the presence of the fermented milk as food matrix, with log numbers >log 7 and 5, respectively, after digestion of the fermented milk. The analyses reported comprise a comprehensive in vitro testing regimen suitable for evaluation of the survival of candidate probiotic bacteria in human digestion as an initial prescreen to clinical trials.     

两株嗜酸乳杆菌体外降低胆固醇的研究

考察两株嗜酸乳杆菌体外降低胆固醇的降低曲线和降低方式,以及不同条件下嗜酸乳杆菌对培养基中胆固醇的降低情况.结果表明,这两株茵对胆固醇有明显的去除作用,其对胆固醇的降低作用是同化吸收和共沉淀的结果,以同化方式为主;随着接种量的增加,降低率也随之增加;不同胆盐对其降低胆固醇效果不同,当胆盐添加量为0.2％(m/V)时,胆固醇降低量最大;当吐温80添加量为0.1％时,胆固醇降低效果最佳.     

Application of in vitro methods for selection of Lactobacillus casei strains as potential probiotics

Probiotics have established their efficacy as dietary adjuncts providing benefits to consumers, but the selection of probiotics before incorporation in diet requires close scrutiny in the form of in vitro as well as in vivo tests. The present study was undertaken to check different in vitro characteristics of seven Lactobacillus casei strains. The characteristics studied include acid and bile tolerance, adhesion and cell surface hydrophobicity, antimicrobial effect on common pathogens and cholesterol reduction. All strains were able to resist pH 3 for 3 h, though resistance to pH 2 was exhibited by NCDC 17, C1 and Y strains only. NCDC 63 and VT strains were able to tolerate 1% and 2% bile concentrations for 12 h. There was wide variation in ability of strains to adhere to isolated rat epithelial cells. The index was highest for C1 at 66%. The electron microscopic adhesion studies on the stainless steel chips did not reveal any specific attachment to surfaces by any of strains. The hydrophobic character for octane was highest for strain C1 at 54.06% and lowest for strain C2 at 4.65%. The ability to antagonize common pathogens was observed in all strains but this activity was attributed to production of organic acids and no specific compound caused the inhibitory effect. The cholesterol reducing ability varied not only for strains but also for time of incubation. NCDC 17 showed maximum reduction in cholesterol level after 48 h of incubation with buffalo plasma as the source of cholesterol. Overall there existed variations in different strains with respect to different characters of significance to be a probiotic.     

Probiotic potential of Lactobacillus delbrueckii strains and their phage resistant mutants

Three commercial phage sensitive strains of Lactobacillus delbrueckii and spontaneous phage resistant mutants isolated from them were studied by focusing on their biological and probiotic features. After incubation in a simulated gastric solution (pH 2.0), viable cell counts decreased moderately, but a protective effect on strains was observed when milk was added. A limited resistance to bile was observed, while most of the strains tolerated lysozyme, and grew in the presence of bile salts and fermented prebiotics. Some strains showed high hydrophobicity values and β-galactosidase activity. The strong antibacterial activity displayed toward pathogens was due to the production of lactic acid. Sensitive strains and their phage resistant variants were able to adhere to Caco-2/TC-7 monolayers and significantly inhibited the invasion of Salmonella enterica serovar Enteritidis into Caco-2/TC-7 cells. All strains tested showed potential probiotic features, suggesting a potential probiotic role of L. delbrueckii.     

Assimilation of cholesterol by yeast strains isolated from infant feces and Feta cheese

Eight yeast strains isolated from infant feces and the traditional Greek Feta cheese, selected for their probiotic properties, were tested along with a commercially available strain of Saccharomyces boulardii for their ability to remove cholesterol from a growth medium (yeast extract glucose peptone broth) supplemented with 0.3% Oxgall. The amount of cholesterol removed during 72 h of growth at 37 degrees C revealed significant variations among the yeast strains examined. Two isolates from infant feces, namely Saccharomyces cerevisiae KK1 and Isaatchenkia orientalis KK5.Y.1 and one isolate from Feta cheese, namely S. cerevisiae 832, along with the commercial strain S. boulardii, were able to remove cholesterol from the growth medium after 48 h of incubation at 37 degrees C. However, Saccharomyces strains proved to be able to remove cholesterol even after 24 h of growth at 37 degrees C. The cholesterol removed from the growth medium was not metabolically degraded but was rather assimilated into the yeast cells. The ability to assimilate cholesterol in vitro and to tolerate low pH levels, gastric juice, and bile indicate that S. cerevisiae 832, and especially S. cerevisiae KK1 and I. orientalis KK5.Y.1 (being more bile and gastric juice tolerant because of their human origin) may be promising candidate strains for use as probiotics.     

Screening of glutamate decarboxylase activity and bile salt resistance of human asymptomatic carriage, clinical, food, and environmental isolates of Listeria monocytogenes

Following consumption, stomach acidity is the first major barrier encountered by the food-borne pathogen Listeria monocytogenes. Analysis of low pH sensitivity and glutamate decarboxylase (GAD) acid resistance system of 14 isolates of L. monocytogenes carried asymptomatically by humans showed that levels of GAD activity were subjected to strain variation. Similar variations were observed for strains responsible for 18 cases of listeriosis, whereas in comparison, 13 strains isolated from food and food-processing plant environments showed lower GAD activity. Following survival of the stomach barrier, L. monocytogenes also has to resist bile salts encountered in the small intestines. Analysis revealed that all strains tested were able to grow in the presence of bile salts with concentrations as high as those encountered in the small intestines and had previously identified bile salt hydrolase (BSH) activity. Strain variation was observed but there was no relationship between the origin of the strains and the ability to degrade bile salts.