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.     

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。     

降胆固醇乳酸菌的筛选及其在内蒙古发酵香肠中的应用

通过对乳酸菌降胆固醇生物学特性及对发酵肉制品中胆固醇降解作用进行研究,结果表明,从内蒙古传统肉肠中分离筛选得到的8 株乳酸菌中,菌株X3-2B有较强的胆固醇降解能力。且在MRS培养基中添加3 g/L胆盐、20 g/L胆固醇和 20 g/L葡萄糖时菌株X3-2B的胆固醇降解能力最大,在不同培养基中发酵不同时间菌株X3-2B对胆固醇的降解能力显著高于标准菌株。在以菌株X3-2B为发酵剂的发酵香肠中,其胆固醇含量显著低于对照组。故菌株X3-2B可作为一株降胆固醇性能较好的肉制品发酵剂。     

Mechanisms of cholesterol removal by lactobacilli under conditions that mimic the human gastrointestinal tract

Five strains of lactobacilli were studied for their ability to remove cholesterol in vitro under conditions that mimic the human gastrointestinal tract. The highest assimilation of cholesterol was observed in media supplemented with oxgall and the lowest in the presence of taurocholic acid. Scanning electron micrographs showed that cholesterol was adhered to the cellular surface of lactobacilli cells. Resting and dead cells were able to remove cholesterol although in small amounts. Additionally, inhibition of cholesterol micelles formation was observed in the presence of bile salts. All strains were able to deconjugate bile salts, where higher deconjugation was observed in the presence of sodium glycocholate compared with other bile salts studied. All strains also exhibited bile salt hydrolase activity and most strains showed higher substrate specificity towards glycine-conjugated bile than towards taurine-conjugated bile. The results indicated that lactobacilli could remove cholesterol in vitro via various mechanisms, and may exert such hypocholesterolaemic effects in vivo.     

六堡茶乳酸菌多样性及其降胆固醇特性分析

探究六堡茶渥堆发酵过程中乳酸菌多样性并筛选具有降胆固醇活性菌株，旨在评价其作为潜在益生菌的体外降胆固醇能力，预防人体因饮食不均衡导致的高血脂症。采用高通量测序、纯培养法分析不同渥堆发酵时期六堡茶中乳酸菌多样性，并对其体外降胆固醇能力、胃肠耐受性进行研究。结果表明，从25 个样品中共检测到乳酸菌4 个属（乳杆菌属（Lactobacillus）、魏斯氏菌属（Weissella）、乳球菌属（Lactococcus）和肠球菌属（Enterococcus））5 个种，其中乳杆菌属为优势菌。共分离得到76 株乳酸菌，包括55 株戊糖乳杆菌（Lactobacillus pentosus）、3 株铅黄肠球菌（Enterococcus casseliflavus）、5 株类肠膜魏斯氏菌（Weissella paramesenteroides）、8 株希腊魏斯氏菌（Weissella hellenica）、5 株台湾乳球菌（Lactococcus taiwanensis）。通过乳酸菌体外降胆固醇实验，发现胆固醇去除率为0%～30%，其中戊糖乳杆菌L131的去除胆固醇率最高，为（29.56±0.37）%；在pH值分别约为3.0、8.0的人工胃液、人工胰液中，L131有最高存活率，分别为（92.70±0.71）%和（77.54±4.81）%，显著高于其他菌株（P＜0.05）。本研究探明六堡茶渥堆发酵过程中乳酸菌的多样性，且发现具有降胆固醇特性的优良乳酸菌株，为健康黑茶产品和茶源益生菌的开发提供理论基础。     

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.     

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.     

Assimilation of cholesterol and probiotic characterisation of yeast strains isolated from raw milk and fermented foods

In this study, cholesterol assimilation ratios and some probiotic characteristics of yeasts were investigated. For this purpose, yeasts were isolated from milk and foods that were naturally fermented and not containing starter culture. In vitro cholesterol assimilation properties were determined in media. The Cholesterol assimilation by yeast strains ranged between 1.36 and 73.33%. Twenty‐one yeast strains showing high assimilation percentage were selected, and their acid tolerance, bile tolerance, bile salt deconjugation activity and survival in simulated gastrointestinal conditions were investigated. Among the strains assessed, 12 of them showed probiotic characteristics.     

具有胆固醇去除能力和胆盐水解酶活性的乳酸菌的体外筛选

对分离自内蒙古传统稀奶油或酸化稀奶油中的10株乳酸菌进行了胆固醇去除能力和胆盐水解酶活性的研究(定性和定量),筛选出了3株具有较高胆固醇去除能力和胆盐水解酶活力的乳酸菌菌株,分别为KLDS6.0330、KLDS6.0333和KLDS6.0335,并对它们进行16SrDNA分子生物学鉴定、酸耐受性和胆盐耐受性的测定。结果表明:这3株菌均为植物乳杆菌(L.Plantarum),并且表现出了较好的酸耐受性和胆盐耐受性。     

Influence of sodium glycocholate on production of conjugated linoleic acid by cells of Lactobacillus reuteri ATCC 55739

ABSTRACT:  The possible influence of a bile salt on production of conjugated linoleic acid (CLA) by Lactobacillus reuteri ATCC 55739 was evaluated. Cells of the lactobacilli grown in MRS broth with and without linoleic acid (LA, 0.2%) were harvested and washed. The washed cells were added to buffer containing 0.2% LA and incubated 18 h at 37 °C. The cells, which had been grown without LA, transformed LA into CLA (mainly c9t11-C18:2) better than did those cells grown with it. When sodium glycocholate (0.3%) was added to the washed cell suspensions, about the same level of CLA was formed as in its absence regardless of whether or not the cells had been grown in broth supplemented with free LA. Thus, glycocholate that occurs in humans did not influence production of CLA by resting cells of the lactobacilli.     

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）。     

Acid Resistance of Twelve Strains of Enterobacter sakazakii, and the Impact of Habituating the Cells to an Acidic Environment

ABSTRACT:  The association of powdered infant formula with cases of severe Enterobacter sakazakii infections in immunocompromised and premature neonates has led to a need to learn about the basic behavior of this emerging pathogen in food systems and the environment. The current study examines the microorganism's stationary-phase acid resistance using 12 strains that had been previously used to characterize its thermal resistance. Acid resistance was determined by initially culturing the isolates for 18 h in brain heart infusion broth (BHI) at 36 °C, transferring the cells to tryptic soy broth (TSB) adjusted to pH 3.0 and 3.5, and determining E. sakazakii survival over the course of 5 h incubation at 36 °C. At pH 3.5, 10 of the 12 strains showed less than a 1 log cycle decline over the 5-h incubation period, with the most acid sensitive strain showing an approximate 3.5 log cycle decline. At pH 3.0, the decline over the 5-h incubation period ranged from 4.9 to >6.3 log cycles; however, substantial diversity was evident when the 1-h/pH 3.0 results were compared. The effect of habituating the cells to a moderately acidic environment was determined by growing the strains in TSB with 0% (nonacidogenic) and 1% glucose (acidogenic), transferring the cells to acidified (pH 3.0) BHI, and determining E. sakazakii survival over the course of 5 h of incubation at 36 °C. While there was diversity observed among the strains, in general the stationary-phase acid resistances of several of the strains were enhanced, at least transitorily, by growth in the acidogenic medium. No apparent correlation between the stationary-phase relative acid resistances of the strains based on the 1-h/pH 3.0 acid inactivation values and the previously reported thermal D-values was observed.     

Fatty Acids in the American Groundnut (Apios americana)

The fatty acid profiles of lipids extracted from groundnut (Apios americana Medikus) seeds and tubers were analyzed using gas-liquid chromatography. Groundnut seeds contained 12.1–18.4% lipids and groundnut tubers 4.1–4.6%, on a dry weight basis. Fatty acid profiles of seed and tuber lipids differred; however, linoleic acid predominated. The fatty acid profiles of seeds and tubers varied between plants collected in two different locations, suggesting genetic variability.     

植物乳杆菌C8的耐受性和体外降胆固醇活性研究

对一株分离自内蒙古奶豆腐中的植物乳杆菌C8进行了酸和胆汁盐耐受实验,抗生素敏感性试验以及体外胆固醇降解实验,并分析了其降胆固醇的作用机理.结果表明,C8能够耐受pH 3和高浓度胆盐(30g/L)环境;对所选10种抗生素均表现出一定的耐药性;该菌具有良好的降胆固醇活性,产生的游离胆酸的量与胆固醇移除能力呈正相关,且以在甘氨胆酸钠存在条件下最佳(5.39mmol/L).植物乳杆菌c8降胆固醇的作用机理主要是菌体细胞的表面吸附作用和胆固醇与胆盐的共沉淀作用.     

8株窖泥链霉菌对酸、乙醇的适应性研究

对分离自窖泥的8株放线菌进行鉴定,并考察其对窖泥环境中pH值、乙醇的耐受性.采用ISP2液体培养基在pH值2.0～3.5、乙醇浓度3%vol ～6%vol条件下培养链霉菌菌株,结果显示,分属于链霉菌属的8个种的8株放线菌均不能耐受pH值2.0及6%vol的乙醇浓度,均能耐受pH值3.5及3%vol的乙醇浓度,5株链霉菌在pH值3.0时可以生长,2株链霉菌可耐受5%vol乙醇,说明分离自窖泥的多数链霉菌对窖泥环境的适应能力较好,但不同种链霉菌的适应能力存在差异,窖泥环境在长期生产过程中对链霉菌属菌株具有一定的选择驯化作用.     

Use of whey and buttermilk based media to obtain biomass of thermophilic LAB

The growth of commercial strains of thermophilic lactic acid bacteria (LAB) was determined in reconstituted dried whey and buttermilk. Kinetic (μ, /h and g, h) and growth (N, cfu/mL; X, cell yield and I_V , percentual variation) parameters were determined using Elliker (streptococci) and MRS (lactobacilli) broths, and a commercial medium as controls. Cheese whey was the least effective substrate for growing streptococci and lactobacilli strains. However, whey and buttermilk with added yeast extract gave the best performance. These media could be efficient and easily available alternatives for producing industrial biomass of thermophilic LAB.     

Selection of potential probiotic lactobacilli for cholesterol-lowering properties and their effect on cholesterol metabolism in rats fed a high-lipid diet

The objectives of this study were to screen probiotic characteristics of lactobacilli isolated from traditionally homemade koumiss products in Xinjiang and Inner Mongolia of China, and to determine and compare the effect of the Lactobacillus strains in vivo on lipid metabolism in rats fed with a high-lipid diet. Three out of 68 strains of Lactobacillus isolated from the koumiss were screened for bile-salt resistance, acid tolerance, and different hypocholesterolemic properties. The cholesterol-lowering effects of the 3 screened strains were estimated in rats fed a high-lipid diet by determination of serum lipids, liver, and fecal cholesterol, fecal total bile acids, and short-chain fatty acids. After a 4-wk feeding period, in comparison with the control group, the groups LIP-1 and MG9-2 had a significant reduction in serum total cholesterol, triglyceride and low-density lipoprotein cholesterol, and significantly increased high-density lipoprotein, the group E7301 had similar effects on serum lipids, but the change was not significant. The groups LIP-1, MG9-2 and E7301 had a significant reduction in liver cholesterol content and an increase in fecal cholesterol content compared with the control group. Total bile acid excretion was significantly higher in rats fed MG9-2 than the other groups. Rats fed diets containing lactic acid bacteria strains had significantly higher propionic acid and butyric acid concentrations in the feces compared with the control. Results indicated that the 3 screened Lactobacillus strains were able to lower cholesterol in vitro, and reduce cholesterol effectively in vivo. The mechanisms behind the hypocholesterolemic effect of 3 strains are likely to be diverse and will need further investigation.     

Characteristics of lactobacilli isolated from dry fermented sausages

A study of the microflora of Spanish dry fermented sausages showed a similar pattern to that observed in other sausages during ripening. Lactic acid bacteria and organisms growing on Mannitol-salt-agar increased during the first five days, remaining at constant levels afterwards. From MRS agar, 368 strains were isolated and characterized. All Lactobacillus strains (343) could be separated into three groups according to their reactions in the API-50 L system. The biochemical characteristics of the Lactobacillus strains analyzed were those typical of isolates from sausages and other meat products. The extracellular and intracellular lipolytic activities of some strains of the three groups were also studied. It is concluded that lipolytic activity of lactobacilli is mainly directed against mono- and diglycerides and short-chain fatty acid triglycerides.     

Characterisation of Lactobacillus fermentum SM-7 isolated from koumiss, a potential probiotic bacterium with cholesterol-lowering effects

BACKGROUND: Lactic acid bacteria (LAB) have been demonstrated to have cholesterol‐reducing effects in many studies. RESULTS: Lactobacillus fermentum SM‐7 screened from ten LAB strains isolated from koumiss, a fermented milk drink, reduced cholesterol by 66.8%. It also showed acid and bile tolerance as well as antimicrobial activity against pathogenic Escherchia coli and Staphylococcus aureus. Lactobacillus fermentum SM‐7 cells assimilated 61.5% and co‐precipitated and absorbed 38.5% of the cholesterol in the media. Co‐precipitation of cholesterol with cholic acid increased rapidly at pH levels below 6. In vivo experiments using L. fermentum SM‐7 on artificially induced hyperlipidaemial ICR mice significantly decreased serum total cholesterol and total triglyceride levels, low‐density lipoprotein cholesterol concentrations and atherogenic index (P < 0.01), while serum high‐density lipoprotein cholesterol concentrations did not increase significantly (P > 0.05). The body weight and liver weight/body weight ratio of SM‐7 groups were lower than those of mice on a high‐cholesterol diet that were not given lactobacilli. There was no bacterial translocation in the liver, spleen or kidney of experimental mice. CONCLUSION: The results suggested that L. fermentum SM‐7 is a potential probiotic bacterium with cholesterol‐lowering effects. Copyright © 2010 Society of Chemical Industry     

乳酸菌胆酸盐耐受性及降胆固醇能力研究

研究了分离自内蒙古传统稀奶油的四株乳酸菌的胆酸盐耐受性和降胆固醇能力。结果表明,供试菌株对牛磺胆酸钠的耐受性与对照菌株相比差异显著(P<0.05),其中屎肠球菌KLDS6.0330的耐受性最强;粪肠球菌KLDS6.0335呈现胆酸盐促进其生长的现象。菌株间胆固醇去除能力不同,在恒定pH5.5条件下去除能力更强(P<0.05);其中KLDS6.0330在不恒定pH和恒定pH5.5条件下分别达到58.47%和61.92%。四株供试菌株均具有共沉淀作用、吸收作用和降解作用。其中,干酪乳杆菌KLDS1.0344以共沉淀作用为主,KLDS6.0330以吸收作用和降解作用最为显著。