Identification of Gibberellin A3 and Abscisic acid from Artemisia capillaris Leaves

The hypocholesterolemic effects of Lactobacillus acidophilus 43121 (43121) and a mixture of Lactobacillus casei and Bifidobacterium longum (MIX) were studied in hypercholesterolemia-induced pigs. Serum total cholesterol was decreased by supplementation of either 43121 or MIX, although, high-density lipoprotein cholesterol was not changed. The hypocholesterolemic effect of 43121 and MIX was mainly due to bile acid dehydroxylation, this effect being supplementation-time dependent.     

Characterization of the cholesterol-reducing activity in a cell-free supernatant of Lactobacillus acidophilus ATCC 43121

This study characterizes the factors responsible for the cholesterol reduction by Lactobacillus acidophilus ATCC 43121. In addition, two-dimensional gel electrophoresis (2-DE) and protein profiling was also used to study the response of ATCC 43121 at the proteome level in the presence of cholesterol. The results show that the cell-free supernatant (CFS) produced by ATCC 43121 in the presence of bile salts could also reduce the cholesterol in the broth, whereas all previous reports have suggested a mechanism by live cells. The active fraction was partially purified by 60% ammonium sulfate precipitation, and subsequent Sephacryl S-100 column chromatography. The molecular weight of the component with cholesterol-reducing activity was estimated to be approximately 12 kDa by SDS-PAGE. These results suggest that the novel protein isolated from CFS may be an important factor in the mechanism for cholesterol reduction by ATCC 43121. In addition, the proteins expressed by ATCC 43121 in the presence of cholesterol micelles were detected by 2-DE, five protein spots with at least a 2.5-fold increase in amounts being identified. The responsible proteins may be involved in the stress-response, translation, and metabolic processes. These results may suggest a new possibility for the mechanism underlying cholesterol reduction by lactic acid bacteria (LAB), differing from the conclusions of previous reports.     

Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation

AIMS: To optimize the conditions for electroporating foreign plasmid DNA into Lactobacillus acidophilus ATCC 43121. METHODS AND RESULTS: The conditions of electroporation were optimized to improve the transformation efficiency. Plasmid pNZ123 containing multicloning site and chloramphenicol resistance was employed to construct a cloning vector. The optimum electroporation conditions for the maximum transformation efficiency were a pulse strength of 12.5 kV cm(-1), a pulse number of 10, a pulse interval of 500 ms, and pNZ123 plasmid DNA concentration of 25 ng microl(-1). Under the optimum conditions the transformation efficiency of L. acidophilus ATCC 43121 was 1.84 +/- 0.13 x 10(4) (+/- standard error of measurements) CFU per mug of plasmid DNA. Other strains of L. acidophilus showed transformation efficiencies ranging from 1.38 +/- 0.02 x 10(4) to 9.32 +/- 0.54 x 10(4) under these conditions. A green fluorescent protein (GFP) was successfully expressed and detected by fluorescence microscopy when the pKU::slpA-GFP, pNZ123 containing GFP gene, was transformed in L. acidophilus ATCC 43121 under the optimum conditions. CONCLUSIONS: The results suggest that electrical parameters, antibiotic concentration, and host specificity play important roles to determine transformation efficiency of lactobacilli. The optimum conditions for the transformation of L. acidophilus ATCC 43121 may be applied to improve transformation efficiency of other lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimized conditions for electrotransformation may provide a mean to improve the introduction of foreign DNA into L. acidophilus to be used as a vehicle for a heterologous protein expression.     

Effect of dietary inclusion of Lactobacillus acidophilus ATCC 43121 on cholesterol metabolism in rats

This study examined the effects of Lactobacillus acidophilus ATCC 43121 (LAB) on cholesterol metabolism in hypercholesterolemia-induced rats. Four treatment groups of rats (n = 9) were fed experimental diets: normal diet, normal diet+LAB (2 x 10(6) CFU/day), hypercholesterol diet (0.5% cholesterol, w/w), and hypercholesterol diet + LAB. Body weight, feed intake, and feed efficiency did not differ among the four groups. Supplementation with LAB reduced total serum cholesterol (25%) and VLDL + IDL + LDL cholesterol (42%) in hypercholesterol diet groups, although hepatic tissue cholesterol and lipid contents were not changed. In the normal diet group, cholesterol synthesis (HMG-CoA reductase expression), absorption (LDL receptor expression), and excretion via bile acids (cholesterol 7 alpha-hydroxylase expression) were increased by supplementation with LAB, and increased cholesterol absorption and decreased excretion were found in the hypercholesterol diet group. Total fecal acid sterols excretion was increased by supplementation with LAB. With proportional changes in both normal and hypercholesterol diet groups, primary bile acids (cholic and chenodeoxycholic acids) were reduced, and secondary bile acids (deoxycholic and lithocholic acids) were increased. Fecal neutral sterol excretion was not changed by LAB. In this experiment, the increase in insoluble bile acid (lithocholic acid) reduced blood cholesterol level in rats fed hypercholesterol diets supplemented with LAB. Thus, in the rat, L. acidophilus ATCC 43121 is more likely to affect deconjugation and dehydroxylation during cholesterol metabolism than the assimilation of cholesterol into cell membranes.     

An in vitro study of the probiotic potential of a bile-salt-hydrolyzing Lactobacillus fermentum strain,and determination of its cholesterol-lowering properties

This study evaluated the use of a bile-salt-hydrolyzing Lactobacillus fermentum strain as a probiotic with potential hypocholesterolemic properties. The effect of L. fermentum on representative microbial populations and overall metabolic activity of the human intestinal microbiota was investigated using a three-stage continuous culture system. Also, the use of galactooligosaccharides as a prebiotic to enhance growth and/or activity of the Lactobacillus strain was evaluated. Administration of L. fermentum resulted in a decrease in the overall bifidobacterial population (ca. 1 log unit). In the in vitro system, no significant changes were observed in the total bacterial, Lactobacillus, Bacteroides, and clostridial populations through L. fermentum supplementation. Acetate production decreased by 9 to 27%, while the propionate and butyrate concentrations increased considerably (50 to 90% and 52 to 157%, respectively). A general, although lesser, increase in the production of lactate was observed with the administration of the L. fermentum strain. Supplementation of the prebiotic to the culture medium did not cause statistically significant changes in either the numbers or the activity of the microbiota, although an increase in the butyrate production was seen (29 to 39%). Results from this in vitro study suggest that L. fermentum KC5b is a candidate probiotic which may affect cholesterol metabolism. The short-chain fatty acid concentrations, specifically the molar proportion of propionate and/or bile salt deconjugation, are probably the major mechanism involved in the purported cholesterol-lowering properties of this strain.     

Lactobacillus plantarum LRCC 5273 isolated from Kimchi ameliorates diet-induced hypercholesterolemia in C57BL/6 mice

ABSTRACT

This study was designed to select potent cholesterol-lowering probiotic strains on HepG2 cell and investigate the effect of selected strain, Lactobacillus plantarum LRCC 5273 and LRCC 5279 in hypercholesterolemic mice. In the results, LP5273 group showed significantly reduced total and LDL cholesterol compared to HCD group. In addition to significantly up-regulated hepatic mRNA expression of LXR-α and CYP7A1, intestinal LXR-α and ABCG5 were significantly up-regulated in LP5273 group. With activation of hepatic and intestinal LXR-α and its target genes, fecal cholesterol and bile acid excretion were increased in LP5273 fed mice. These results suggest that LP5273 ameliorates hypercholesterolemia in mice through the activation of hepatic and intestinal LXR-α, resulting in enhancement of fecal cholesterol and bile acids excretion in the small intestine. The results of present study suggest mechanistic evidences for hypocholesterolemic effects of L. plantarum spp., and may contribute to future researches for prevention of hypercholesterolemia and cardiovascular disease.     

Effects of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> MA2 isolated from Tibet kefir on lipid metabolism and intestinal microflora of rats fed on high-cholesterol diet

The objective of this study was to evaluate the effects of Lactobacillus plantarum MA2, an isolate from Chinese traditional Tibet kefir, on cholesterol-lowering and microflora of rat in vivo. Rats were fed on cholesterol-enriched experimental diet, supplemented with lyophilized L. plantarum MA2 powder, with a dose of 10¹¹ cells/day per mice. The results showed that L. plantarum MA2 feeding significantly lowered serum total cholesterol, low-density lipoprotein cholesterol, and triglycerides level, while there was no change in high-density lipoprotein cholesterol. In addition, liver total cholesterol and triglycerides was also decreased. However, fecal cholesterol and triglycerides was increased significantly (P < 0.05) in comparison with the control. Also, L. plantarum MA2 increased the population of lactic acid bacteria and bifidobacteria in the fecal, but it did not change the number of Escherichia coli as compared to control. Moreover, pH, moisture, and organic acids in the fecal were also measured. The present results indicate the probiotic potential of the L. plantarum MA2 strain in hypocholesterolemic effect and also increasing the probiotic count in the intestine.     

Assimilation of cholesterol by Lactobacillus acidophilus

Considerable variation was found among strains of Lactobacillus acidophilus isolated from the fecal flora of pigs with regard to the ability to grow well in the presence of bile and to assimilate cholesterol from a laboratory growth medium. The uptake of cholesterol occurred only when the culture(s) was growing in the presence of bile under anaerobic conditions. Consumption of L. acidophilus RP32, which was selected for its ability to grow well in the presence of bile and to assimilate cholesterol from the laboratory medium, significantly inhibited increases in serum cholesterol levels of pigs (P less than 0.05) fed a high-cholesterol diet. Consumption of L. acidophilus P47, which was selected for its ability to grow in the presence of bile and lack of ability to remove cholesterol from the growth medium, failed to have a similar effect. This indicates that certain strains of L. acidophilus act directly on cholesterol in the gastrointestinal tract, and thus may be beneficial in reducing serum cholesterol levels.     

Assimilation of cholesterol by Lactobacillus acidophilus.

Considerable variation was found among strains of Lactobacillus acidophilus isolated from the fecal flora of pigs with regard to the ability to grow well in the presence of bile and to assimilate cholesterol from a laboratory growth medium. The uptake of cholesterol occurred only when the culture(s) was growing in the presence of bile under anaerobic conditions. Consumption of L. acidophilus RP32, which was selected for its ability to grow well in the presence of bile and to assimilate cholesterol from the laboratory medium, significantly inhibited increases in serum cholesterol levels of pigs (P less than 0.05) fed a high-cholesterol diet. Consumption of L. acidophilus P47, which was selected for its ability to grow in the presence of bile and lack of ability to remove cholesterol from the growth medium, failed to have a similar effect. This indicates that certain strains of L. acidophilus act directly on cholesterol in the gastrointestinal tract, and thus may be beneficial in reducing serum cholesterol levels.     

降胆固醇乳酸菌的筛选和分析比较

目的比较7株乳酸菌各项生理特性,筛选高效、安全降解胆固醇的益生菌株,为后期开发应用奠定基础。方法对选定菌株依次进行体外降胆固醇、胆盐水解酶(BSH)活力、人工胃肠液及胆盐耐受性、细胞粘附性能的测定和比较,并分析抗生素耐药性和抑制病原菌活性,最后利用16S rRNA基因测序确定细菌种属。结果不同菌株各项生理指标存在差异。其中菌株ZL-2、JQI-7和PC-26的BSH酶活分别为0.010、0.015和0.030 U,可高效清除体外胆固醇,3株菌体外降解胆固醇率在58%以上;并且耐酸、耐胆盐,具备较好的HT--29细胞粘附性,不存在抗生素耐药性,可有效抑制大肠埃希菌、金黄色葡萄球菌、铜绿假单胞菌和阴沟肠杆菌生长。结论菌株ZL-2、JQI-7和PC-26满足降胆固醇益生菌株的生物学特性,可用于进一步研究和开发。     

模拟人体胃肠道环境筛选益生乳杆菌

【目的】筛选具有益生特性的乳杆菌作为保健型酸奶的候选菌株。【方法】从健康人肠道和奶豆腐中分离筛选出耐受人工胃液的乳杆菌,对其进行体外益生特性(人工胃肠液耐受性、胆盐耐受性、抑菌活性及胆固醇降解能力)研究。【结果】从在乳杆菌分离培养基上有溶钙圈的41株菌株中筛选出5株耐酸、耐人工胃液较强的菌株,经16S rR NA基因测序鉴定,其中3株为乳杆菌,分别命名为植物乳杆菌Lp MT-3、植物乳杆菌Lp MT-5和唾液乳杆菌LsA F-7。在人工胃液中3株菌的耐受力均强于商品化的对照菌株LGG(鼠李糖乳杆菌GG);转入肠液4 h后直至26 h,Lp MT-5存活率基本稳定在45%左右,仅次于LGG。胆盐浓度为0.10%时,3株乳杆菌的耐胆盐能力均强于LGG;胆盐浓度为0.20%时,Lp MT-3和LsA F-7仍能存活。3株乳杆菌均具有抑菌活性,对粪肠球菌的抑制最明显,其次是金黄色葡萄球菌,对大肠杆菌、沙门氏菌的抑制作用较差。3株乳杆菌对胆固醇的清除效力依次为Lp MT-3LpM T-5Ls AF-7;清除率依次为Ls AF-7Lp MT-3LpM T-5。【结论】筛选出3株适应人体胃肠液环境、耐胆盐、抑菌及降胆固醇活力强的乳杆菌,可作为进一步开发新的益生菌产品和保健型酸奶的菌株。     

罗氏乳杆菌无细胞上清培养液移除胆固醇能力的研究

本文探讨了罗氏乳杆菌DSM122460无细胞上清培养液(Cell-Free Supernatant,CFS)移除胆固醇的能力。采用邻苯二甲醛法测定DSM122460和对照菌株ST-III发酵过程中及其CFS对胆固醇的移除能力,并研究不同CFS浓度下的移除能力。并采用HPLC法测定CFS对照、热处理组和pH7.0组的胆盐水解酶活力,同时测定其移除胆固醇能力。结果显示,DSM122460不仅在发酵过程中具有较高的移除胆固醇能力,其CFS也表现出较高的移除能力,CFS中含有除胆盐水解酶以外的可移除胆固醇的蛋白类成分。这提示可能存在一种乳酸菌移除胆固醇的新机制。     

The effects of Lactobacillus-fermented milk on lipid metabolism in hamsters fed on high-cholesterol diet

The objective of this study was to evaluate the effects of local Lactobacillus strains (NTU 101 and 102) on cholesterol-lowering effects in vivo. Thirty male hamsters were housed, divided into five groups, and fed on a cholesterol diet (5 g/kg diet) to induce hypercholesterolemia. Milk fermented by Lactobacillus paracasei subsp. paracasei NTU 101, Lactobacillus plantarum NTU 102, and Lactobacillus acidophilus BCRC 17010 was administrated for this study. After treatment with different fermented milk, blood was taken and liver was removed for the determination of lipoproteins, including total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglyceride. Lactobacilli and bifidobacteria decreased (10⁵) in the control group; when hamsters were fed on fermented milk, the number of lactobacilli (10⁷–10⁸) and bifidobacteria (10⁵–10⁷) was increased. Serum and liver total cholesterol levels were significantly reduced by about 26.4, 23.5, and 30.1% and by about 17.7, 15.9, and 13.4% when hamsters were given fermented milk. However, serum HDL-C and LDL-C were also reduced. The results of this study showed that the hypocholesterolemic effect of local Lactobacillus strains was attributed to its ability to lower serum and liver total cholesterol levels. Thus, local Lactobacillus strains could significantly increase probiotic count.     

嗜酸乳杆菌同化MRS培养基中胆固醇能力的研究

目的 对嗜酸乳杆菌在MRS液体培养基中同化胆固醇的能力进行初步研究。方法模拟人体不同胆固醇水平。结果 嗜酸乳杆菌对低胆固醇或正常水平胆固醇同化作用不明显,而对高胆固醇水平的同化作用比较明显。结论 嗜酸乳杆菌具有同化胆固醇的能力。     

喂饲Lactobacillus casei Zhang对大鼠体内脂质代谢的影响

从内蒙古锡林郭勒盟正蓝旗传统酸马奶中分离筛选到1株降胆固醇功效较好的乳杆菌Lactobacillus．casei Zhang。用Lb．casei Zhang灌胃高脂血症模型大鼠研究其对血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)的影响。结果表明:灌胃14 d后,Lb．casei Zhang热致死菌体组大鼠血清中TC浓度较对照组显著降低(P<0．01),TG浓度虽有降低但不呈显著性,同时HDL-C浓度显著升高(P<0．05);Lb．casei Zhang活菌体组大鼠血清中TC、TG浓度均有所降低,HDL-C浓度升高,但未呈显著性。灌胃28 d后,Lb．casei Zhang热致死菌体组大鼠血清中TC浓度较对照组显著降低(P<0．05),Lb．casei Zhang活菌体组TC浓度及两组TG、LDL-C浓度降低,但差异不显著,HDL-C未发生明显的改变。Lb．casei Zhang热致死菌体组和活菌体组粪便中的胆酸含量均增高,但只有热致死菌体组差异显著(P<0．05)。通过实验表明Lb．casei Zhang通过菌体对胆固醇的吸收吸附及减少肝肠循环中的胆酸来达到降血脂的功效。     

Effects of Lactobacillus amylovorus and Bifidobacterium breve on cholesterol

To determine the validity of the hypothesis of assimilation and/or precipitation of cholesterol by Lactobacillus and Bifidobacterium species, culture were undertaken in TPY medium containing oxgall or taurocholic acid. In the case of growing cells, both strains were able to remove cholesterol in the presence of bile salts. Nevertheless, the behaviour was different according to the kind of bile salt. In the presence of taurocholic acid, the removal of cholesterol was due to both bacterial uptake and precipitation. In the presence of Oxgall, bacterial uptake and precipitation were observed for Lactobacillus but only precipitation occurred for Bifidobacterium.     

Probiotic Properties of Lactobacillus Strains Isolated from Tibetan Kefir Grains

The objective of this study was to evaluate the functional properties of lactic acid bacteria (LAB) isolated from Tibetan kefir grains. Three Lactobacillus isolates identified as Lactobacillus acidophilus LA15, Lactobacillus plantarum B23 and Lactobacillus kefiri D17 that showed resistance to acid and bile salts were selected for further evaluation of their probiotic properties. The 3 selected strains expressed high in vitro adherence to Caco-2 cells. They were sensitive to gentamicin, erythromycin and chloramphenicol and resistant to vancomycin with MIC values of 26 µg/ml. All 3 strains showed potential bile salt hydrolase (BSH) activity, cholesterol assimilation and cholesterol co-precipitation ability. Additionally, the potential effect of these strains on plasma cholesterol levels was evaluated in Sprague-Dawley (SD) rats. Rats in 4 treatment groups were fed the following experimental diets for 4 weeks: a high-cholesterol diet, a high-cholesterol diet plus LA15, a high-cholesterol diet plus B23 or a high-cholesterol diet plus D17. The total cholesterol, triglyceride and low-density lipoprotein cholesterol levels in the serum were significantly (P<0.05) decreased in the LAB-treated rats compared with rats fed a high-cholesterol diet without LAB supplementation. The high-density lipoprotein cholesterol levels in groups B23 and D17 were significantly (P<0.05) higher than those in the control and LA15 groups. Additionally, both fecal cholesterol and bile acid levels were significantly (P<0.05) increased after LAB administration. Fecal lactobacilli counts were significantly (P<0.05) higher in the LAB treatment groups than in the control groups. Furthermore, the 3 strains were detected in the rat small intestine, colon and feces during the feeding trial. The bacteria levels remained high even after the LAB administration had been stopped for 2 weeks. These results suggest that these strains may be used in the future as probiotic starter cultures for manufacturing novel fermented foods.     

Identification of novel hypocholesterolemic peptides derived from bovine milk beta-lactoglobulin

This study was designed to clarify the mechanisms of hypocholesterolemic action of beta-lactoglobuline tryptic hydrolysate (LTH) and to identify the novel hypocholesterolemic peptide derived from LTH by screening using Caco-2 cells and animal studies. Serum and liver cholesterol levels were significantly lower in rats fed LTH than in those fed casein tryptic hydrolysate (CTH). The present study suggests that the inhibition of micellar solubility of cholesterol which causes the suppression of cholesterol absorption by a direct interaction between cholesterol mixed micelles, and LTH in the jejunal epithelia is part of the mechanism underlying the hypocholesterolemic action of LTH. Though no one could trace the hypocholesterolemic peptide to any protein origin, we identified, for the first time, a novel hypocholesterolemic peptide, Ile-Ile-Ala-Glu-Lys (IIAEK). Surprisingly, the present study provides the first direct evidence that a new hypocholesterolemic peptide derived from beta-lactoglobuline can powerfully influence serum cholesterol levels and exhibit a greater hypocholesterolemic activity in comparison with that of medicine, beta-sitosterol, in animal studies.     

Identification of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains from breast-fed infant and investigation of their cholesterol-reducing effects

Hypercholesterolemia has been reported to be the main cause of cardiovascular diseases and the leading cause of death. Therefore, decreasing serum cholesterol level is very important for preventing the cardiovascular diseases. It has been supposed that probiotics in human gastrointestinal tract have the ability to decrease serum cholesterol level by reducing the absorption of cholesterol from the intestinal tract and the bile salt deconjugation. In this study, 28 strains of Lactobacillus spp., isolated from breast-fed infant’s feces, were identified and investigated for their bile salt deconjugation ability. The deconjugation ability of the strains was determined by the release of cholic acid resulting from the deconjugation of conjugated bile salts. Research results showed that four of the strains had bile salt deconjugation ability. The strains with deconjugation ability have been identified in species level by using biochemical test, and molecular techniques, API 50CHL test and 16S rRNA gene sequence analysis respectively. LP1, E3, and E9 strains with deconjugation activity were identified as Lactobacillus rhamnosus and GD2 strain as Lactobacillus plantarum. Even if oxgall decreases the viability of bacteria, the highest amount of cholesterol precipitation (42%) was performed by GD2 strain in the presence of 0.3% (w/v) bile. This study demonstrated that the identified Lactobacillus strains had an excellent ability to survive at low pH, a high bile deconjugation ability, and hypocholesterolemic effect in in vitro conditions.     

Bile salts and cholesterol induce changes in the lipid cell membrane of Lactobacillus reuteri

AIMS: The objective of this study was to evaluate the effect of bile salts and cholesterol in the lipid profile of Lactobacillus reuteri CRL 1098 and to determine the relationship existing between these changes: the in vitro removal of cholesterol and the tolerance of the cells to acid and cold stress. METHODS AND RESULTS: Lactobacillus reuteri CRL 1098 was grown in the following media: MRS (deMan Rogosa Sharpe; MC, control medium), MB (MC with bile salts), MCH (MC with sterile cholesterol) and MBCH (MC with bile salts and cholesterol). Fatty acids were determined by analytical gas-liquid chromatography, and phospholipids and glycolipids by colorimetric techniques. The cells from different culture media were subjected to cold and acid stress. The MB cultures displayed a decrease in phospholipids and a low ratio of saturated : unsaturated fatty acids. The presence of the unusual C18 : 0,10-OH and C18 : 0,10-oxo fatty acids was the prominent characteristic of the bile salts growing cells. The relative increase in glycolipids and the changes in the fatty acids profiles of the MB cells would be responsible for the cholesterol remotion. The changes induced by bile salts in the lipid profile did not improve the tolerance of L. reuteri CRL 1098 to freezing and acid stress. CONCLUSIONS: The changes in lipid profiles reported in this study would play a key role in the response of Lactobacilli to environmental stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides useful information about the effect of bile salts on the cell membrane of L. reuteri, a probiotic enterolactobacillus. The steady-state response of the cells subjected to bile stress seems to be the appropriate model for evaluating the bacterial behaviour in detergent-containing gastrointestinal tracts, where the bile salts stress would presumably be continuous.