超声波处理对淫羊藿苷生物转化的影响

利用超声波技术分别对淫羊藿苷粗品及淫羊藿苷转化发酵液进行处理,探讨超声波处理对淫羊藿苷生物转化效果的影响。经过超声波处理的实验组与未经超声波处理的对照组相比,转化效果显著提高;高效液相色谱图显示经超声波处理后淫羊藿苷峰几乎消失,苷元峰突出,超声波处理对淫羊藿苷生物转化效果影响很大。本研究确定超声波的最佳处理条件为频率40kHz,时间30min。     

沼泽红假单胞菌粗酶液生物转化柚皮苷

目的初步探索沼泽红假单胞菌粗酶液生物转化柚皮苷的特点。方法利用高效液相色谱技术,以柚皮苷的降解率为指标,考察不同温度、pH、底物浓度和培养时间对粗酶液降解柚皮苷的影响。在适宜转化条件下,探索底物和产物的含量变化。结果最适转化条件:40℃~50℃,pH 7.0~8.0,最大有效转化底物浓度750μg/mL,15 h时柚皮苷降解率较高为55.31%。在最适转化条件下,底物浓度500μg/mL,培养至17 h柚皮苷被降解完全,柚皮素浓度达到最大值204μg/mL,转化率为85.39%;10~19 h,转化率均大于80.00%,13 h达到最大值94.83%。结论本研究首次发现沼泽红假单胞菌粗酶液能够生物转化柚皮苷,并阐明了不同培养条件对柚皮苷降解的影响,以及转化过程中物质含量的变化。     

一株发酵转化黄芩苷菌株的筛选及鉴定

为实现生物发酵法转化黄芩苷生产活性产物黄芩素,本研究从自然发毛的新鲜黄芩植株根部筛选发酵转化黄芩苷生成黄芩素的菌株,并进行产物分析和菌株鉴定。通过马铃薯葡萄糖培养液(PDB)富集,黄芩药粉培养液初筛,马铃薯葡萄糖琼脂(PDA)平板分离后转入PDB黄芩苷培养液复筛,高效液相色谱法定性及定量分析,得到一株能够高效转化黄芩苷为黄芩素的菌株RM3,该菌在添加0. 1%黄芩苷的PDB培养液中能够将黄芩苷转化为黄芩素,未经任何优化条件下,28℃,150 rpm培养5天后摩尔转化率达到83. 87%。通过对菌落的形态、显微结构观察及ITS序列分析比对,鉴定菌株RM3为青霉菌(Penicillium sp. RM3)。该菌转化黄芩苷生成黄芩素效率高,且清洁环保,可缓解中药资源紧张,满足市场和临床需求,是一株很有前途的黄芩素生产菌。     

不同动物肠道优势需氧菌对黄豆苷原转化菌株转化能力的影响

摘要: 【目的】探讨不同动物肠道优势需氧菌对黄豆苷原转化菌株转化能力的影响。【方法】有氧条件下,采用稀释涂布法分别从ICR 小鼠、芦花鸡、长白猪和獭兔等4 种健康动物肠道中分离优势需氧菌,将不同动物的优势需氧菌分别与不同类型黄豆苷原转化菌株进行厌氧混合培养,高效液相色谱检测培养液中黄豆苷原的转化情况。【结果】16S rRNA 基因序列分析,结合形态学及相关理化特性分析表明,分离的22 株优势需氧菌分属埃希氏菌属(10 株) 、变形菌属(5 株) 、肠球菌属(4 株) 、芽胞杆菌属(2 株) 和假单胞菌属(     

大肠癌细胞分泌的β—葡萄糖醛酸苷酶与其侵袭力关系的研究

以Fischman法检测了6种培养的人大肠癌细胞系分泌至培养液的β-葡萄糖醛酸苷酶活力。结果显示高侵袭力细胞系分泌的该酶活力显著高于低侵袭力细胞系,提示培养的人大肠癌细胞分泌的β-葡萄糖醛酸苷酶水平可作为判断其侵袭力高低的有用指标。     

黄芩苷抗炎作用机制的研究进展

黄芩苷(baicalin)是由黄芩(Scutellaria baicalensis Georgi)根部分离而来的黄酮类物质,为黄芩的主要活性成分。黄芩苷药理作用广泛,具有良好的抗炎效果,其主要机制包括调节肠道菌群、抑制核因子κB(NF-κB)核转位、增加相关microRNA的表达、抑制自噬、调节Treg/Th17平衡等。现综述黄芩苷抗炎症作用机制的最新研究进展,为其深入研究及在临床上的应用提供依据。     

重组β-葡萄糖醛酸苷酶键选择性的半理性改造

为了提高Escherichia coli重组表达的β-葡萄糖醛酸苷酶(PGUS-E)的键选择性,本研究以PGUS-E结构与功能关系的推测为指导,选择了可能影响PGUS-E的键选择性的R329、T369、N467位点进行定点饱和突变,利用薄层层析(TLC)和高效液相色谱(HPLC)对键选择进行筛选,得到优势突变酶R329K、T369V。结果显示:与PGUS-E酶相比,突变酶R329K、T369V键选择性分别提高26.9%、34.3%。突变酶的酶学性质研究表明,突变酶的最适p H和温度与PGUS-E一致,但其酶催化效率下降。由此可见,R329、T369对酶催化的键选择性和酶的活性有显著影响。综上结果,本文应用饱和突变方法改善了PGUS-E的键选择性,为酶的结构和功能关系理解提供了实验依据。     

基于膨润土的层柱黏土固定β-葡萄糖醛酸苷酶的研究

以膨润土制备的层柱黏土为载体,考察给酶量、固定化pH、温度和时间对固定化β-葡萄糖醛酸苷酶活性的影响,并对其操作稳定性进行研究。结果表明：给酶量为2700U／g,最适pH为3．6,固定化温度40℃,固定化60min条件下固定化酶催化活性较高;酶经固定化后其热稳定性及储存稳定性显著提高。     

黄芩的结构与黄芩苷含量的关系

应用解剖学、组织化学定位和植物化学技术,研究了一、二年生黄芩(Scutellaria baicalensis Georgi)营养器官的结构特征与黄芩苷积累的关系.结果表明:黄芩各营养器官的结构类似于一般草本双子叶植物,黄芩苷主要分布在各器官的薄壁组织细胞内,其中以维管组织的薄壁细胞为多.器官间含量的不同表现为根>叶>茎,这与其结构特征相对应,根中维管组织所占比例最大,其中薄壁组织细胞又占主要部分,故根中黄芩苷含量最高.说明根部是黄芩苷的主要贮存部位,符合黄芩以根入药的传统.不同生长期黄芩苷含量测定结果表明,两年生营养生长前期春季根内含量最高,所以春季采挖为好.     

黄芩的结构与黄芩苷含量的关系

应用解剖学、组织化学定位和植物化学技术,研究了一、二年生黄芩（Scutellaria baicalensis Georgi）营养器官的结构特征与黄芩苷积累的关系。结果表明：黄芩各营养器官的结构类似于一般草本双子叶植物,黄芩苷主要分布在各器官的薄壁组织细胞内,其中以维管组织的薄壁细胞为多。器官间含量的不同表现为根〉叶〉茎,这与其结构特征相对应,根中维管组织所占比例最大,其中薄壁组织细胞又占主要部分,故根中黄芩苷含量最高。说明根部是黄芩苷的主要贮存部位,符合黄芩以根入药的传统。不同生长期黄芩苷含量测定结果表明,两年生营养生长前期春季根内含量最高,所以春季采挖为好。     

Biotransformation of 1-Nitropyrene in Intestinal Anaerobic Bacteria

Mutagenic nitroaromatic compounds have recently been found in photocopies, urban atmosphere, automobile exhaust and wastewater. 1-Nitropyrene (1-NP) is readily formed when pyrene, ubiquitous in the environment, is exposed to nitrogen dioxide in the urban atmosphere or in automobile exhaust, and is highly mutagenic, inducing 449 his⁺ revertants/plate/nmol from Salmonella typhimurium strain TA98 in the absence of S9 fraction in the Salmonella-microsome test. It is possible to swallow sputum or some food containing 1-NP and it would come into contact with the normal bacterial flora. We determined the 1-NP nitroreductase activity in environmental and laboratory bacterial strains. We found that the mutagenicity of 1-NP mixed with the feces of a healthy man or a culture of anaerobic bacteria was decreased. The product proved to be 1-aminopyrene (1-AP), based on its fluorescence spectrum, its mass spectrum, and its characteristic thin layer chromatographic and high performance liquid chromatographic patterns. The 1-NP nitroreductase activity of aerobic bacteria was low, but crude extracts from the anaerobic bacteria, i.e., Bacteroides fragilis, B. thetaiotaomicron, B. vulgatus, Fusobacterium mortiferum, F. nucleatum, Clostridium perfringens, C. sporogenes, Bifidobacterium adolescentis, B. bifidum, Eubacterium lentum, E. limosum, and Peptostreptococcus anaerobius, all easily converted 1-NP to 1-AP and proportionally decreased the mutagenic activity of 1-NP.     

Exopolysaccharides Produced by Intestinal Bifidobacterium Strains Act as Fermentable Substrates for Human Intestinal Bacteria

Eleven exopolysaccharides (EPS) isolated from different human intestinal Bifidobacterium strains were tested in fecal slurry batch cultures and compared with glucose and the prebiotic inulin for their abilities to act as fermentable substrates for intestinal bacteria. During incubation, the increases in levels of short-chain fatty acids (SCFA) were considerably more pronounced in cultures with EPS, glucose, and inulin than in controls without carbohydrates added, indicating that the substrates assayed were fermented by intestinal bacteria. Shifts in molar proportions of SCFA during incubation with EPS and inulin caused a decrease in the acetic acid-to-propionic acid ratio, a possible indicator of the hypolipidemic effect of prebiotics, with the lowest values for this parameter being obtained for EPS from the species Bifidobacterium longum and from Bifidobacterium pseudocatenulatum strain C52. This behavior was contrary to that found with glucose, a carbohydrate not considered to be a prebiotic and for which a clear increase of this ratio was obtained during incubation. Quantitative real-time PCR showed that EPS exerted a moderate bifidogenic effect, which was comparable to that of inulin for some polymers but which was lower than that found for glucose. PCR-denaturing gradient gel electrophoresis of 16S rRNA gene fragments using universal primers was employed to analyze microbial groups other than bifidobacteria. Changes in banding patterns during incubation with EPS indicated microbial rearrangements of Bacteroides and Escherichia coli relatives. Moreover, the use of EPS from B. pseudocatenulatum in fecal cultures from some individuals accounted for the prevalence of Desulfovibrio and Faecalibacterium prausnitzii, whereas incubation with EPS from B. longum supported populations close to Anaerostipes, Prevotella, and/or Oscillospira. Thus, EPS synthesized by intestinal bifidobacteria could act as fermentable substrates for microorganisms in the human gut environment, modifying interactions among intestinal populations.     

Biotransformation of Ginsenosides (Rb1, Rb2, Rb3, Rc) in Human Intestinal Bacteria and Its Effect on Intestinal Flora

Ginsenosides, including Rb₁, Rb₂, Rb₃ and Rc, belong to protopanaxadiol-type saponins in Panax ginseng C. A. Mey. Their contents are high in P. ginseng. They could inhibit oxidant stress, enhance immunity, lower blood sugar, resist tumor cells and facilitate other physiological activities. This study aimed to explore the interaction between ginsenosides Rb₁, Rb₂, Rb₃ and Rc and the intestinal flora of healthy people. It also sought to analyse the biotransformation products and pathways of these ginsenosides in in-vitro human intestinal bacteria and their effects on the diversity of human intestinal flora. Human intestinal bacteria were incubated with ginsenosides Rb₁, Rb₂, Rb₃ and Rc at 37 °C under anaerobic conditions. Samples were taken at different timepoints. The transformed products were identified by rapid high-resolution liquid chromatography-quadrupole time-of-flight mass spectrometry. After 48 h of transformation, the transformed product of ginsenosides Rb₁, Rb₂, Rb₃ and Rc was ginsenoside compound K. The transformation rates were 83.5 %, 88.7 %, 85.6 %, and 84.2 %. 16S rRNA sequencing technology was applied to the bioinformatic analysis of faecal samples incubated for 48 h. Relative to the blank control, the relative abundance of Firmicutes and Proteobacteria significantly increased at the phylum level. Moreover, the relative abundance of Bacteroidetes significantly decreased in ginsenosides Rb₁, Rb₂, Rb₃ and Rc. At the genus level, the relative abundance of Escherichia significantly increased, whereas that of Dorea, Prevotella and Megasphaera significantly decreased in all groups. These results showed that Rb₁, Rb₂, Rb₃ and Rc could improve the structure and diversity of human intestinal flora and balance the metabolic process.     

Biotransformation Patterns of 2,4,6-Trinitrotoluene by Aerobic Bacteria

2,4,6-Trinitrotoluene (TNT), a toxic nitroaromatic explosive, accumulates in the environment, making necessary the remediation of contaminated areas and unused materials. Although bioremediation has been utilized to detoxify TNT, the metabolic processes involved in the metabolism of TNT have proven to be complex. The three aerobic bacterial strains reported here (Pseudomonas aeruginosa, Bacillus sp., and Staphylococcus sp.) differ in their ability to biotransform TNT and in their growth characteristics in the presence of TNT. In addition, enzymatic activities have been identified that differ in the reduction of nitro groups, cofactor preferences, and the ability to eliminate-NO₂ from the ring. The Bacillus sp. has the most diverse bioremediation potential owing to its growth in the presence of TNT, high level of reductive ability, and capability of removing-NO₂ from the nitroaromatic ring. Received: 16 May 1997 / Accepted: 19 July 1997     

Degradation of N-Nitrosamines by Intestinal Bacteria

A major proportion of bacterial types, common in the gastrointestinal tract of many animals and man, were active in degrading diphenylnitrosamine and dimethylnitrosamine, the former being degraded more rapidly than the latter. At low nitrosamine concentrations (<0.05 μmol/ml), approximately 55% of added diphenylnitrosamine, 30% of N-nitrosopyrrolidine, and 4% of dimethylnitrosamine were degraded. The route of nitrosamine metabolism by bacteria appears to be different from that proposed for breakdown by mammalian enzyme systems in that carbon dioxide and formate were not produced. In bacteria, the nitrosamines were converted to the parent amine and nitrite ion and, in addition, certain unidentified volatile metabolites were produced from dimethylnitrosamine by bacteria. The importance of bacteria in reducing the potential hazard to man of nitrosamines is discussed.     

Biotransformation of 2,4,6-Trinitrotoluene by Pure Culture Ruminal Bacteria

Twenty-one ruminal bacteria species were tested for their ability to degrade 2,4,6-trinitrotoluene (TNT) within 24 h. Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Lactobacillus vitulinus, Selenomonas ruminantium, Streptococcus caprinus, and Succinivibrio dextrinosolvens were able to completely degrade 100 mg/L TNT, with <5% of the original TNT recovered as diaminonitrotoluene metabolites. Eubacterium ruminantium, Lactobacillus ruminis, Ruminobacter amylophilus, Streptococcus bovis, and Wolinella succinogenes were able to completely degrade 100 mg/L TNT, with 23–60% of the TNT recovered as aminodinitrotoluene and/or diaminonitrotoluene metabolites. Clostridium polysaccharolyticum, Megasphaera elsdenii, Prevotella bryantii, Prevotella ruminicola, Ruminococcus albus, and Ruminococcus flavefaciens were able to degrade 80–90% of 100 mg/L TNT. Desulfovibrio desulfuricans subsp. desulfuricans, Prevotella albensis, and Treponema bryantii degraded 50–80% of the TNT. Anaerovibrio lipolytica was completely inhibited by 100 mg/L TNT. These results indicate that a variety of rumen bacteria is capable of transforming TNT.     

Biotransformation of Trichloroethylene Using Butane-Oxidizing Bacteria

Mixed butane-utilizing cultures were obtained through sequential batch enrichment under 6% (vol/vol) butane in air using one sediment and four different soil samples with varying histories of contamination as inocula. Subsamples of each environmental sample were subjected to one of three pretreatments prior to inoculation: saturation with 30% ethanol, a 15-min exposure to 60°C, or no treatment. Thirteen of the 14 mixed cultures that were obtained appeared to cometabolize trichloroethylene (TCE) while growing at the expense of butane. All 13 caused a loss of at least one-third of TCE from initial aqueous levels between 4 and 25?µg/ml during 6 days of growth on butane provided at initial aqueous concentrations between 90 and 160?µg/ml. Two cultures cometabolized essentially all the available TCE during this test. One culture, which was obtained from an ethanol-pretreated inoculum, vigorously consumed butane while leaving TCE levels essentially unchanged. However, two other mixed cultures originally derived from the same environmental sample as the ineffective culture were moderately active in TCE cometabolism. Thus, TCE-cometabolizing butane oxidizers appeared to be present in all five of the environmental samples used in these studies.     

Stereospecific Biotransformation of Dihydrodaidzein into (3S)-Equol by the Human Intestinal Bacterium Eggerthella Strain Julong 732

Stereochemical course of isoflavanone dihydrodaidzein (DHD) reduction into the isoflavan (3S)-equol via tetrahydrodaidzein (THD) by the human intestinal anaerobic bacterium Eggerthella strain Julong 732 was studied. THD was synthesized by catalytic hydrogenation, and each stereoisomer was separated by chiral high-performance liquid chromatography. Circular dichroism spectroscopy was used to elucidate the absolute configurations of four synthetic THD stereoisomers. Rapid racemization of DHD catalyzed by Julong 732 prevented the substrate stereospecificity in the conversion of DHD into THD from being confirmed. The absolute configuration of THD, prepared by reduction of DHD in the cell-free incubation, was assigned as (3R,4S) via comparison of the retention time to that of the authentic THD by chiral chromatography. Dehydroequol (DE) was unable to produce the (3S)-equol both in the cell-free reaction and in the bacterial transformation, negating the possible intermediacy of DE. Finally, the intermediate (3R,4S)-THD was reduced into (3S)-equol by the whole cell, indicating the inversion of stereochemistry at C-3 during the reduction. A possible mechanism accounting for the racemization of DHD and the inversion of configuration of THD during reduction into (3S)-equol is proposed.Isoflavones are natural dietary phytoestrogens mainly occurring in the leguminous plants, such as soybean. Daidzein and genistein, two major isoflavones in soybean, have received a considerable attention due to their bioactivities beneficial to the human health, including estrogenic (9), anticancer (14), antioxidant (1, 21), and cardioprotective (11) activities. Recently, special interest has been focused on the biological effects of the daidzein metabolites, which are being actively studied for drug development (5, 16).Daidzein is known to be metabolized in the human intestine by the resident microflora, and various metabolites, such as dihydrodaidzein (DHD), 7,4′-dihydroxyisoflavan-4-ol (tetrahydrodaidzein; THD), 7,4′-dihydroxyisoflav-3-ene (dehydroequol; DE), O-desmethylangolensin (O-DMA), and equol, are detected in the human urine (Fig. ​(Fig.1)1) (6, 7, 10). Among the metabolites, (3S)-equol has about 100 times higher estrogenic activity than daidzein itself (15). However, only about 30 to 50% of humans can produce equol from daidzein (12). In addition, a high correlation was found between the beneficial effects on females by soy food intake and the presence of equol in their urine (4). Therefore, the ability to metabolize daidzein into equol conferred by the intestinal microflora in human is regarded as a hallmark of daidzein responsiveness (3, 34).Open in a separate windowFIG. 1.Proposed pathway for isoflavone daidzein reduction by intestinal microflora leading to equol formation. The absolute configuration of THD is depicted as (3R,4S) according to the conclusion of the present study.The daidzein metabolic sequence has been proposed based on the presence of various metabolites of daidzein produced by the human intestinal bacteria; daidzein is reduced into DHD, then into THD and DE, and finally into (3S)-equol in sequential reactions (Fig. ​(Fig.1)1) (7, 10). However, the pathway and the individual reactions in the pathway have not been fully elucidated partly due to the unavailability of pure microbial isolates.To confirm the proposed metabolic pathway of the human intestinal microflora, attempts have been made to isolate the daidzein-metabolizing bacterial phenotype from human feces. The reduction of daidzein into equol through the cooperation of the microfloral community in the human intestine is thought to be likely and was demonstrated by using the whole microflora from human (2) and monkey (23) feces. However, daidzein metabolism by the whole-rat intestinal flora results in the formation of DHD, and further reaction leading to the formation of unknown aliphatic compounds was implied (24).Various bacterial phenotypes have been suggested to have a responsible role in daidzein metabolism in the small intestines of animals. An anaerobic bacterium, Clostridium sp. strain HGH6 (8), and a Clostridium-like strain, TM-40 (27), were found to reduce daidzein into DHD, and the C-ring cleavage was executed by a strain of Eubacterium (25). A human intestinal bacterium that could produce equol was first reported in 2005. Eggerthella strain Julong 732, which could not reduce daidzein into DHD, was found to reduce DHD into equol (28), thus establishing the aforementioned reduction sequence leading to the biologically active (S)-equol from daidzein via DHD in the human intestine (7, 10). Eggerthella species are normal residents of the human gut, and some species are implicated as causative agents of bacteremia (13). The microbial phenotypes that can reduce daidzein all the way into equol were recently isolated from mice (19), rats (20), pigs (33), and humans (18, 32). Nevertheless, the enzymology of the reduction, such as the nature of the enzyme responsible and the reaction mechanism, has yet to be established.In the present study, the enzyme reaction mechanisms of two consecutive reduction reactions converting DHD into (3S)-equol were stereochemically assessed. To this end, four stereoisomers of THD were first synthesized, and their absolute configurations were determined. With the correlation of the absolute configuration of the synthetic THD isomers and the circular dichroism (CD) spectra at hand, the absolute configuration of THD produced through the cell-free bacterial reduction of DHD was determined. Each synthetic THD stereoisomer was then tested as a metabolic feedstock for (3S)-equol production during the growth of Julong 732. We found that only one of the THD steroisomers, (3R,4S)-THD, the very stereoisomer produced by the bacterial DHD reduction, was converted into (3S)-equol and that the final reduction accompanied the inversion of the configuration at C-3 of THD.