降胆固醇乳酸菌的应用及其机理研究进展

乳酸菌除了能够改善肠道微生态环境促进人体健康外,有些乳酸菌还具有能降低人体血清胆固醇的特性。近年来许多研究都已证明某些乳杆菌、双歧杆菌及肠球菌等具有调节血脂代谢、降低人体血清胆固醇的能力。本文综述了降胆固醇乳酸菌的体外筛选模型及其降胆固醇的作用机理,以期为进一步开发相关产品提供技术依据。     

乳酸菌的益生作用及其应用研究进展

乳酸菌是一类有益于人类健康的重要微生物,它的菌体、无细胞提取物和胞外分泌物都有益生作用,其益生作用主要表现在清除自由基、抗脂质过氧化性、降低胆固醇含量、延缓机体老化和消除病变等方面。因而,含有乳酸菌的益生菌产品在饲料生产、食品和临床上都有着广泛的应用。     

Effect of gastric and small intestinal digestion on lactic acid bacteria activity in a GIS1 simulator

The selection of probiotic strains resistant to gastrointestinal transit is an important stage when developing supplements that contain viable biomass. A total of six strains belonging to different genotypes were tested and compared with both a positive and negative control (Lactobacillus plantarum 5s). Significant differences were found between strains as a result of gastrointestinal transit using the in vitro GIS1 static simulator. The Lactobacillus rhamnosus 428ST strain showed maximum viability as a result of in vitro transit, featuring a survival capacity value, Cs, of over 50 ± 0.01%. The remaining genotypes that were tested showed significant reductions in the enzymes and bile salts at the time of action. The value of the survivability capacity was directly correlated with the synthesis of exopolysaccharides and lactic acid. The test results of the GIS1 system have been compared with those of other studies on gastrointestinal transit resistance that used dynamic models.     

Microbiology of the malolactic fermentation: Molecular aspects

Abstract Malolactic fermentation conducted by lactic acid bacteria follows alcoholic fermentation during winemaking, and several positive effects make it indispensable for most wines. Research has focused on the growth and physiology of lactic acid bacteria in wine; resulting in the design of malolactic starter cultures. Future work on these starters will concentrate on aromatic changes as additional criteria for strain selection. Although the main features of the malolactic enzyme and its gene are known, the detailed mechanism of the malolactic reaction remains unclear. Cloning and expression of this activity in enological strains of Saccharomyces cereuisiae might be one of the next most important advances in the control of malic acid degradation in wine.     

益生菌地衣芽孢杆菌治疗鸡腹泻机理初探

目的：探讨益生菌地衣芽孢杆菌饲喂鸡腹泻的治疗效果以及分子机理。方法：从养鸡场土壤中选择性分离芽孢杆菌,通过优 化条件提高产生芽孢的比例;利用芽孢和大米混合后饲喂腹泻鸡,通过克隆测序分析腹泻鸡肠道微生物群落在饲喂前后的差异。 结果：我们分离到一株地衣芽孢杆菌,通过优化条件使产芽孢的比例提高到60 %。利用芽孢饲喂一周后能够有效地治疗鸡腹泻。 分析鸡肠道微生物群落发现：鸡腹泻后其肠道细菌群落的多样性和均匀度降低。造成腹泻鸡肠道主要菌群为动胶杆菌（Zoogloea sp.）;经芽孢饲喂后,主要菌群转变为拟杆菌（Bacteroides sp.）,与健康鸡的肠道微生物主要菌群相似。结论：我们筛选到一株可能 治疗鸡腹泻的芽孢杆菌,通过优化培养条件能够提高其产芽孢比例;腹泻鸡经芽孢饲喂后,其肠道中致病菌被益生菌所代替,形 成正常的肠微生物菌群而恢复健康。     

In Vitro Comparison of the Effects of Probiotic,Commensal and Pathogenic Strains on Macrophage Polarization

Macrophages are important with respect to both innate and adaptive immune responses and are known to differentiate into pro-inflammatory M1- or anti-inflammatory M2-phenotypes following activation. In order to study how different bacteria affect macrophage polarization, we exposed murine RAW 264.7 macrophages to sixteen different strains representing probiotic strains, pathogens, commensals and strains of food origin. Increased inducible nitric oxide synthase (iNOS) or arginase-1 gene expression indicates M1 or M2 polarization, respectively, and was quantified by qRT-PCR. Strains of Escherichia and Salmonella elevated iNOS expression more so than strains of Enterococcus, Lactobacillus and Lactococcus, indicating that Gram-negative strains are more potent M1 inducers. However, strain-specific responses were observed. For instance, Escherichia coli Nissle 1917 was a poor inducer of iNOS gene expression compared to the other E. coli strains, while Enterococcus faecalis Symbioflor-1 was more potent in this respect compared to all the eleven Gram-positive strains tested. Macrophage polarization was further characterized by quantifying secreted pro- and anti-inflammatory cytokines. Exposure to the pathogen E. coli 042 produced a cytokine profile indicating M1 differentiation, which is in accordance with the PCR data. However, exposure to most strains resulted in either high or low secretion levels of all cytokines tested, rather than a clear M1 or M2 profile. In general, the Gram-negative strains induced high levels of cytokine secretion compared to the Gram-positive strains. Interestingly, strains of human origin had a higher impact on macrophages compared to strains of food origin.     

Impact of fermentation pH and temperature on freeze-drying survival and membrane lipid composition of <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> Si3

During the industrial stabilization process, lactic acid bacteria are subjected to several stressful conditions. Tolerance to dehydration differs among lactic acid bacteria and the determining factors remain largely unknown. Lactobacillus coryniformis Si3 prevents spoilage by mold due to production of acids and specific antifungal compounds. This strain could be added as a biopreservative in feed systems, e.g. silage. We studied the survival of Lb. coryniformis Si3 after freeze-drying in a 10% skim milk and 5% sucrose formulation following different fermentation pH values and temperatures. Initially, a response surface methodology was employed to optimize final cell density and growth rate. At optimal pH and temperature (pH 5.5 and 34 °C), the freeze-drying survival of Lb. coryniformis Si3 was 67% (±6%). The influence of temperature or pH stress in late logarithmic phase was dependent upon the nature of the stress applied. Heat stress (42 °C) did not influence freeze-drying survival, whereas mild cold- (26 °C), base- (pH 6.5), and acid- (pH 4.5) stress significantly reduced survival. Freeze-drying survival rates varied fourfold, with the lowest survival following mild cold stress (26 °C) prior to freeze-drying and the highest survival after optimal growth or after mild heat (42 °C) stress. Levels of different membrane fatty acids were analyzed to determine the adaptive response in this strain. Fatty acids changed with altered fermentation conditions and the degree of membrane lipid saturation decreased when the cells were subjected to stress. This study shows the importance of selecting appropriate fermentation conditions to maximize freeze-drying viability of Lb. coryniformis as well as the effects of various unfavorable conditions during growth on freeze-drying survival.     

Fermentation of wheat: Effects of backslopping different proportions of pre-fermented wheat on the microbialand chemical composition

Abstract

The objective of the study was to examine effect of backslop on the chemical and microbiological characteristics of fermented wheat (FW). Coarsely ground wheat was mixed with water (1:3 wt/wt) and inoculated with 6 log cfu ml^?1 each of an overnight culture of Lactobacillus plantarum and Pediococcus pentosaceus. Four fermentation treatments were conducted in 45 l, closed, PVC containers over 48 hours. Three treatments investigated the benefits of the addition of previously fermented wheat (backslopping, BSL) at different proportions (0.20, 0.33 or 0.42 kg) to freshly prepared wheat. The control treatment contained no addition of BSL. Elimination of coliforms from the FW within 48 h was only achieved through backslopping; where coliform bacteria counts decreased from approximately 6.5 log₁₀ cfu ml^?1 to less than 3 log₁₀ cfu ml^?1. There was no apparent advantage in increasing the backslop proportion above 0.20. However, the exclusion of coliform bacteria required the pH to remain below 4.0 for at a minimum of 24 h. The results of these studies indicate that fermentation of wheat has the potential to reduce the risk of feed-borne colibacillosis and provides a practical alternative to producers that cannot ferment multiple diets or have limited fermentation capacity.     

Antagonistic,Anti-oxidant,Anti-inflammatory and Anti-diabetic Probiotic Potential of Lactobacillus agilis Isolated From the Rhizosphere of the Medicinal Plants

Potential probiotic bacteria can be used as a biotherapeutic agent and a sustainable alternative to antibiotics, as an anti-oxidative, anti-inflammatory, and anti-diabetic agent without causing any serious side effects. Mostly human-friendly Lactic acid bacteria (LAB) have been isolated from the animal-human origin to be used as biotherapeutic agents or to produce useful metabolites (nutraceutical). However, less information is known about the role of medicinal plants associated LAB as biotherapeutic agents. The isolation of 115 human-friendly Lactobacillus strains was done from the rhizosphere of the medicinal plants Ocimum tenuiflorum, Azadirachta indica, Ficus carica. The obtained bacteria were then tested for their safe status before being using it for a beneficial purpose. Out of 115 strains, 29 (25%) were negative for blood hemolytic activities. Among these 29 isolates, three isolates did not show a breakdown of gelatin and were recognized as safe. Antibiotic resistance assay showed resistance of two of them against antibiotics discs of Streptomycin (10 µg), Ciprofloxacin (20 µg), Vancomycin (30 µg), Metronidazole (10 µg), Ampicillin (5 µg), Chloramphenicol (30 µg), Kanamycin (30 µg), Erythromycin (15 µg), Penicillin (10 µg) and Tetracycline (30 µg). The bacterial isolate (T-2) was found safe that was identified as Lactobacillus agilis by sequence analysis of 16 s rRNA gene and processed in vitro as an anti-bacterial, anti-oxidant, anti-diabetic, and anti-inflammatory agent. Free radical scavenging activities and inhibition of α-amylase activities for Lactobacillus agilis were found relative to standard drug values as 68% and 73% and 51.3% and 65.3%, respectively. The in-vitro anti-inflammatory assay showed 61.6% (Lactobacillus agilis) while showed 69% (aspirin) activity for denaturation albumin protein. The results suggested that Lactobacillus agilis can be used as a potential probiotic strain as well as can be used to produce nutraceuticals.     

几株乳酸菌益生潜力及降胆固醇的研究

采用体外试验方法研究7株乳酸菌和双歧杆菌对人体消化道不良环境的抵抗能力、在肠道上皮的黏附定植能力及其降胆固醇潜力。结果表明:受试菌对CCL-187细胞粘附能力存在显著差异(P<0.05),粪肠球菌M1粘附能力最强;各菌对胃液和胰液都有较强耐受能力,在模拟胃液和胰液中分别培养3h和4h后仍能保证95%以上存活;在含胆汁培养基中受到不同程度抑制,但都能存活;受试菌在生长过程中能够使环境中胆固醇降低从25%～54%不等,粪肠球菌A30和A31和嗜酸乳杆菌A878和PB1能使胆固醇降低40%以上,是良好的降胆固醇备选菌株。     

Probiotic potential of lactic acid bacteria isolated from fermented dairy milks on antiproliferation of colon cancer cells

Fifty-four strains of lactic acid bacteria obtained from fermented dairy milks were investigated for possible use as probiotics and for colon cancer biological products. Five of these strains inhibited growth of eight food-borne pathogens including Helicobacter pylori, Escherichia coli, and Salmonella typhimurium. Three of these strains survived at pH 2.5 and in 0.3% bile salts. Additionally they produced no haemolysis, were resistant to kanamycin and adhered to Caco-2 cells. 16S rRNA gene sequences of probiotic strains indicated that RM11 and RM28 were Enterococcus faecium and Lactobacillus fermentum, respectively. Both the cultured medium and live whole cells from probiotic strains were tested for antiproliferation of colon cancer cells through MTT and Trypan Blue exclusion assays. The probiotic strains of E. faecium RM11 and L. fermentum RM28 also triggered antiproliferation of colon cancer cells at the rates of 21–29%, and 22–29%, respectively. This suggested that both strains could be used as potential probiotics in functional food or for colon cancer biological products. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Immobilization of Infant Fecal Microbiota and Utilization in an <Emphasis Type="Italic">in vitro</Emphasis> Colonic Fermentation Model

Bacteria isolated from infant feces were immobilized in polysaccharide gel beads (2.5% gellan gum, 0.25% xanthan gum) using a two-phase dispersion process. A 52-day continuous culture was carried out in a single-stage chemostat containing precolonized beads and fed with a medium formulated to approximate the composition of infant chyme. Different dilution rates and pH conditions were tested to simulate the proximal (PCS), transverse (TCS), and distal (DCS) colons. Immobilization preserved all nine bacterial groups tested with survival rates between 3 and 56%. After 1 week fermentation, beads were highly colonized with all populations tested (excepted Staphylococcus spp. present in low numbers), which remained stable throughout the 7.5 weeks of fermentation, with variations below 1 log unit. However, free-cell populations in the circulating liquid medium, produced by immobilized cell growth, cell-release activity from gel beads, and free-cell growth, were altered considerably by culture conditions. Compared to the stabilization period, PCS was characterized by a considerable and rapid increase in Bifidobacterium spp. concentrations (7.4 to 9.6 log CFU/mL), whereas Bifidobacterium spp., Lactobacillus spp., and Clostridium spp. concentrations decreased and Staphylococcus spp. and coliforms increased during TCS and DCS. Under pseudo-steady-state conditions, the community structure developed in the chemostat reflected the relative proportions of viable bacterial numbers and metabolites generally encountered in infant feces. This work showed that a complex microbiota such as infant fecal bacteria can be immobilized and used in a continuous in vitro intestinal fermentation model to reproduce the high bacterial concentration and bacterial diversity of the feces inoculum, at least at the genera level, with a high stability during long-term experiment.     

In Vitro and In Vivo Survival and Transit Tolerance of Potentially Probiotic Strains Carried by Artichokes in the Gastrointestinal Tract

The ability of potentially probiotic strains of Lactobacillus plantarum and Lactobacillus paracasei to survive on artichokes for at least 90 days was shown. The anchorage of bacterial strains to artichokes improved their survival in simulated gastrointestinal digestion. L. paracasei IMPC2.1 was further used in an artichoke human feeding study involving four volunteers, and it was shown that the organism could be recovered from stools.     

Progress and potential in the biotechnology of lactic acid bacteria

Abstract: Current activities and future prospects for the biotechnology of lactic acid bacteria are reviewed. Genetic engineering technology, including advances and limitations of plasmid vectors and chromosomal integration strategies are discussed together with the status of gene expression and the importance of in vivo gene transfer systems and transposition. Areas of biotechnological application considered include proteolysis and flavour generation, bacteriophage resistance, antimicrobials, metabolic engineering and the possible uses of lactic acid bacteria in relation to health.     

Microbes versus microbes: immune signals generated by probiotic lactobacilli and their role in protection against microbial pathogens

Probiotic lactic acid bacteria can signal the immune system through innate cell surface pattern recognition receptors or via direct lymphoid cell activation. In some cases, this action has been shown to be sufficient to modulate local- and systemic-level in vivo immune responses. Practical applications of probiotics include their use in anti-tumour and anti-allergy immunotherapy, but there is also increasing evidence that some probiotics can stimulate a protective immune response sufficiently to enhance resistance to microbial pathogens. This review outlines the experimental and clinical evidence for enhanced anti-microbial immune protection by probiotic lactic acid bacteria, focussing on those studies where a correlative or suggestive link has been shown between immune modulation and enhanced protection.     

一株寡营养细菌胞外多糖的摇瓶发酵研究

从新疆的寡营养环境——古尔班通古特沙漠中分离到一株寡营养细菌Azotobacter sp．(1～15mg碳/L培养基),通过进行Azotobacter sp．菌的单因子优化培养基的试验、摇瓶培养工艺条件的优化试验(培养温度、培养时间、初始pH值、溶氧量),确定了菌种生长与营养需求等主要因子与胞外多糖产量、粘度的关系,结果表明,摇瓶发酵的最适宜条件为:以蔗糖为碳源,碳酸钙含量为2g/L,初始pH值为7左右,种龄72～84h,磷酸二氢钾、硫酸镁的含量分别为0．3g/L、0．1g/L,接种体积分数15%,于37℃摇瓶培养72h,250mL摇瓶装液量为50mL,在适宜条件下粘多糖的产量最大可达到1145．94μg/mL,粘性可达9200 mPa·s。     

In Vitro Continuous Fermentation Model (PolyFermS) of the Swine Proximal Colon for Simultaneous Testing on the Same Gut Microbiota

In vitro gut modeling provides a useful platform for a fast and reproducible assessment of treatment-related changes. Currently, pig intestinal fermentation models are mainly batch models with important inherent limitations. In this study we developed a novel in vitro continuous fermentation model, mimicking the porcine proximal colon, which we validated during 54 days of fermentation. This model, based on our recent PolyFermS design, allows comparing different treatment effects on the same microbiota. It is composed of a first-stage inoculum reactor seeded with immobilized fecal swine microbiota and used to constantly inoculate (10% v/v) five second-stage reactors, with all reactors fed with fresh nutritive chyme medium and set to mimic the swine proximal colon. Reactor effluents were analyzed for metabolite concentrations and bacterial composition by HPLC and quantitative PCR, and microbial diversity was assessed by 454 pyrosequencing. The novel PolyFermS featured stable microbial composition, diversity and metabolite production, consistent with bacterial activity reported for swine proximal colon in vivo. The constant inoculation provided by the inoculum reactor generated reproducible microbial ecosystems in all second-stage reactors, allowing the simultaneous investigation and direct comparison of different treatments on the same porcine gut microbiota. Our data demonstrate the unique features of this novel PolyFermS design for the swine proximal colon. The model provides a tool for efficient, reproducible and cost-effective screening of environmental factors, such as dietary additives, on pig colonic fermentation.     

In vitro Effects of Prebiotics and Synbiotics on Apis cerana Gut Microbiota

This study aimed to investigate in vitro effects of the selected prebiotics alone, and in combination with two potential probiotic Lactobacillus strains on the microbial composition of Apis cerana gut microbiota and acid production. Four prebiotics, inulin, fructo-oligosaccharides, xylo-oligosaccharides, and isomalto-oligosaccharides were chosen, and glucose served as the carbon source. Supplementation of this four prebiotics increased numbers of Bifidobacterium and lactic acid bacteria while decreasing the pH value of in vitro fermentation broth inoculated with A. cerana gut microbiota compared to glucose. Then, two potential probiotics derived from A. cerana gut at different dosages, Lactobacillus helveticus KM7 and Limosilactobacillus reuteri LP4 were added with isomalto-oligosaccharides in fermentation broth inoculated with A. cerana gut microbiota, respectively. The most pronounced impact was observed with isomalto-oligosaccharides. Compared to isomalto-oligosaccharides alone, the combination of isomalto-oligosaccharides with both lactobacilli strains induced the growth of Bifidobacterium, LAB, and total bacteria and reduced the proliferation of Enterococcus and fungi. Consistent with these results, the altered metabolic activity was observed as lowered pH in in vitro culture of gut microbiota supplemented with isomalto-oligosaccharides and lactobacilli strains. The symbiotic impact varied with the types and concentration of Lactobacillus strains and fermentation time. The more effective ability was observed with IMO combined with L. helveticus KM7. These results suggested that isomalto-oligosaccharides could be a potential prebiotic and symbiotic with certain lactobacilli strains on A. cerana gut microbiota.     

ClpL is essential for induction of thermotolerance and is potentially part of the HrcA regulon in Lactobacillus gasseri

Stress-inducible proteins are likely to contribute to the survival and activity of probiotic bacteria during industrial processes and in the gastrointestinal tract. The recently published genome sequence of probiotic Lactobacillus gasseri ATCC 33323 suggests the presence of ClpC, ClpE, ClpL, and ClpX from the Clp ATPase family of stress proteins. The heat-shock response of L. gasseri was studied using 2-D DIGE. A total of 20 protein spots showing significant (p<0.05) increase in abundance after 30 min heat-shock were identified, including DnaK, GroEL, ClpC, ClpE, and ClpL. To study the physiological role of ClpL, one of the most highly induced proteins during heat-shock, its corresponding gene was inactivated. The DeltaclpL mutant strain had growth characteristics that were indistinguishable from wild-type under several stress conditions. However, in the absence of functional ClpL, L. gasseri exhibited drastically reduced survival at a lethal temperature and was unable to induce thermotolerance. Genome sequences indicate that the expression of clp genes in several Lactobacillus species is regulated by HrcA, instead of CtsR, the conserved clp gene regulator of low G+C Gram-positive bacteria. Electrophoretic mobility shift assays using L. gasseri HrcA protein and clpL upstream fragments revealed, for the first time, a direct interaction between HrcA and the promoter of a clp gene from a Lactobacillus.