益生菌制剂对抗生素诱导腹泻模型小鼠肠道菌群的恢复

本论文采用肠上皮细胞模型HT-29细胞系研究8株植物乳杆菌在HT-29细胞上的黏附性,并研究黏附性较强的菌株及其胞外多糖（ExopolySaccharides,EPS）抑制大肠杆菌(E?coliATCC25922)在HT-29细胞上黏附及刺激HT-29细胞产生炎性因子的作用。结果表明8株植物乳杆菌在HT-29细胞上的黏附性差异较大：黏附性最强的植物乳杆菌35通过取代、竞争和排阻方式抑制大肠杆菌在HT-29细胞上黏附,抑制黏附率分别为30%、33%和59%,其EPS在作用浓度为500 μg/mL时对大肠杆菌的抑制黏附率为32%。植物乳杆菌35可抑制大肠杆菌刺激HT-29细胞产生IL-8,通过取代、竞争、排阻方式抑制大肠杆菌刺激HT-29细胞产生IL-8,抑制率分别为3%、28%和40%;其EPS抑制大肠杆菌刺激HT-29细胞产生IL-8具有浓度效应,浓度为500 μg/mL时抑制率最高,为50%。而对于IL-10表达量的影响均不显著。结果表明植物乳杆菌35具有抑制大肠杆菌引起肠炎的潜在益生功能。     

植物乳杆菌35对大肠杆菌在肠上皮细胞HT-29上黏附及刺激产生促炎细胞因子的影响

本论文采用肠上皮细胞模型HT-29细胞系研究8株植物乳杆菌在HT-29细胞上的黏附性,并研究黏附性较强的菌株及其胞外多糖(Exopoly Saccharides,EPS)抑制大肠杆菌(E.coli ATCC25922)在HT-29细胞上黏附及刺激HT-29细胞产生炎性因子的作用。结果表明8株植物乳杆菌在HT-29细胞上的黏附性差异较大:黏附性最强的植物乳杆菌35通过取代、竞争和排阻方式抑制大肠杆菌在HT-29细胞上黏附,抑制黏附率分别为30%、33%和59%,其EPS在作用浓度为500μg/mL时对大肠杆菌的抑制黏附率为32%。植物乳杆菌35可抑制大肠杆菌刺激HT-29细胞产生IL-8,通过取代、竞争、排阻方式抑制大肠杆菌刺激HT-29细胞产生IL-8,抑制率分别为3%、28%和40%;其EPS抑制大肠杆菌刺激HT-29细胞产生IL-8具有浓度效应,浓度为500μg/mL时抑制率最高,为50%。而对于IL-10表达量的影响均不显著。结果表明植物乳杆菌35具有抑制大肠杆菌引起肠炎的潜在益生功能。     

乙醇胁迫对植物乳杆菌膜生理及黏附性的影响

目的：探讨植物乳杆菌在乙醇胁迫下黏附性变化及其抗乙醇胁迫的生理响应机制。方法：用不同体积分数的乙醇胁迫植物乳杆菌,测定菌体存活率、细胞膜完整性及超微结构的变化;采用人体结肠癌细胞HT-29作为体外黏附模型,测定乙醇胁迫后菌体对HT-29细胞的黏附率;通过气相色谱-串联质谱法分析菌体细胞膜脂肪酸的变化。结果：随着乙醇体积分数的增加,植物乳杆菌的存活率逐渐下降,菌体细胞膜完整性及其表面结构遭到破坏,对HT-29细胞黏附率降低,同时,细胞膜中脂肪酸的不饱和脂肪酸比例升高。结论：乙醇胁迫可降低菌体黏附HT-29细胞的能力,植物乳杆菌细胞膜中脂肪酸的组成及其完整性在抗乙醇胁迫过程中发挥重要作用。     

1株具有拮抗空肠弯曲杆菌作用的唾液乳杆菌的研究

对分离自泡菜、发酵奶酒以及新鲜婴儿粪便的11株乳酸菌(已证明其发酵上清液对空肠弯曲杆菌生长具有明显抑制作用)进行研究。经对其人工胃、肠液的耐受能力测定,表明这11株乳酸菌通过胃肠道后均能存活。经对全部乳酸菌抑制共培养空肠弯曲杆菌生长能力的测定及其抑菌物质的鉴定,得到对共培养空肠弯曲杆菌生长具有强烈抑制作用的1株唾液乳杆菌ZX5,该菌发酵上清液的抑菌物质很可能为有机酸、过氧化氢及细菌素类物质。测定该株唾液乳杆菌抑制空肠弯曲杆菌黏附及侵袭HT-29细胞的能力,结果表明ZX5可将空肠弯曲杆菌对HT-29细胞的黏附及侵袭率降低38%~55%。其中,以排阻作用最为明显。     

乳杆菌胞外多糖抗氧化活性研究

从本实验室保藏的8株产胞外多糖的乳杆菌:干酪乳杆菌-Y3,干酪乳杆菌-Y4,干酪乳杆菌-Y16,植物乳杆菌-Y41,植物乳杆菌-Y42,植物乳杆菌-Y44,干酪乳杆菌-Y35,鼠李糖乳杆菌LGG中,筛选出1株产抗氧化活性多糖的菌株,评价其抗氧化性并分析其结构。采用酸沉醇提法从8株乳杆菌的MRS培养液中提取胞外多糖,并用苯酚硫酸法和考马斯亮蓝法测定粗多糖和蛋白含量,同时测定胞外多糖的1,1-二苯基-2-三硝基苯肼自由基(DPPH)清除率、2,2’-连氮基-双-(3-乙基苯并二氢噻唑啉-6-磺酸)自由基(ABTS)清除率、羟自由基(·OH)清除率、铁离子还原力(FRAP)、氧自由基吸收能力(ORAC)及对ABAP氧化损伤HT-29细胞的保护作用,以评价8株菌胞外多糖抗氧化活性。通过主成分分析法得到产高抗氧化活性胞外多糖的菌株为Y42。进一步研究发现:随着菌株Y42胞外多糖作用浓度的降低,对ABAP氧化损伤HT-29细胞的保护作用降低,然而都显著高于氧化损伤组(P0.05)。菌株Y42胞外多糖显著上调HT-29细胞过氧化物酶和超氧化物歧化酶的表达量(P0.05),HT-29细胞内谷胱甘肽过氧化物酶活性也显著高于氧化组(P0.05)。菌株Y42胞外多糖由中性多糖和酸性多糖组成,其单糖组成为甘露糖、葡萄糖醛酸、氨基葡萄糖、氨基半乳糖、半乳糖,它们的物质的量比为11.30∶3.51∶10.64∶7.53∶7.19。红外光谱扫描显示菌株Y42胞外多糖具有典型的特征吸收峰,属于吡喃糖环的骨架模式。     

乳杆菌耐消化应激能力及消化应激对其肠道黏附能力的影响

首先通过体外模拟唾液-胃液-肠液应激实验研究乳杆菌的耐消化应激能力,然后研究其对肠道黏蛋白和Caco-2细胞的黏附及抑制肠道病原菌黏附的能力,最后探讨消化应激对乳杆菌黏附能力的影响。结果表明,副干酪乳杆菌W125、m111和发酵乳杆菌146在依次经过模拟唾液-胃液-肠液应激后存活率分别为2.70%、3.53%及11.15%,活菌数分别为7.46、7.24（lg（CFU/mL））及 8.35（lg（CFU/mL））,且对黏蛋白和Caco-2细胞的黏附率显著高于其他菌株（P＜0.05）,分别为15.67%、8.75%、8.38%和11.47%、21.34%、10.44%;3 株菌株均可通过排除、竞争和替代的方式抑制大肠杆菌CICC10899和沙门菌WX29对肠道的黏附,黏附抑制率均大于13.51%;消化应激显著降低了副干酪乳杆菌W125和发酵乳杆菌146对肠道的黏附能力（P＜0.05）,但显著增加了副干酪乳杆菌m111的黏附能力（P＜0.05）,黏附率由17.60%增加到30.45%,且主要黏附素由消化应激前的表层蛋白变为应激后的蛋白和多糖;消化应激前后副干酪乳杆菌m111均为长梭状、圆润的杆状,表层物质的厚度也没有发生显著变化（P＞0.05）。副干酪乳杆菌m111具有较强的耐消化应激以及对肠道黏附的能力,且消化应激可提高其黏附肠道的能力,是1 株潜在的具有良好应用价值的益生乳杆菌。     

乳酸菌胞外多糖对结肠癌HT-29细胞增殖的影响

从15株大连工业大学大连市益生菌功能研究重点实验室保藏植物乳杆菌中筛选出对人结肠癌细胞HT-29抑制率最高的乳杆菌胞外多糖,对其进行分离纯化并研究多糖对HT-29细胞增殖的影响。通过苯酚硫酸法测定15种乳酸菌胞外多糖产量、3-(4,5-二甲基吡啶-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四唑[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,MTS]测定不同浓度的粗多糖对HT-29细胞的抑制率,最终筛选出高产胞外多糖、对HT-29细胞增殖抑制率高的菌株Lactobacillus plantarum12所产胞外多糖,其胞外多糖的糖含量为54%、蛋白含量为3.6%。对其胞外多糖进行DEAE-Sepharose Fast Flow离子柱和Sepharose CL-6B凝胶柱的分离纯化,得到中性多糖组分EPS12-1和酸性多糖组分EPS12-2,2个多糖组分的糖含量分别为31.82%和35.21%,通过MTS法测定L. plantarum12菌所产粗多糖、多糖组分EPS12-1和EPS12-2分别在50、100、250、500μg/mL浓度时对HT-29细胞的抑制率,发现多糖浓度为250μg/mL时,对HT-29细胞增殖达到了最佳抑制效果,抑制率分别为27.58%、7.11%、12.00%。当多糖浓度为250μg/mL时,HT-29细胞凋亡率、活性氧(reactive oxygen species,ROS)水平、caspase 8活性,均处在较高水平。L. plantarum12胞外多糖通过增加ROS酶活和激活caspase 8来抑制HT-29增殖。     

产胞外多糖乳杆菌的筛选及多糖功能的研究

本论文从大连地区海产品消化腺中筛选产胞外多糖(EPS)的乳杆菌属菌株,并研究其所产EPS的功能特性,选出一株目标菌,对其EPS分离纯化,测定纯化后各多糖组分的相对分子量。获得EPS产量相对较高的菌株经16S r RNA序列测定鉴定为Lactobacillus plantarum subsp.plantarum-4,Lactobacillus plantarum-12,Lactobacillus plantarumsubsp.plantarum-49;对3株植物乳杆菌及其EPS进行清除DPPH自由基的测定,Lactobacillus plantarum-12的EPS浓度为0.5 mg/mL时,清除率为48.82±3.88%,显著高于另外2株菌(p0.05);测定植物乳杆菌的EPS抑制E.coli ATCC25922在HT-29细胞上的粘附效果,Lactobacillus plantarum-12的EPS浓度为0.2mg/mL时,抑制率为78.23%±2.46%,显著高于另外两株菌(p0.05);Lactobacillus plantarum-12的EPS可显著抑制E·coli ATCC25922刺激HT-29细胞产生IL-8(p0.05)。Lactobacillus plantarum-12的EPS经DEAE-Sepharose Fast Flow离子交换柱、Sepharose CL-6B凝胶柱和Sephacryl HR S200凝胶柱层析分离得到两组分,测定两组分的相对分子质量分别为8.5×10~4 u和7.4×10~4 u。     

具有抑制HT-29细胞增殖及益生功能乳酸杆菌的筛选

以15株乳酸杆菌为实验菌株,研究其抑制HT-29细胞增殖效果,初筛选抑制效果较好的8株菌进行黏附HT-29细胞实验和抗氧化能力实验。结果表明:抑制HT-29细胞增殖效果较好的8株菌抑制率为10.02%～15.63%;对HT-29细胞的黏附率为3.9%～19.1%。抗氧化能力实验结果显示活菌悬液、菌体细胞破碎液、菌体发酵上清液还原能力为23.43～271.76μmol/L的L-半胱氨酸盐酸盐量,DPPH自由基清除率为10.89%～50.42%,羟自由基清除率为7.46%～33.16%,超氧阴离子自由基清除率为17.64%～46.13%,脂质抗氧化能力为10.45%～51.91%。经主成分分析表明嗜酸乳杆菌KLDS1.0901综合性能最佳,嗜酸乳杆菌KLDS1.1003其次。模拟胃肠道的实验结果显示上述2株菌具有较好的耐受胃肠液能力,经过模拟胃肠道后活菌数可在10~8 CFU/m L以上,存活率可达50%。总体上嗜酸乳杆菌KLDS1.0901和KLDS1.1003具有较高的抑制HT-29细胞增殖能力和良好的益生功能,其中嗜酸乳杆菌KLDS1.0901更佳。     

植物乳杆菌胞外多糖的脱色及其体外抑瘤效应

研究大孔吸附树脂对植物乳杆菌胞外多糖的脱色工艺,并采用Cell Counting Kit-8(CCK-8)法检测植物乳杆菌胞外多糖EPS及单一组分EPS-1、EPS-2对大肠癌细胞株HT-29的体外抑瘤效应。结果表明,选择S-8树脂,树脂用量为4g/100mL,调节胞外多糖溶液的pH值为6,在25℃条件下,糖液质量浓度为2mg/mL,静态吸附4h后脱色率为72.58%,糖保留率为69.15%。植物乳杆菌胞外多糖EPS、EPS-1和EPS-2对大肠癌细胞HT-29的体外增殖均具有显著的抑制作用(P＜0.01),且呈现出良好的量效关系。     

In vitro adherence properties of Lactobacillus rhamnosus DR20 and Bifidobacterium lactis DR10 strains and their antagonistic activity against an enterotoxigenic Escherichia coli

Adhesion and colonisation properties of three probiotic strains namely, Lactobacillus rhamnosus DR20, L. acidophilus HN017, and Bifidobacterium lactis DR10, were determined in vitro using the differentiated human intestinal cell-lines including HT-29, Caco-2, and HT29-MTX, and compared with properties of L. acidophilus LA-1 and L. rhamnosus GG (two commercial probiotic strains). Two independent methods were employed to quantitate the "adhesiveness" of each strain. In the first method, the bacteria adhered to human cells were detected by Gram staining and counted in different fields under a microscope. Bacteria were also radio-labelled and extent of adhesion determined by scintillation counting. All three strains showed strong adhesion with the human intestinal cell lines in vitro. Adhesion indices of the three strains to two cell lines, i.e. HT-29, and Caco-2 varied between 99 +/- 17 and 219 +/- 36. With mucus-secreting cell-line HT29-MTX, the adhesion indices of all the strains were 2-3 times higher. The adhesion indices of L. acidophilus LA-1 and L. rhamnosus GG were comparable to the other three probiotic strains. We also investigated the inhibitory effect of adhering strains against the intestinal cell monolayer colonization by a known enterotoxigenic strain of Escherichia coli (strain O157:H7). Pre-treatment of E. coli O157:H7 with 2.5-fold concentrated cell-free culture supernatants from L. acidophilus HN017, L. rhamnosus DR20 and B. lactis DR10 reduced the culturable E. coli numbers on TSB plates and also reduced the invasiveness and cell association characteristics of this toxic strain. The inhibitory molecules secreted into the spent media by these strains were partially affected by treatments with lactate dehydrogenase, trypsin and proteinase K suggesting that overall inhibition may be due to a synergistic action of lactic acid and proteinaceous substances.     

The effect of calcium ions on adhesion and competitive exclusion of Lactobacillus ssp. and E. coli O138

The adhesion abilities of 11 strains of Lactobacillus were determined in vitro using the IPEC-J2 cell line as a model system. Bacteria cultures included the probiotic strains L. rhamnosus GG, L. reuteri ATCC 55730, L. johnsonii NCC 533 and L. reuteri DSM 12246, and new isolates of Lactobacillus ssp. Adhesion was quantified by scintillation counting of radiolabelled bound bacteria. The highest adhesion of 38%, was determined for L. reuteri DSM 12246 followed by L. plantarum Q47 with an adhesion level of 24%. Other strains showed moderate to low binding of less than 16%. Competitive adhesion experiments on IPEC-J2 cells demonstrated that strongly adhesive strains, as L. reuteri DSM 12246 and L. plantarum Q47, significantly reduced the attachment of the less adhesive strains, such as L. rhamnosus GG and L. johnsonii NCC 533, both under condition of co-incubation and in displacement assays, indicating that bacteria may share the same binding sites for attachment to intestinal cells. Furthermore, it was revealed that calcium ions significantly increased the binding of tested lactobacilli to IPEC-J2 cells; and therefore, added calcium may be useful in enhancing the adhesion of normally weakly adhesive probiotic cultures. In contrast, no significant change in adhesion of lactobacilli was observed in the presence of Mg and Zn ions. Displacement assays performed with pathogenic E. coli O138 showed that all tested Lactobacillus strains reduced the attachment of E. coli O138 to IPEC-J2 by more than 2-fold both in the presence and the absence of calcium ions. The strains of Lactobacillus did not differ significantly in the extent of their inhibition of E. coli O138 adhesion, indicating that the reduced adhesion of E. coli O138 was due to steric hindrance of the binding sites rather than to specific interactions.     

Lactobacillus菌株的益生功能评价

本研究评价了分离自传统发酵乳制品中4株L．paracasei supsp．paracasei菌株在模拟胃、肠液中的存活性,在HT-29细胞上的粘附性,对致病菌的抑制能力,以及刺激外周血单核细胞（PBMCs）增殖的能力。其中菌株Lactobacillus paracasei supsp．paracasei M5AL、J23AL在模拟胃液存活率较高,而菌株Lactobacillus paracasei supsp．paracaseiGl5AL、T3AL在胃液中暴露3h即失去活性;4株菌在模拟肠液中存活率较高。菌株L．paracasei supsp．paracasei M5AL和G15AL在HT．29细胞上有较强粘附能力f〉40bacteria cells per 100HT-29cells）,并抑制病原微生物Ecoli ATCC25922、styphimurium ATCC14028、S.sonnei ATCC25931生长。4株菌活菌体刺激PBMCs增殖,具有潜在免疫调节功能。     

Adhesive and chemokine stimulatory properties of potentially probiotic Lactobacillus strains

Five Lactobacillus plantarum strains and two Lactobacillus johnsonii strains, stemming either from African traditionally fermented milk products or children's feces, were investigated for probiotic properties in vitro. The relationship between the hydrophobic-hydrophilic cell surface and adhesion ability to HT29 intestinal epithelial cells was investigated, and results indicated that especially the L. johnsonii strains, which exhibited both hydrophobic and hydrophilic surface characteristics, adhered well to HT29 cells. Four L. plantarum and two L. johnsonii strains showed high adherence to HT29 cells, generally higher than that of the probiotic control strain Lactobacillus rhamnosus GG. Most strains with high adhesion ability also showed high autoaggregation ability. The two L. johnsonii strains coaggregated well with the intestinal pathogens Listeria monocytogenes Scott A, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Salmonella enterica serovar Typhimurium ATCC 14028. The L. plantarum BFE 1685 and L. johnsonii 6128 strains furthermore inhibited the adhesion of at least two of these intestinal pathogens in coculture with HT29 cells in a strain-dependent way. These two potential probiotic strains also significantly increased interleukin-8 (IL-8) chemokine production by HT29 cells, although modulation of other cytokines, such as IL-1, IL-6, IL-10, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor alpha (TNF-alpha), and transforming growth factor beta (TGF-beta), did not occur. Altogether, our results suggested that L. plantarum BFE 1685 and L. johnsonii BFE 6128 showed good adherence, coaggregated with pathogens, and stimulated chemokine production of intestinal epithelial cells, traits that may be considered promising for their development as probiotic strains.     

产胞外多糖植物乳杆菌的筛选及粗多糖的活性研究

通过观察乳酸菌菌落拉丝状况并测定其胞外多糖（exopolysaccharide,EPS）的产量,筛选出1 株所产EPS黏性好、产量高的乳酸菌AR307,经鉴定为植物乳杆菌。为得到更多的胞外多糖,对植物乳杆菌AR307的发酵条件进行优化,确定其在发酵温度32 ℃、发酵时间20 h条件下的产糖量为389 mg/L。在体外实验中,所得胞外多糖具有抑制HT-29肿瘤细胞活性、降低血糖水平的作用。     

Characterization and bioactivities of the exopolysaccharide from a probiotic strain of Lactobacillus plantarum WLPL04

Exopolysaccharide (EPS) was extracted and purified from Lactobacillus plantarum WLPL04, which has been confirmed previously as a potential probiotic for its antagonistic and immune-modulating activity. It has a molecular weight of 6.61 × 10⁴ Da, consisting of xylose, glucose, and galactose in an approximate molar ratio of 3.4:1.8:1. Microstructural studies demonstrated that the EPS appeared as a smooth sheet structure with many homogeneous rod-shaped lumps. The preliminary in vitro assays indicated that the EPS could significantly inhibit the adhesion of Escherichia coli O157:H7 to HT-29 cells in competition, replacement, and inhibition assays at a dose of 1.0 mg/mL, with an inhibition rate of 20.24 ± 2.23, 29.71 ± 1.21, and 30.57 ± 1.73%, respectively. Additionally, the EPS exhibited strong inhibition against biofilm formation by pathogenic bacteria, including Pseudomonas aeruginosa CMCC10104, E. coli O157:H7, Salmonella Typhimurium ATCC13311, and Staphylococcus aureus CMCC26003. Furthermore, the EPS showed good inhibitory activity against the proliferation of HT-29 cells. The characteristics and bioactivities of this EPS may make it a promising candidate in developing functional food.     

Screening probiotics from Lactobacillus strains according to their abilities to inhibit pathogen adhesion and induction of pro-inflammatory cytokine IL-8

Probiotics can be screened according to their abilities to inhibit pathogen adhesion and inhibit the production of pro-inflammatory cytokines. Eleven Lactobacillus strains isolated from traditional fermented dairy foods in Xinjiang, China, were studied for their potential to inhibit adhesion of Escherichia coli to intestinal epithelial cells and to inhibit E. coli–induced production of interleukin (IL)-8 by intestinal epithelial cells. The results showed that the 11 strains could inhibit adhesion of E. coli to Caco-2 cell monolayers and inhibit the induction of IL-8 production by E. coli in HT-29 cells. The inhibiting activities of the Lactobacillus strains against E. coli adhesion and IL-8 induction were strain-specific and not positively correlated, whereas the excluding activity of the strains against E. coli adhesion and their coaggregation with E. coli were positively correlated. The effector molecules of the strains with probiotic potential should be identified to explain the mechanism behind these observations.