Extracellular Polysaccharides Matrix—An Often Forgotten Virulence Factor in Oral Biofilm Research

Oral diseases related to dental biofilms continue to afflict the majority of the world＇s population. Among them, dental caries continues to be the single most prevalent and costly oral infectious disease （Marsh, 2003; Dye et al., 2007）. Dental caries results from the interaction of specific bacteria with constituents of the diet within a dental biofilm known as plaque （Bowen, 2002）. Sucrose is considered to be the ＂arch criminal＂ from the dietary aspect because it serves as a substrate for synthesis of extracellular （EPS） and intracellular （IPS） polysaccharides in dental biofilm and is also fermentable （Bowen, 2002）.

In Vitro Antioxidant Function of Extracellular Polysaccharides from Tremella aurantialba

Extracellular polysaccharide from Tremella aurantialba (TEP) significantly inhibited H2O2-induced haemolysis of RBCs, significantly decreased the amount of malondialdehyde (MDA) produced in mice liver homogenates as a result of autoxidation, and inhibited VitC-Fe2+-induced swelling of liver mitochondria.

Screening of Toxin Mutant of Dickeya zeae and Its Biological Characters

[Objective]This study aimed to screen toxin mutant of Dickeya zeae(Erwinia chrysanthemi pv.zeae)and investigate its biological characters.[Method]We obtained a toxin mutant strain D.zeae Ech7-3-42 by using acridine orange as a mutagenic agent and compared their biological characteristics and virulence between the toxin mutant and wild strain.[Result]There was no significant difference in pectin lyase,protease,cellulase and the production of extracellular polysaccharide and lipopolysaccharide,but significant difference in toxin biological activities and virulence.Ech7-3-42 mutant did not produce toxin,as well as the loss of virulence on rice and HR on tobacco,but did not lose the ability to soft rot on potato.Mutant strain Ech7-3-42 can infect rice root and then enriched in the root neck and stalk,but it could not cause rice foot rot.Dickeya zeae(wild and mutant strain)could be detected by PCR in the root neck and below the 1-2 cm long stem area,but could not be detected in the leaves.[Conclusion]We believed that toxin may be one of the important factors for D.zeae virulence on rice.

Effects of sand burial on biomass, chlorophyll fluores-cence and extracellular polysaccharides of man-made cyanobacterial crusts under experimental conditions

Soil cyanobacterial crusts occur throughout the world, especially in the semiarid and arid regions. It always encounters sand burial, which is an important feature of mobile sand dunes. A greenhouse study was conducted to determine the effects of sand burial on biomass, chlorophyll fluorescence and extracellular polysaccharides of man-made cyanobacterial crusts in six periods of time (0, 5, 10, 15, 20 and 30 d after burying) and at five depths (0, 0.2, 0.5, 1 and 2cm). The results indicated that with the increase of the burial time and burial depth extracellular polysaccharides content and Fv/Fm decreased correspondingly and there were no significant differences between 20 and 30 burial days under dif-ferent burial depths. The degradation of chlorophyll a content appeared only at 20 and 30 burial days and there was also no significant difference between them under different burial depths. It was also observed a simultaneous decrease of the values of the Fv/Fm and the content of extracellular poly-saccharides happened in the crusted cyanobacterium Microcoleus vaginatus Gom. It may suggest that there exists a relationship between extracellular polysaccharides and recovery of the activity of pho-tosystem II (PS II) after rehydration.

Effect of proteins,polysaccharides,and particle sizes on sludge dewaterability

Four batch experiments of hydrolysis and acidification were carried out to investigate the distributions of proteins （PN） and polysaccharides （PS） in the sludge, the PN/PS ratio, the particle sizes, and their relationship with sludge dewaterability （as determined by capillary suction time, CST）. The sludge flocs were stratified through centrifugation- and ultrasound-based method into four fractions： （1） slime, （2） loosely bound extracellular polymeric substances （LB-EPS）, （3） tightly bound EPS （TB-EPS）, and （4） pellet. The results showed that PN was mainly partitioned in the pellet （80.7%） and TB-EPS （9.6%） fractions, while PS distributed evenly in the four fractions. During hydrolysis and acidification, PN was transferred from the pellet and TB-EPS fractions to the slime fraction, but PS had no significant transfer trends. The mean particle sizes of the sludge flocs decreased with hydrolysis and acidification. The pH had a more significant influence on the dewaterability of sludge flocs than temperature. Sludge dewaterability during hydrolysis and acidification processes greatly deteriorated from 9.7 s at raw sludge to 340-450 s under alkaline conditions. However, it was just slightly increased under acidic conditions. Further investigation suggested that CST was affected by soluble PN, soluble PN/PS, and particle sizes of sludge flocs, but was affected slightly by total PN, PS, or PN/PS in the whole sludge flocs and other fractions （except slime）.

Inhibitory Effect of Extracellular Polysaccharide EPS-II from Pseudoalteromonas on Candida adhesion to Cornea in vitro

Objective Fungal keratitis（FK） is a vision-threatening infection,whose treatment requires more effective and safer anti-fungal agent exploitation urgently.With this aim,we focused on the effect of an extracellular polysaccharide on fungal adhesion to human corneal epithelial cells.Methods We performed the cytotoxicity assays of the extracellular polysaccharide EPS-II from an antarctic bacterium Pseudoaltermonas and evaluated its inhibitory effect on Candida albicans cells＇ adherence to human corneal epithelial cells（HCECs）.Results EPS-II,which displayed minor cytotoxicity but also promoted proliferation of HCECs,could inhibit the adherence of yeast cells to HCECs in a dose-dependent manner.EPS-II could also suppress the subsequent PI3K/AKT signaling pathway,and thereby decrease the expression of early inflammatory cytokines.Conclusions ExtracellularpolysaccharideEPS-IIwassuggestedasanewnaturalagentforattenuatingFK.

Production and characterization of an extracellular polysaccharide of antarctic marine bacteria Pseudoalteromonas sp. S-15-13

Twenty-seven antarctic bacteria producing extracellular polysaccharide （EPS） were selected from 57 strains by staining technology. The effects of major environmental factors on the growth and EPS production of Pseudoalteromonas sp. S - 15 - 13 were investigated, and the EPS was separated and purified for characterization analysis. The results showed that the optimal conditions for the EPS production were culture period, 56 h; growth temperature, 8 ℃ ; carbon source, 1.0% glucose; NaCI concentration, 3.0% ; pH 6.0 - 7.0. The EPS was purified by cold ethanol precipitation, proteins removal, ion exchange chromatography and gel chromatography technology. The molecular mass of EPS - H was 62 kDa as determined by the high performance gel permeation chromatography. Its sugar composition was a homopolymer of marmose analyzed by gas chromatograph spectroscopy. After repeated freezing and thawing of the bacteria hiomass in the presence of EPS, the bacterial growth was much higher than that observed after freezing in the absence of EPS and the difference augmented with the increase of freeze-thaw cycles. It is hypothesized that the adaptation of Pseudoalteromonas sp. S- 15 - 13 to the antarctic marine conditions, characterized by low temperature, high NaCl concentration and repeated freeze-thaw cycles, might be related to the EPS production ability.

A Polysaccharide-Degrading Marine Bacterium Flammeovirga sp.MY04 and Its Extracellular Agarase System

Bacteria of the genus Flammeovirga can digest complex polysaccharides (CPs), but no details have been reported regarding the CP depolymerases of these bacteria. MY04, an agarolytic marine bacterium isolated from coastal sediments, has been identified as a new member of the genus Flammeovirga. The MY04 strain is able to utilize multiple CPs as a sole carbon source and grows well on agarose, mannan, or xylan. This strain produces high concentrations of extracellular proteins (490 mg L-1 ± 18.2 mg L-1 liquid culture) that exhibit efficient and extensive degradation activities on various polysaccharides, especially agarose. These proteins have an activity of 310 U mg-1 ± 9.6 U mg-1 proteins. The extracellular agarase system (EAS) in the crude extracellular enzymes contains at least four agarose depolymerases, which are with molecular masses of approximately 30-70 kDa. The EAS is stable at a wide range of pH values (6.0-11.0), temperatures (0-50℃), and sodium chloride (NaCl) concentrations (0- 0.9 mol L-1). Two major degradation products generated from agarose by the EAS are identified to be neoagarotetraose and neoagarohexaose, suggesting that β-agarases are the major constituents of the MY04 EAS. These results suggest that the Flammeovirga strain MY04 and its polysac-charide-degradation system hold great promise in industrial applications.

Isolation, chemical characteristics and immunity activity of an extracellular polysaccharide EPS I isolated from Antarctic bacterium Pseudoalteromonas sp.S-15-13

A new extracelluar polysaccharide （EPS） was isolated and purified from Antarctic bacterium S-15-13, identified as Pseudoalteromonas sp. After being separated and purified by DEAE-Sephadex A-50 ionexchange and Sephadex G-100 gel chromatography, two mains fractions （EPS I and EPS Ⅱ ） were ob-tained. EPS I was composed of mannose, glucose and galactose with a molecular weight of 23kDa and EPS Ⅱ was composed of mannose only with a molecular weight of 62kDa. The effect of the polysaccharide EPS I on the cellular immune response of mice was investigated. Results demonstrated that EPS I could markedly facilitate lymphocyte proliferation, and might be a strong immunomodulator.

The regulating role of mutant IKBα in expression of TIMP-2 and MMP-9 in human glioblastoma multiform

Background Our previous studies demonstrated that mutant IKBα （IKBαM） inhibited the occurrence, growth and angiogenesis of human glioblastoma multiform （GBM）. However, the specific mechanism by which IKBaM regulates protein-degrading enzymes secreted from GBM to inhibit invasion and metastasis has remained unclear. The aim of the present study was to investigate the regulatory role and significance of IKBαM genes in the expression of tissue inhibitor of metalloproteinase （TIMP）-2 and matrix metalloproteinase （MMP）-9 in human GBM. Methods We established the following four GBM cell lines stably expressing IKBaM by plasmid construction, gene transfection and screening for IKBαM protein expression： mutant IKBa-transfected cells （G36A-M）, wild-type IKBa-transfected cells （G36A-W）, empty plasmid transfected cells （G36A-P） and untransfected cells （G36A）. The TIMP-2 and MMP-9 expression was detected by RT-PCR and Western blotting. Tumor cells were then implanted subcutaneously into nude mice to establish an animal model of ectopic tumor growth, and TIMP-2 and MMP-9 expression was determined by immunohistochemical methods. Results The results showed that there was a significant increase in TIMP-2 expression and a significant decrease in MMP-9 expression in the G36A-M group at both the RNA and protein levels compared with the G36A-W group, G36A-P group and G36A group. Similar results were observed in the immunohistochemical staining analysis of tumor tissues. In the G36A-M group, TIMP-2 expression was significantly higher while MMP-9 expression was significantly lower than in the other three groups. Conclusions Our findings indicate that IKBaM inhibits the activation of NF-KB. It significantly up-regulates TIMP-2 expression in human malignant glioma cells and down-regulates the expression of MMP-9. Thus, IKBαM maintains the integrity of the extracellular matrix and further inhibits the growth and metastasis of tumor tissues. Chin Med J 2009; 122（2）：205-211

Effect of Sludge Retention Time on the Fate of Proteins and Polysaccharides in AAO Process

Effect of sludge retention time( SRT) on the removal of potential and polysaccharides in an anaerobic / anoxic / aerobic( AAO) process was investigated. The Lowry method and anthrone method were used to detect proteins and polysaccharides. Total removal efficiency of proteins at SRT of 10 to 25 d in the AAO system was higher than 90%. Polysaccharides removal efficiencies were above 80% when SRT was increased from 15 to 25 d,whereas only 81% of polysaccharides was removed at SRT of 10 d. The biodegradation part of proteins and polysaccharides increased from87. 40% to 93% and from 74. 22% to 86. 94% with increasing SRTs.The ratios of polysaccharides and proteins in extracellular polymer substances( EPSs) were around 1. 5-3 in different SRTs. As SRT increasing,polysaccharides and proteins discharged with residual sludge decreased gradually. The amount of EPSs decreased with increasing SRTs.

Optimization of Production and Preliminary Characterization of New Exopolysaccharides from <i>Gluconacetobacter hansenii</i>LMG1524

The influence of different carbon and nitrogen sources, of ethanol concentration, the optimal pH, temperature and medium composition were evaluated on extracellular polysaccharides (EPS) synthesis and bacterial growth of Gluconacetobacter hansenii LMG1524, and preliminary characterization of EPS was investigated. The highest EPS yields were obtained using glycerol and ammonium sulphate as carbon and nitrogen sources, respectively. The increase of ethanol concentration in the medium did not influence the EPS synthesis but reduced the bacterial growth. The optimum temperature and pH for polysaccharides production were respectively 25℃ and 5;whereas for cell growth were respectively 30℃ and 4. The optimal culture medium composition was determined as follows: 10 g/L sucrose, 0.892 g/L (NH4)2SO4, 0.34 g/L NaNO3, 3 mL acetic acid, 1.5 g/L KH2PO4, 1.5 g/L K2HPO4 and 0.6 g/L MgSO4. The polysaccharides produced were of 14 and 10 polymerization degrees (DP) and constituted mainly of glucose, galactose and mannose, in relative percent of 36.36, 33.94 and 22.42, respectively.

基于MAPK/ERK通路探讨茯苓多糖对卵巢早衰模型大鼠的作用及其分子机制

目的基于丝裂原活化蛋白激酶/细胞外调节蛋白激酶(MAPK/ERK)通路探讨茯苓多糖对卵巢早衰(POF)模型大鼠的作用及其分子机制。方法取50只SD雌性大鼠腹腔注射环磷酰胺诱导复制POF模型,随机分为5组:模型组、茯苓多糖低剂量组(100 mg/kg)、茯苓多糖高剂量组(200 mg/kg)、茴香霉素组(MAPK激活剂,10 mg/kg)、茯苓多糖高剂量+茴香霉素组,每组10只;另取10只SD雌性大鼠腹腔注射等剂量生理盐水作为对照组。分组处理后分别测定各组大鼠子宫指数及卵巢指数;酶联免疫吸附试验(ELISA)测定各组大鼠血清性激素[雌二醇(E2)、促黄体生成素(LH)、卵泡刺激素(FSH)]水平;HE染色观察各组大鼠卵巢形态;酶联免疫吸附试验检测各组大鼠血清及卵巢组织肿瘤坏死因子-α(TNF-α)、白细胞介素-4(IL-4)、白细胞介素-18(IL-18)水平;Western blotting检测各组大鼠卵巢组织MAPK/ERK通路相关蛋白表达。结果与对照组比较,模型组大鼠卵巢间质结构有一定程度的出血和纤维化损伤,卵泡发育失常,血清FSH和LH水平、血清和卵巢组织TNF-α、IL-4、IL-18水平、卵巢组织p-p38 MAPK/p38 MAPK及p-ERK1/2/ERK1/2相对表达量均升高(P<0.05),子宫指数和卵巢指数、E_(2)水平均降低(P<0.05)。与模型组比较,茯苓多糖低剂量组、茯苓多糖高剂量组大鼠卵巢形态损伤及卵泡发育失常均减轻,血清FSH及LH水平、血清和卵巢组织TNF-α、IL-4、IL-18水平、卵巢组织p-p38 MAPK/p38 MAPK及p-ERK1/2/ERK1/2相对表达量均降低(P<0.05),子宫指数和卵巢指数、E_(2)水平均升高(P<0.05),且茯苓多糖高剂量组效果更优(P<0.05)。茴香霉素组大鼠卵巢形态损伤及卵泡发育失常加重,血清FSH及LH水平、血清和卵巢组织TNF-α、IL-4、IL-18水平、卵巢组织p-p38 MAPK/p38 MAPK及p-ERK1/2/ERK1/2相对表达量均升高(P<0.05),子宫指数及卵巢指数、E_(2)水平均降低(P<0.05)。茯苓多糖高剂量+茴香霉素组逆转茯苓多糖作用,呈相反趋势(P<0.05)。结论茯苓多糖可通过抑制MAPK/ERK通路的激活减轻POF大鼠炎症,从而改善其卵巢功能和性激素分泌,缓解大鼠POF症状。

胞外多糖对盐胁迫下荆芥种子萌发、幼苗生长及生理特性的影响

为提高盐渍地中荆芥的种植效益,探究植物乳杆菌(Lactobacillus plantarum)胞外多糖对盐胁迫下荆芥种子萌发、幼苗生长的影响,同时从幼苗根系生理特性上分析其具体作用机制,设置6个处理[空白对照组(CK1)、盐胁迫对照组(CK2)、50 mg/kg胞外多糖组(T1)、100 mg/kg胞外多糖组(T2)、200 mg/kg胞外多糖组(T3)、400 mg/kg胞外多糖组(T4)]进行试验。结果表明,胞外多糖对荆芥种子萌发和幼苗生长具有显著的促进作用。其中,添加200 mg/kg胞外多糖(T3)效果最显著,与CK2相比,其种子发芽率、发芽势、发芽指数和活力指数分别显著增加76.13%、144.16%、137.45%、265.28%,幼苗株高、茎粗、地下干质量和地上干质量分别显著升高60.49%、14.73%、40.08%、85.39%,脯氨酸、可溶性蛋白、可溶性糖含量分别提高21.02%、84.56%、40.02%,Na+含量降低54.63%,K+含量升高95.24%,Na+/K+值降低76.76%,根系电解质渗出率、丙二醛(MDA)含量分别降低44.54%、43.24%,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)活性分别显著提高30.39%、34.59%、24.31%。综上所述,植物乳杆菌胞外多糖可以通过根系渗透调节和激活抗氧化酶防御系统来提高幼苗的抗盐性,进而促进荆芥种子萌发和幼苗生长。

基于高产胞外粗多糖的白灵芝发酵茶培养基配方优化

为探究白灵芝在液体茶基质上分泌胞外粗多糖的最佳营养条件及其影响因素,本研究在单因素实验的基础上,通过正交试验和方差分析,优化了该菇在茶培养基上胞外粗多糖形成的营养条件,并采用相关性与通径分析,探究了其液体培养特性与胞外粗多糖分泌的关系。结果表明,在马铃薯50 g/L、葡萄糖40 g/L、红土3 g/L、普洱茶20 g/L、茶粉颗粒大小0.075 mm的茶培养基上,白灵芝的胞外粗多糖产量最高(1.49±0.33 g/L)且受各营养条件的影响顺序为葡萄糖>茶叶种类>红土>马铃薯>茶粉颗粒大小。此外,该菇胞外粗多糖的分泌受发酵液可溶性固形物、菌球数量的影响显著,其中前者对其间接影响最大,后者对其直接作用与综合作用最大。总之,茶培养基的营养条件(种类、浓度、颗粒大小等)与白灵芝的液体培养特性对其胞外粗多糖的分泌影响较大且存在交互作用,上述结果可为该菇多糖的提取及功能性食品的开发提供理论参考。

细菌根际黏附的界面作用机制研究

界面作用是细菌在环境中黏附、定殖、形成生物膜和发挥生态功能的基础和前提,对植物吸收养分、病原微生物拮抗具有重要意义。微生物-植物根际相互作用研究大多数从生态学和分子生物学角度出发,应用多种组学手段研究根系分泌物对于根际微生物定殖数量、群落组成和生理功能的影响,忽略了细菌定殖过程中物理、化学的界面作用机制及其对黏附的贡献。本文从界面作用机制出发,探讨了不同类型根系分泌物对细菌表面性质、细菌胞外聚合物(EPS)分子组成以及黏附功能的调控作用;梳理了黏附过程中细菌-植物生物大分子相互作用的主要模式及其微观机制;归纳了根际定殖过程可视化研究方法和微生物-植物生物大分子相互作用的分析手段;提出了根际生物分子组成分析、黏附蛋白质功能预测、根际定殖原位观测方法等亟需加强的研究方向。

美人鱼发光杆菌美人鱼亚种dly基因缺失株构建及其生物学特性研究

美人鱼发光杆菌美人鱼亚种(Photobacterium damselae subsp.damselae,PDD)是广泛分布于全球海洋环境中的一种致病菌。本研究以实验室保存的一株高致病性PDD菌株(PDD1608)作为对象,初步探究毒力基因dly对PDD菌株的生物学特性和致病力的影响。利用λRed重组技术成功构建毒力基因dly缺失株Δdly PDD1608::Cm,比较野生株和缺失株的生长、涌动性、药物敏感性、生理生化特性、生物被膜形成能力、菌株及胞外产物(extracellular products,ECP)的溶血性和磷脂酶活性等生物学特性。选用海水青鳉鱼(Oryzias melastigma)作为实验动物,通过人工感染实验测定野生株和缺失株及其ECP对海水青鳉鱼的致病性。结果显示,毒力基因dly缺失后导致PDD菌株生长变慢,涌动性、溶血性和磷脂酶活性均降低;野生株和缺失株的药物敏感性和生理生化特性未产生变化;与野生株相比,缺失株的生物被膜形成能力有显著差异(P<0.05);人工感染实验表明,缺失株及其ECP对海水青鳉鱼的致病性降低。毒力基因dly影响PDD菌株的生长、涌动性、溶血性和磷脂酶活性等多种生物学特性,并且与PDD菌株及其ECP的致病力强弱密切相关。

巴马地区产胞外多糖益生乳酸菌的筛选及鉴定

采用含碳酸钙的快速初筛培养基,结合形态学观察、革兰氏染色、过氧化氢酶接触实验、菌落拉丝法和硫酸-苯酚法,从巴马自然发酵食品中筛选产胞外多糖乳酸菌,对其耐酸、耐胆盐、细胞表面疏水性和生物安全性进行研究,并采用分子生物学技术对筛选益生菌株进行鉴定。结果表明,从巴马自然发酵食品中筛选到6株产胞外多糖乳酸菌,编号分别为R2、R3、R4、R5、R6和R9;其中菌株R6多糖产量最高(106.73 mg/L),p H 3时耐酸性最佳,0.05%胆盐时延滞期最短(1.50 h),细胞表面疏水性最突出(在十六烷中的细胞表面疏水性达到了93.28%),对沙门氏菌、大肠杆菌和金黄色葡萄球菌的抑制作用最显著,且对多种常见抗生素敏感和不具有溶血活性,菌株R6被鉴定为植物乳杆菌(Lactiplantibacillus plantarum),可作为潜在益生菌株。

葡聚糖蔗糖酶在食品级乳酸菌中重组表达

为研究葡聚糖在乳酸菌中的合成情况以及结构分析,本研究将葡聚糖蔗糖酶DsrB与带有SP45分泌信号肽的乳酸乳球菌表达载体pNZ8148质粒载体进行连接,电转入乳酸乳球菌NZ9000中进行异源表达。经由一系列醇沉、三氯乙酸去除蛋白质、二次醇沉、透析以及纯化等步骤得到多糖溶液。采用高效液相色谱法检测单糖组成;利用HPSEC测定葡聚糖的分子量;通过傅里叶红外技术、核磁共振技术以及扫描电镜分析多糖的结构并观察多糖表面特征。结果表明,将葡聚糖蔗糖酶在乳酸菌中进行表达,在蔗糖浓度为10%的条件下,葡聚糖产量为10.54 g/L;单糖组成显示仅含有葡萄糖一种单糖;胞外多糖分子量测定结果为2.4×10^(6) Da;由傅里叶红外和核磁联合表明体外合成的胞外多糖仅含有α-(1,6)糖苷键;扫描电镜表示胞外多糖表面呈现为多孔状结构。在乳酸菌中异源表达,实现了葡聚糖的食品级生产,为葡聚糖于食品中应用提供了理论基础。

黄褐假单胞菌Y11产胞外多糖发酵条件优化及其抗氧化活性研究

为提高黄褐假单胞菌(Pseudomonas syringae)液态发酵产胞外多糖(EPS)量,以胞外多糖产量为评价指标,通过单因素及响应面试验对菌株发酵产胞外多糖条件进行优化,并对其胞外多糖的抗氧化性进行研究。结果表明,黄褐假单胞菌最佳产胞外多糖条件为麦芽糖44 g/L、大豆蛋白胨11 g/L、磷酸二氢钠8 g/L、接种量4%、转速160 r/min、发酵温度28℃、发酵时间72 h、初始pH 7.0。在此优化条件下,胞外多糖的产量为4.56 g/L,是优化前的3.7倍。抗氧化性研究结果表明,在一定浓度范围内,EPS对DPPH自由基、ABTS自由基、羟自由基、超氧阴离子自由基具有较好的清除活性,当胞外多糖质量浓度为2.5 mg/mL时,EPS对DPPH自由基、ABTS自由基、羟自由基、超氧阴离子自由基的清除能力分别为(70.83±1.80)%、(45.00±1.73)%、(65.83±1.80)%和(44.6±2.11)%,说明黄褐假单胞菌液态发酵产胞外多糖具有一定的抗氧化活性。