【作者】 艾连中；

【导师】 张灏； 郭本恒； 汤坚；

【作者基本信息】 江南大学 ， 食品科学， 2007， 博士

【摘要】 近年来,乳酸菌胞外多糖已成为研究的热点,它不仅具有改善发酵食品流变、口感、和质构的重要作用,而且具有促进人体健康的重要生理活性,如抗诱变、抗肿瘤、免疫调节、降胆固醇等活性。本文对干酪乳杆菌LC2W胞外多糖的产生条件、分离纯化、生理功能、物化性质以及结构特征等进行了系统研究。比较了不同乳酸菌培养基对干酪乳杆菌LC2W产生胞外多糖的影响,选择胞外多糖产量较高(83 mg/L)的脱脂奶粉为基础培养基。研究了以脱脂奶粉为基础培养基,添加不同碳源、氮源和其他盐类对胞外多糖产量的影响,其中大豆蛋白胨、葡萄糖和K2HPO4能显著提高胞外多糖产量,通过正交实验确定干酪乳杆菌LC2W优化培养基组成为:脱脂奶粉12.0%,大豆蛋白胨1.0%,葡萄糖1.5%,K2HPO4 0.1%。采用单因素分析接菌量、培养温度和培养时间对干酪乳杆菌LC2W产生胞外多糖的影响,并通过响应面分析法确定高产胞外多糖工艺优化参数:接种量4.0%,培养温度32.5 oC,培养时间26 hr。最适培养基和最优培养条件(不控制培养基pH)下EPS产量约为137 mg/L。研究发现控制pH 6.0发酵,EPS的产量最大为160 mg/L,较未控制pH时的产量提高了约17%。发酵液通过加热灭酶后冷却离心除去凝结蛋白和菌体,然后用终浓度4.0%(w/v)的三氯乙酸除上清液中蛋白质,超滤浓缩后,用终浓度75%(v/v)乙醇沉淀得粗多糖LCP,得率为153.4 mg/L。粗多糖LCP经过DEAE-Sepharose FF离子交换柱层析分级纯化得到三个组分,由pH 7.6 Tris-HCl缓冲液洗脱得到组分LCP1和LCP2,由0.2～1.0 mol/L NaCl的Tris-HCl缓冲液线性梯度洗脱得到组分LCP3,LCP1、LCP2和LCP3分别占LCP质量的35.74%,12.61%和33.34%,总回收率为81.7%。LCP1、LCP2和LCP3在Sepharose CL-6B和HPLC上都显示均一组分,结合紫外、红外图谱和其化学组成分析确定LCP1和LCP2是两种中性多糖,LCP3为一种蛋白质-多糖复合物,其中蛋白质含量约为60%。胞外多糖LCP1、LCP2和LCP3分别以15 mg/kg体重剂量灌胃自发性高血压大鼠(SHR),每天一次,连续灌胃7天,对照组灌胃生理盐水。结果显示LCP1能显著降低SHR的动脉收缩压(P<0.01),最大降压幅度(约20 mmHg)出现在给药后2~4 h,LCP2和LCP3降血压效果不明显。LCP1、LCP2和LCP3对SHR心率均没有影响,说明这三组分对SHR的循环系统未产生不利影响。胞外多糖LCP1对SHR降高血压的机理研究发现SHR连续灌胃胞外多糖LCP1可显著降低主动脉ACE活性,而对肺、肾及血清中ACE活力没有明显影响,对照组和LCP1组SHR血清中ET-1和CO含量没有明显不同。体外淋巴细胞培养实验显示,LCP1、LCP2和LCP3均无细胞毒性,体外免疫活性实验可知,胞外多糖LCP1、LCP2和LCP3能显著抑制T/B淋巴细胞的增殖反应和混合淋巴的增殖反应,显示较强的免疫抑制活性。利用毛细管电泳进一步鉴定LCP1的纯度,通过旋光仪测定LCP1水溶液中的比旋光度[α]52859=+62.0°(H2O;浓度1.12),采用乌氏粘度计测得LCP1水溶液固有粘度[η]为1.930 dL/g。示差折光检测仪测得LCP1在0.1 mol/L的NaNO3溶液中比折光指数增量(dn/dc)为1.20。通过高效凝胶色谱测定LCP1重均分子量Mw为1.236×106 Da、固有粘度[η]为1.875 dL/g、多分散性指数Pd为1.202和Mark-Houwink指数为0.654,Mark-Houwink指数表明LCP1在0.1 mol/L的NaNO3溶液中呈无规卷曲构象。通过动态激光光散射研究表明LCP1分子在水溶液中聚集比较严重,达到45%,而在NaNO3溶液中几乎不发生聚集,故确定0.1 mol/L NaNO3溶液为研究LCP1溶液分子信息的理想溶剂。采用静态激光光散射测得LCP1在0.1 mol/L的NaNO3溶液中的重均分子量Mw为1.276×106 Da、回旋半径Rg为52.9 nm、第二维里系数A2为2.44×10-4 cm3mol/g2;动态激光光散射测定LCP1水力半径Rh为34.8 nm,ρ值(Rg/Rh)为1.61表明LCP1分子在溶液中呈无规卷曲的构象。通过对LCP1溶液流变性质研究发现,高浓度LCP1溶液呈剪切变稀,低浓度时,剪切速率对粘度没有影响,溶液粘度随温度升高下降明显,pH对LCP1溶液粘度影响很小,盐可以降低LCP1溶液粘度,粘度降低与盐浓度有关,通过粘弹性研究发现LCP1溶液不能形成胶体。离子色谱测定LCP1由鼠李糖、葡萄糖和半乳糖组成,摩尔比约为3:5:2。通过高碘酸氧化、Smith降解、甲基化分析和部分酸水解的化学方法与GC-MS、GC、HPAEC-PAD、1D和2D NMR技术相结合,分析推测出LCP1的结构如下:更多还原

【Abstract】 The exopolysaccharides （EPS） produced by the food-grade lactic acid bacteria （LAB） have been extensively studied during the last decades. These LAB EPS are considered to not only play an important role in the rheology, mouthfeel and texture of fermentation products, but also provide beneficial physiological effects on human health, such as antimutagenic, antitumour, immunomodulating and cholesterol-lowering activity. The objectives of the current work are to study the fermenting conditions of EPS produced by Lactobacillus casei LC2W, to isolate and purify EPS, and to study the bioactivity, physicochemical properties and structure characterization of EPS.The effects of different culture media on EPS produced by Lactobacillus casei LC2W were studied. Skim milk in which the yield of EPS was 83 mg/mL was chosen as a basic medium of Lactobacillus casei LC2W. Addition of soybean peptone as nitrogen, glucose as carbon and K2HPO4 to skim milk could remarkably increase the yield of EPS. The culture media of Lactobacillus casei LC2W were optimized by the orthogonal experiment design as follows: skim milk powder 12%, soybean peptone1.0%, glucose1.5% and K2HPO4 0.1%.The effects of inoculum concentration, incubation temperature and time on EPS produced by Lactobacillus casei LC2W were studied by single factor analysis. Optimal parameters of the fermenting procedure of EPS by RSM were obtained: inoculum concentration 4.0%, incubation temperature 32.5°C and incubation time of 26 h. The yield of EPS was 137 mg/L under the optimum culture medium and fermenting conditions without controlling pH, but if controlling pH 6.0 the largest yield 160 mg/L was obtained and increased by 17% compared with the yield of no controlling pH.The fermentation broth was pretreated by heating to inactivate enzymes and then centrifuging to remove cells and coagulated proteins, and trichloroacetic acid was added into the supernatant to a final concentration of 4.0% （w/v） to remove proteins. The EPS of which the yield was 153.4 mg/L were precipitated from the supernatant with three volumes of cold ethanol.LCP were fractionated by anion exchange chromatography of DEAE-Sepharose FF column, LCP1 and LCP2 were first obtained by the elution of 0.05 mol/L Tris-HCl buffer （pH 7.6） and LCP3 was followed by a linear gradient of NaCl concentration （0-1.2 mol/L）. The total recovery of LCP was 81.7%, and the contents of LCP1, LCP2 and LCP3 were 35.74%, 12.61% and 33.34% based on LCP, respectively. Three polysaccharides identified by gel filtration chromatography on Sepharose CL-6B column and HPLC were homogeneous. Combined with UV-spectroscopy, FT-IR and monosaccharide composition analysis, LCP1 and LCP2 were thought to be neutral polysaccharides, and LCP3 was a complex of protein-polysaccharide which protein content was 60%.SHR were continuously fed respectively with the test samples （LCP1, LCP2 and LCP3） at the daily dosage of 15 mg kg–1 and the control sample （normal saline） for seven days. The results showed that LCP1 produced a significant decrease in the systolic blood pressure （SBP） of SHR （P<0.01）, the maximal decreases （20 mmHg） were noticed from 2 h to 4 h after the oral administration, and no significant changes were found in SBP of SHR fed with LCP2 and LCP3. Meanwhile, LCP1, LCP2 and LCP3 showed no effect on heart rate of SHR, indicating they had no disadvantage effect on the circulatory system of SHR.The study on the blood-pressure-lowering mechanism showed that LCP1 reduced obviously ACE activity in aorta while no effects on ACE activity in the lung, kidney and blood serum, and that the ET-1 and CO contents in SHR blood serum made no difference between the control and LCP1 groups.Lymphocyte culture test in vitro showed that LCP1, LCP2 and LCP3 had no cytotoxicity. Immunocompetence test in vitro indicated these polysaccharides all inhibit the proliferation of T- and B-lymphocytes and mixed lymphocytes and had the obvious immunosuppressive bioactivity.The physicochemical properties of LCP1 were as follows: the purity of LCP1 was further confirmed by capillary electrophoresis （CE） analysis; the specific rotation measured by the polarimeter was [α]52859 = +62.0°（H2O, c 1.12）; the intrinsic viscosity in aqueous solution determined by Ubbelohde viscometer was 1.930 dL/g; the refractive index increment （dn/dc） in 0.1 mol/L NaNO3 solution tested by Differential Refractometer was 1.20; and the molecular weight, intrinsic viscosity, polydispersity and Mark-Houwink exponent of LCP1 determined using HPSEC were 1.236×106 Da,1.875 dL/g, 1.202, and 0.654, respectively. Mark-Houwink exponent 0.654 indicated LCP1 molecule in NaNO3 solution was a random coil conformation.LCP1 molecules in aqueous solution formed the aggregates of which the aggregation rate was 45% while in NaNO3 solution there was only one population without aggregation. Therefore, 0.1 mol/L NaNO3 solution was a good solvent for studying the molecule information of LCP1. The Zimm plot from static light scattering measurement for LCP1 in 0.1 mol/L NaNO3 solution was obtained, and weight average molecular weight （Mw）, radius of gyration （Rg） and the second virial coefficient （A2） measured were 1.276×106 Da, 52.9 nm, 2.44×10-4 cm3 mol/g2, respectively. Hydrodynamic radius （Rh） from dynamic light scattering obtained was 34.8 nm by extrapolating to angle zero. LCP1 molecules in NaNO₃ solution was found to be a random coil conformation by analyzingρ=Rg/Rh （1.61）.Steady-shear rheological measurements of LCP1 showed a non-Newtonian shear-thinning flow behavior at high concentrations and a Newtonain flow behavior at low concentrations. The apparent viscosity decreased with the increase of temperature, and pH had a little effects. Salt might reduce the viscosity of LCP1 but the decrease was relative to salt concentration. LCP1 was found not to form a gel by viscoelastic tests.LCP1 was composed of rhamnose, glucose and galactose in mol rate of Rha:Glc:Gal=3:5:2. The structure of LCP1 was carried out by combining periodate oxidation, Smith degradation, methylation analysis and partial acid hydrolysis with GC-MS, GC, HPAEC-PAD and 1D 2D NMR. The possible structure was as follows:更多还原