植物乳杆菌胞外多糖发酵、结构鉴定及其功能特性研究
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摘要
乳酸菌(Lactic acid bacteria, LAB)胞外多糖(exopolysaccharides, EPS)是LAB在生长代谢过程中分泌到细胞壁外的黏液多糖和荚膜多糖的总称。由于其独特的理化性质及流变学特性,LAB EPS被作为增稠剂、乳化剂、胶凝剂和稳定剂,广泛的用于食品企业的生产中。目前的一些研究表明,LAB EPS具有抗肿瘤、增强免疫力、抗炎性、抗病毒、抗突变和抗氧化等功效。由于LAB被公认为是安全的(generally regarded as safe, GRAS)食品级微生物,因此其产生的EPS也相应地被认为是安全的,可以直接用于食品中。
本论文对从自然发酵泡菜中筛选到的一株产EPS的LAB进行了鉴定,并对其EPS进行了较系统的研究。主要结果如下：
1.采用菌落拉丝法这一简便的筛选技术从植物源性的天然发酵制品(泡菜)中筛选到一株高产EPS的LAB70810,通过形态学观察、生理生化鉴定、以及分子生物学的手段对该菌株进行了鉴定,最终将菌株70810判定为植物乳杆菌(Lactobacillus plant arum)。以Lb. plantarum70810EPS的产量为指标,通过单因素优化得出结果分别为：MRS培养基,最佳碳源为蔗糖,蔗糖30g/L,最佳氮源为大豆蛋白胨,大豆蛋白胨10g/L,接种量4%,pH6,发酵时间24h,发酵温度30℃。通过Plackett-Burman设计对Lb. plantarum70810生产EPS的培养基成分和培养条件共计14个因素进行了优化,通过SAS软件分析得到影响EPS产量的三个主要因素是蔗糖、接种量以及发酵温度。对由Plackett-Burman设计筛选出的蔗糖、接种量以及发酵温度,采用中心组合设计(CCD)及响应面分析法,得到Lb. plantarum70810发酵产EPS的最优条件为：蔗糖34g/L、接种量5%、发酵温度31℃。在此条件下发酵Lb. plantarum70810实际所得EPS产量为425.16mg/L,与预测值基本吻合。
2.通过Sevag法、三氯乙酸法脱蛋白的效果比较,认为终浓度4%的三氯乙酸法脱蛋白的效果好,蛋白质脱除率可达到86.4%。通过单因素试验分析了Lb.plantarum70810EPS醇沉的最佳条件；采用Box-Behnken的中心组合设计及响应面分析,建立了Lb.plantarum70810EPS醇沉条件的二次多项式数学模型。通过模型分析并修正后得到醇沉的最佳工艺参数为：静置时间13h、乙醇添加倍数4倍、发酵液浓缩倍数3.50倍。在此条件下进行验证实验所得EPS平均提取量为320.17mg/L。大孔吸附树脂对Lb.plantarum70810EPS脱色纯化的结果表明：选择S-8树脂,树脂用量为4%,多糖溶液浓度为2mg/mL,调节多糖溶液的pH值为6.0,在25℃条件下,静态吸附4h,脱色率为72.58%,糖保留率为69.15%。经S-8树脂脱色后的粗多糖样品依次使用DEAE-52和Sephadex G-100进行分级纯化后主要得到两个组分(EPS-1和EPS-2),采用HPLC法对EPS-1和EPS-2进行纯度鉴定和分子量测定,结果表明,EPS-1和EPS-2为均一的多糖组分,平均相对分子量(Mw)分别为1.75×104和1.20×105Da。分别采用硫酸-苯酚法、硫酸-间羟基联苯比色法、考马斯亮蓝法和氯化钡-明胶比色法,测定粗多糖、EPS-1和EPS-2中的总糖、糖醛酸、蛋白质和硫酸基的含量,结果表明,粗多糖、EPS-1、EPS-2的总糖含量分别为76.75%、94.45%、90.55%；糖醛酸含量分别为3.19%、2.09%、1.71%；蛋白质含量分别为0.32%、0.82%、1.58%；硫酸基未检出。
3.通过多种方法对EPS-1和EPS-2的结构特征进行了测定。采用三甲基硅醚衍生化法及气相色谱法对多糖样品的单糖组成进行分析,结果表明,EPS-1单糖组成及摩尔比为葡萄糖：甘露糖：半乳糖=53.24：10.41：1.00。EPS-2单糖组成及摩尔比为葡萄糖：甘露糖：岩藻糖=3.48：9.61：1.00。综合单糖组成分析、红外扫描、甲基化反应和NMR分析的结果,基本可以确定EPS-1是以1→4连接的α-型吡喃葡萄糖、甘露糖为主链并含有少量半乳糖的中性多糖。EPS-2是以1→4连接的α-型吡喃葡萄糖为主链、以1→2,6连接的甘露糖为支链,并含有少量岩藻糖的酸性多糖。
4.透析吸附试验结果表明,Lb. plantarum70810EPS对Pb(Ⅱ)离子的最佳吸附pH为5.0,吸附平衡时间为6h,吸附温度为30℃。当EPS溶液浓度为5g/L、Pb(Ⅱ)离子浓度在0.1mg/L到1000mg/L之间时,随着初始Pb(Ⅱ)离子浓度的增加,EPS对Pb(Ⅱ)离子的吸附量也增加。EPS用量从0.0005g(浓度为0.1g/L)增加到0.001g(浓度为0.2g/L)时,其对Pb(Ⅱ)离子的吸附量随着EPS用量的增加而增加；当EPS用量进一步增加时,其对Pb(Ⅱ)离子的吸附量则逐渐降低。EPS吸附Pb(Ⅱ)离子前后的红外光谱分析表明,羧基是EPS起吸附作用的主要官能团。
5.采用CCK-8法探讨了Lb.plantarum70810胞外粗多糖(Crude EPS)及纯化组分EPS-1和EPS-2对人前列腺癌细胞PC-3,人结肠癌细胞HCT-116,人肝癌细胞HepG2,及人大肠癌细胞HT-29的体外增殖抑制作用。结果表明,Crude EPS. EPS-1和EPS-2对人前列腺癌细胞PC-3,人结肠癌细胞HCT-116,人肝癌细胞Hep G2,及人大肠癌细胞HT-29的生长均具有明显的体外抑制作用,且呈现出一定的量效关系和时间-效能关系。当多糖样品浓度为400μg/mL时,处理72h后,Crude EPS、EPS-1和EPS-2对人前列腺癌细胞PC-3的增殖抑制率分别为93.74%、38.54%和39.40%；对人结肠癌细胞HCT-116的增殖抑制率分别为57.35%、55.01%和57.48%；对人肝癌细胞Hep G2的抑制率分别为96.76%、52.73%和59.76%；对人大肠癌细胞HT-29的抑制率分别为46.10%、39.78%和57.45%。
Lactic acid bacteria (LAB) exopolysaccharides (EPS) are long-chain polysaccharides secreted by LAB into the extracellular environment during growth. Due to their unique physical and rheological properties, EPS are widely used in the food industry as thickeners, emulsifiers, gelling agents and stabilizers. In addition, these polysaccharides have been demonstrated to possess important biological properties, such as antitumor, immunomodulatory, immunostimulatory, antinociceptive and antiinflammatory, antiviral, antimutagenicity and antioxidant activities. EPS produced by LAB have gained considerable attention mainly because LAB are food-grade bacteria known as GRAS and their EPS could be easily utilized in foods in a juridical point of view.
The isolation and identification of this EPS-producing Lb. plantarum strain70810from Chinese Paocai and the analysis of its EPS were carried out systemically. The main results are as follows:
1. LAB isolated from various traditional Chinese fermented foods were screened for the production of EPS. The rod-shaped strain70810from Chinese Paocai, which was identified as Lb. plantarum (HQ259238) by morphological, physiological, biochemical and16S rDNA tests, was selected due to its highest EPS production capability for further study. Effects of various culture conditions (media, carbon, nitrogen, inoculation volume, initial pH, temperature, and culture time) on EPS production were investigated firstly by single factor method. For EPS production with, the preferable culture conditions were30℃and pH6for24h with sucrose30g/L, soybean peptone10g/L, as the carbon, nitrogen of MRS meadium, respectively. The fermentation conditions of exopolysaccharides produced by Lb. plantarum70810were optimized. Three main factors were screened by Plackett-Burman (PB) design from fourteen nutritional and cultural factors affecting Lb. plantarum70810EPS yield. The screened significant factors were then optimized by response surface methodology (RSM). The PB experimental results showed that sucrose, inoculation volume and temperature were significant factors for the production of EPS. The optimal conditions were confirmed by RSM as follows:sucrose34g/L, inoculation volume5%(V/V) and temperature31℃. Under these optimal conditions, the70810EPS yield reached425.16mg/L. Conclusion:It is feasible to apply PB design and RSM to optimize the fermentation conditions of exopolysaccharides from Lb. plantarum70810.
2. To remove proteins, two methods were tested:Sevage method and trichloroacetic acid (TCA) method. The results indicated that TCA method is better. TCA final concentration4%was selected and the deproteinization rate was86.4%. The optimal conditions of ethanol precipitation of EPS produced by Lb. plantarum70810were investigated by single-factor experiments and response surface methodology. A mathematical model was then developed to show the significant effect of each factor and their interactions on the yield of EPS. The optimal parameters for ethanol precipitation of Lb. plantarum70810EPS were confirmed as follows:precipitation time12.56h, volume of ethanol3.9times, concentration multiple of original fermentation supernatant3.54times. Under the optimal condition, the model-predicted and experimental values of EPS yield were323.11mg/L and320.17mg/L, respectively, revealing0.91%relative error between them. The optimal decolorization conditions of EPS from Lb. plantarum70810were studied with macroporous adsorptive resin by single-factor experiments. The results showed that resin S-8demonstrated the highest decolorization ratio among various resins tested. The optimized decolorization conditions were determined as follows:resin S-8of4%(W/V), pH value of EPS solution6, concentration of EPS solution2mg/mL, for4h at25℃in static state. Under these conditions, the decolorization ratio and retention ratio of EPS were72.58%and69.15%, respectively. The decolorized and deproteinized EPS obtained from S-8resin was further purified by chromatography of DEAE-52and Sephadex G-100to afford two polysaccharide fractions (EPS-1and EPS-2). EPS-1and EPS-2both showed only one symmetrical peak on HPLC with the molecular weights estimated to be1.75×104and1.20×105Da, respectively. As to crude EPS, EPS-1, and EPS-2, polysaccharides content, measured by employing the method of sulfuric acid-phenol coloration, was76.75%、94.45%and90.55%, respectively. Uronic acid content, determined by using the method of cartazole-sulfuric acid assay, was3.19%、2.09%and1.71%, respectively. Protein content, measured by applying the method of coomassie brilliant blue coloration, was0.32%、0.82%and1.58%, respectively. Sulfuric radical content, determined by employing the method of gelatin-barium chloride assay, was not detected, respectively.
3. The structural characterizations of EPS-1and EPS-2were investigated by various methods. The analysis of monosaccharide compositions of EPS-1and EPS-2were carried out by GC. EPS-1was composed of glucose, mannose and galactose with a molar ratio of53.24:10.41:1.00. EPS-2was composed of glucose, mannose and fucose with a molar ratio of3.48:9.61:1.00. Spectra of FTIR implied that there were a-glycosidic bond and pyranose rings in EPS-1and EPS-2. On the base of the results of monosaccharide compositions, FTIR, methylation analysis, GC/MS,1H and13C NMR, it was possible to conclude that the repeated unit of EPS-1contained a backbone composed of1→4-linked-glucose and mannose, which attached to galactose. EPS-2contained a backbone composed of1→4-linked-glucose and branched by1→2,6-linked-mannose, which attached to fucose.
4. Biosorption of Pb(II) from aqueous solutions by70810EPS was studied with parameters of initial pH, contact time, initial Pb(II) concentration, adsorbent dosage, and temperature, respectively. Maximum adsorption of Pb (II) was observed at pH5,30℃, and contact time6h, respectively. The adsorption capacity was also found to be dependent on initial Pb(II) concentration and adsorbent dosage. The adsorption capacity increased when initial Pb(II) concentration increased from0.1to1000mg/L. The metals uptake increased from25.44to160.62mg/g with70810EPS dosage between0.5and1mg in5mL volume, but decreased as70810EPS dosage further increased. Fourier transform infrared spectroscopy (FT-IR) spectra analysis indicated that some functional groups (e.g.,-OH, COO-, C=O and-NH) of70810EPS were involved in Pb(II) biosorption process.
5. The inhibition effects of crude EPS, EPS-1and EPS-2on the growth of PC-3cells, HCT-116cells, Hep G2cells and HT-29cells were evaluated in vitro by using Cell Counting Kit-8(CCK-8) method. The results showed that, crude EPS, EPS-1and EPS-2all presented significantly higher antitumor activity against the PC-3cells, HCT-116cells, Hep G2cells and HT-29cells in vitro than blank control groups, and the inhibition ability was dose-dependent and time-dependent. At the concentration of400μg/ml for72h, the inhibition rates of crude EPS, EPS-1and EPS-2against the PC-3cells were93.74%,38.54%and39.40%, respectively. The inhibition rates of crude EPS, EPS-1and EPS-2against the HCT-116cells were57.35%,55.01%and57.48%, respectively. The inhibition rates of crude EPS, EPS-1and EPS-2against the Hep G2cells were96.76%,52.73%and59.76%, respectively. The inhibition rates of crude EPS, EPS-1and EPS-2against the HT-29cells were46.10%,39.78%and57.45%, respectively.
本论文对从自然发酵泡菜中筛选到的一株产EPS的LAB进行了鉴定,并对其EPS进行了较系统的研究。主要结果如下：
1.采用菌落拉丝法这一简便的筛选技术从植物源性的天然发酵制品(泡菜)中筛选到一株高产EPS的LAB70810,通过形态学观察、生理生化鉴定、以及分子生物学的手段对该菌株进行了鉴定,最终将菌株70810判定为植物乳杆菌(Lactobacillus plant arum)。以Lb. plantarum70810EPS的产量为指标,通过单因素优化得出结果分别为：MRS培养基,最佳碳源为蔗糖,蔗糖30g/L,最佳氮源为大豆蛋白胨,大豆蛋白胨10g/L,接种量4%,pH6,发酵时间24h,发酵温度30℃。通过Plackett-Burman设计对Lb. plantarum70810生产EPS的培养基成分和培养条件共计14个因素进行了优化,通过SAS软件分析得到影响EPS产量的三个主要因素是蔗糖、接种量以及发酵温度。对由Plackett-Burman设计筛选出的蔗糖、接种量以及发酵温度,采用中心组合设计(CCD)及响应面分析法,得到Lb. plantarum70810发酵产EPS的最优条件为：蔗糖34g/L、接种量5%、发酵温度31℃。在此条件下发酵Lb. plantarum70810实际所得EPS产量为425.16mg/L,与预测值基本吻合。
2.通过Sevag法、三氯乙酸法脱蛋白的效果比较,认为终浓度4%的三氯乙酸法脱蛋白的效果好,蛋白质脱除率可达到86.4%。通过单因素试验分析了Lb.plantarum70810EPS醇沉的最佳条件；采用Box-Behnken的中心组合设计及响应面分析,建立了Lb.plantarum70810EPS醇沉条件的二次多项式数学模型。通过模型分析并修正后得到醇沉的最佳工艺参数为：静置时间13h、乙醇添加倍数4倍、发酵液浓缩倍数3.50倍。在此条件下进行验证实验所得EPS平均提取量为320.17mg/L。大孔吸附树脂对Lb.plantarum70810EPS脱色纯化的结果表明：选择S-8树脂,树脂用量为4%,多糖溶液浓度为2mg/mL,调节多糖溶液的pH值为6.0,在25℃条件下,静态吸附4h,脱色率为72.58%,糖保留率为69.15%。经S-8树脂脱色后的粗多糖样品依次使用DEAE-52和Sephadex G-100进行分级纯化后主要得到两个组分(EPS-1和EPS-2),采用HPLC法对EPS-1和EPS-2进行纯度鉴定和分子量测定,结果表明,EPS-1和EPS-2为均一的多糖组分,平均相对分子量(Mw)分别为1.75×104和1.20×105Da。分别采用硫酸-苯酚法、硫酸-间羟基联苯比色法、考马斯亮蓝法和氯化钡-明胶比色法,测定粗多糖、EPS-1和EPS-2中的总糖、糖醛酸、蛋白质和硫酸基的含量,结果表明,粗多糖、EPS-1、EPS-2的总糖含量分别为76.75%、94.45%、90.55%；糖醛酸含量分别为3.19%、2.09%、1.71%；蛋白质含量分别为0.32%、0.82%、1.58%；硫酸基未检出。
3.通过多种方法对EPS-1和EPS-2的结构特征进行了测定。采用三甲基硅醚衍生化法及气相色谱法对多糖样品的单糖组成进行分析,结果表明,EPS-1单糖组成及摩尔比为葡萄糖：甘露糖：半乳糖=53.24：10.41：1.00。EPS-2单糖组成及摩尔比为葡萄糖：甘露糖：岩藻糖=3.48：9.61：1.00。综合单糖组成分析、红外扫描、甲基化反应和NMR分析的结果,基本可以确定EPS-1是以1→4连接的α-型吡喃葡萄糖、甘露糖为主链并含有少量半乳糖的中性多糖。EPS-2是以1→4连接的α-型吡喃葡萄糖为主链、以1→2,6连接的甘露糖为支链,并含有少量岩藻糖的酸性多糖。
4.透析吸附试验结果表明,Lb. plantarum70810EPS对Pb(Ⅱ)离子的最佳吸附pH为5.0,吸附平衡时间为6h,吸附温度为30℃。当EPS溶液浓度为5g/L、Pb(Ⅱ)离子浓度在0.1mg/L到1000mg/L之间时,随着初始Pb(Ⅱ)离子浓度的增加,EPS对Pb(Ⅱ)离子的吸附量也增加。EPS用量从0.0005g(浓度为0.1g/L)增加到0.001g(浓度为0.2g/L)时,其对Pb(Ⅱ)离子的吸附量随着EPS用量的增加而增加；当EPS用量进一步增加时,其对Pb(Ⅱ)离子的吸附量则逐渐降低。EPS吸附Pb(Ⅱ)离子前后的红外光谱分析表明,羧基是EPS起吸附作用的主要官能团。
5.采用CCK-8法探讨了Lb.plantarum70810胞外粗多糖(Crude EPS)及纯化组分EPS-1和EPS-2对人前列腺癌细胞PC-3,人结肠癌细胞HCT-116,人肝癌细胞HepG2,及人大肠癌细胞HT-29的体外增殖抑制作用。结果表明,Crude EPS. EPS-1和EPS-2对人前列腺癌细胞PC-3,人结肠癌细胞HCT-116,人肝癌细胞Hep G2,及人大肠癌细胞HT-29的生长均具有明显的体外抑制作用,且呈现出一定的量效关系和时间-效能关系。当多糖样品浓度为400μg/mL时,处理72h后,Crude EPS、EPS-1和EPS-2对人前列腺癌细胞PC-3的增殖抑制率分别为93.74%、38.54%和39.40%；对人结肠癌细胞HCT-116的增殖抑制率分别为57.35%、55.01%和57.48%；对人肝癌细胞Hep G2的抑制率分别为96.76%、52.73%和59.76%；对人大肠癌细胞HT-29的抑制率分别为46.10%、39.78%和57.45%。
Lactic acid bacteria (LAB) exopolysaccharides (EPS) are long-chain polysaccharides secreted by LAB into the extracellular environment during growth. Due to their unique physical and rheological properties, EPS are widely used in the food industry as thickeners, emulsifiers, gelling agents and stabilizers. In addition, these polysaccharides have been demonstrated to possess important biological properties, such as antitumor, immunomodulatory, immunostimulatory, antinociceptive and antiinflammatory, antiviral, antimutagenicity and antioxidant activities. EPS produced by LAB have gained considerable attention mainly because LAB are food-grade bacteria known as GRAS and their EPS could be easily utilized in foods in a juridical point of view.
The isolation and identification of this EPS-producing Lb. plantarum strain70810from Chinese Paocai and the analysis of its EPS were carried out systemically. The main results are as follows:
1. LAB isolated from various traditional Chinese fermented foods were screened for the production of EPS. The rod-shaped strain70810from Chinese Paocai, which was identified as Lb. plantarum (HQ259238) by morphological, physiological, biochemical and16S rDNA tests, was selected due to its highest EPS production capability for further study. Effects of various culture conditions (media, carbon, nitrogen, inoculation volume, initial pH, temperature, and culture time) on EPS production were investigated firstly by single factor method. For EPS production with, the preferable culture conditions were30℃and pH6for24h with sucrose30g/L, soybean peptone10g/L, as the carbon, nitrogen of MRS meadium, respectively. The fermentation conditions of exopolysaccharides produced by Lb. plantarum70810were optimized. Three main factors were screened by Plackett-Burman (PB) design from fourteen nutritional and cultural factors affecting Lb. plantarum70810EPS yield. The screened significant factors were then optimized by response surface methodology (RSM). The PB experimental results showed that sucrose, inoculation volume and temperature were significant factors for the production of EPS. The optimal conditions were confirmed by RSM as follows:sucrose34g/L, inoculation volume5%(V/V) and temperature31℃. Under these optimal conditions, the70810EPS yield reached425.16mg/L. Conclusion:It is feasible to apply PB design and RSM to optimize the fermentation conditions of exopolysaccharides from Lb. plantarum70810.
2. To remove proteins, two methods were tested:Sevage method and trichloroacetic acid (TCA) method. The results indicated that TCA method is better. TCA final concentration4%was selected and the deproteinization rate was86.4%. The optimal conditions of ethanol precipitation of EPS produced by Lb. plantarum70810were investigated by single-factor experiments and response surface methodology. A mathematical model was then developed to show the significant effect of each factor and their interactions on the yield of EPS. The optimal parameters for ethanol precipitation of Lb. plantarum70810EPS were confirmed as follows:precipitation time12.56h, volume of ethanol3.9times, concentration multiple of original fermentation supernatant3.54times. Under the optimal condition, the model-predicted and experimental values of EPS yield were323.11mg/L and320.17mg/L, respectively, revealing0.91%relative error between them. The optimal decolorization conditions of EPS from Lb. plantarum70810were studied with macroporous adsorptive resin by single-factor experiments. The results showed that resin S-8demonstrated the highest decolorization ratio among various resins tested. The optimized decolorization conditions were determined as follows:resin S-8of4%(W/V), pH value of EPS solution6, concentration of EPS solution2mg/mL, for4h at25℃in static state. Under these conditions, the decolorization ratio and retention ratio of EPS were72.58%and69.15%, respectively. The decolorized and deproteinized EPS obtained from S-8resin was further purified by chromatography of DEAE-52and Sephadex G-100to afford two polysaccharide fractions (EPS-1and EPS-2). EPS-1and EPS-2both showed only one symmetrical peak on HPLC with the molecular weights estimated to be1.75×104and1.20×105Da, respectively. As to crude EPS, EPS-1, and EPS-2, polysaccharides content, measured by employing the method of sulfuric acid-phenol coloration, was76.75%、94.45%and90.55%, respectively. Uronic acid content, determined by using the method of cartazole-sulfuric acid assay, was3.19%、2.09%and1.71%, respectively. Protein content, measured by applying the method of coomassie brilliant blue coloration, was0.32%、0.82%and1.58%, respectively. Sulfuric radical content, determined by employing the method of gelatin-barium chloride assay, was not detected, respectively.
3. The structural characterizations of EPS-1and EPS-2were investigated by various methods. The analysis of monosaccharide compositions of EPS-1and EPS-2were carried out by GC. EPS-1was composed of glucose, mannose and galactose with a molar ratio of53.24:10.41:1.00. EPS-2was composed of glucose, mannose and fucose with a molar ratio of3.48:9.61:1.00. Spectra of FTIR implied that there were a-glycosidic bond and pyranose rings in EPS-1and EPS-2. On the base of the results of monosaccharide compositions, FTIR, methylation analysis, GC/MS,1H and13C NMR, it was possible to conclude that the repeated unit of EPS-1contained a backbone composed of1→4-linked-glucose and mannose, which attached to galactose. EPS-2contained a backbone composed of1→4-linked-glucose and branched by1→2,6-linked-mannose, which attached to fucose.
4. Biosorption of Pb(II) from aqueous solutions by70810EPS was studied with parameters of initial pH, contact time, initial Pb(II) concentration, adsorbent dosage, and temperature, respectively. Maximum adsorption of Pb (II) was observed at pH5,30℃, and contact time6h, respectively. The adsorption capacity was also found to be dependent on initial Pb(II) concentration and adsorbent dosage. The adsorption capacity increased when initial Pb(II) concentration increased from0.1to1000mg/L. The metals uptake increased from25.44to160.62mg/g with70810EPS dosage between0.5and1mg in5mL volume, but decreased as70810EPS dosage further increased. Fourier transform infrared spectroscopy (FT-IR) spectra analysis indicated that some functional groups (e.g.,-OH, COO-, C=O and-NH) of70810EPS were involved in Pb(II) biosorption process.
5. The inhibition effects of crude EPS, EPS-1and EPS-2on the growth of PC-3cells, HCT-116cells, Hep G2cells and HT-29cells were evaluated in vitro by using Cell Counting Kit-8(CCK-8) method. The results showed that, crude EPS, EPS-1and EPS-2all presented significantly higher antitumor activity against the PC-3cells, HCT-116cells, Hep G2cells and HT-29cells in vitro than blank control groups, and the inhibition ability was dose-dependent and time-dependent. At the concentration of400μg/ml for72h, the inhibition rates of crude EPS, EPS-1and EPS-2against the PC-3cells were93.74%,38.54%and39.40%, respectively. The inhibition rates of crude EPS, EPS-1and EPS-2against the HCT-116cells were57.35%,55.01%and57.48%, respectively. The inhibition rates of crude EPS, EPS-1and EPS-2against the Hep G2cells were96.76%,52.73%and59.76%, respectively. The inhibition rates of crude EPS, EPS-1and EPS-2against the HT-29cells were46.10%,39.78%and57.45%, respectively.
引文
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