莲子低聚糖对双歧杆菌增殖效应的研究
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摘要
莲(Nelumbo nucifera Gaertn.)属睡莲科莲属植物，为福建省特色经济作物之一，具有丰富的营养及药用功效。本研究以福建建宁产莲子作为原料提取低聚糖，研究莲子低聚糖对双歧杆菌的体外增殖效应、莲子低聚糖对提升双歧杆菌耐模拟胃肠道逆环境能力的影响、响应面法优化莲子低聚糖为碳源的双歧杆菌培养基配方、莲子低聚糖的分离并对分离产物进行初步结构分析，为拓展莲子低聚糖应用，丰富莲子功能学领域研究提供理论依据。
以莲子作为原料提取低聚糖，研究莲子低聚糖对青春双歧杆菌的体外增殖作用。结果表明：与葡萄糖(GLU)、低聚木糖(XOS)和低聚果糖(FOS)相比，莲子低聚糖(LSO)对青春型双歧杆菌增殖效果极为显著(P<0.01)。培养基中莲子低聚糖最佳添加浓度为7g/L。菌体生长曲线的研究表明：在莲子低聚糖培养基中生长的双歧杆菌停滞期较葡萄糖培养基更短，最终菌体浓度较高，说明莲子低聚糖是一种有效的双歧杆菌增殖因子。
以葡萄糖、低聚木糖和低聚果糖为对照，研究莲子低聚糖对双歧杆菌耐模拟胃肠道逆环境特性的影响。结果表明：pH2.0环境下保持1.5h和pH2.0、pH3.0环境下保持3h，莲子低聚糖能显著提高双歧杆菌的酸耐受性(P<0.05)。1%牛胆盐环境下，莲子低聚糖相较于低聚木糖和低聚果糖能显著提高双歧杆菌的牛胆盐耐受性(P<0.05)。在模拟胃肠液环境中，莲子低聚糖具有提高双歧杆菌模拟胃肠液耐受性的能力，此能力与葡萄糖、低聚木糖和低聚果糖对比无差异(P>0.05)。说明莲子低聚糖能有效提高双歧杆菌在模拟胃肠道逆环境中的耐受能力，提示莲子低聚糖可能具有较好的耐消化特性。
采用部分因子和中心组合试验设计法结合响应面分析法优化双歧杆菌培养基配方，结果表明：影响培养双歧杆菌菌体干重的培养基因子为莲子低聚糖、胰蛋白胨和pH。最佳培养基配方为：大豆蛋白胨5g/L，莲子低聚糖7.91g/L，胰蛋白胨3.66g/L，酵母粉10g/L，吐温-801mL，L-半胱氨酸盐酸盐0.5g/L，无机盐溶液40mL，pH7.04。配方优化后的培养基可获得菌体干重为4.722g/L，菌体干重的回归模型为：
Y=19.327x_1+15.896x_2+17.720x_3-1.008x_1x_1-0.515x_1x_2-0.212x_1x_3-2.103x_2x_2+0.509x_2x_3-1.271x_3x_3-163.217
莲子低聚糖经DEAE纤维素-52、葡聚糖凝胶G-15和G-25分离后，得到三种组分：LSO1、LSO2和LSO3-2。紫外全波段扫描分析结果表明，低聚糖溶液中的蛋白质已基本除尽。高效液相色谱分析结果表明，LSO1、LSO2和LSO3-2的糖链都由D-果糖组成。红外光谱指纹区特征吸收峰分析结果表明，莲子低聚糖分离出的三个低聚糖组分都含有羧基，其中LSO1和LSO2组分的单糖基为α-糖苷键构型，以吡喃环形式存在。
Lotus-Seed (Nelumbo nucifera Gaertn.) is one of the special economical resourcein Fujian Province, which contains rich nutrition and special health value. In thispaper, oligosaccharides were extracted as the material from lotus-seed, theproliferative effects of Bifidobacterium in vitro, the effects of Bifidobacteriumadolescence with acquired tolerance to simulated human gastrointestinal tract, theRSM was used to optimize Bifidobacterium medium components, the separatedtechnology and the structure information were studied. The application of oligo-saccharides is expanded, the basis data were offered for the lotus-seed’s functionresearch.
The proliferative effects of lotus-seed oligosaccharides on Bifidobacterium ado-lescentis in vitro were studied. The results showed that: the proliferative effects ofLSO was considerable significant difference with GLU, XOS and FOS (P<0.01). Thebest concentration of LSO in medium was7g/L. The retardate time of Bifidobacter-ium adolescence in LSO medium was shorter than GLU medium, which can be shownthat LSO was an effective Bifidobacterium factor proliferation.
The simulated gastric tolerance of Bifidobacterium adolescence with LSO wasassayed and compared with GLU, XOS and FOS. The results showed that: Bifido-bacterium adolescence was incubated at pH2.0for1.5h or pH2.0and pH3.0for3h,the acid tolerance of Bifidobacterium adolescence can be improved signifycantly byLSO in this environment (P<0.05). The bile tolerance of Bifidobacterium adolescencecan be improved significantly by LSO in the1%bovine bile salt and compared withXOS and FOS (P<0.05). The simulated gastric juices tolerance of Bifidobacteriumadolescence with LSO was not significant difference with the others by in thesimulated digestive juice (P>0.05). The results indicated that the ability ofBifidobacterium adolescence in simulated human gastrointestinal tract can beenhanced by LSO, suggesting that the resistance to digestion of LSO may beperformed.
Fractional factorial and central composite experimental design combined with response surface methodology was used to optimize the composition of Bifido-bacterium adolescence medium, the results show that: the important factors of theBifidobacterium medium which made influence for the dry weight of Bifidobacteriumadolescence was lotus-seed oligosaccharides, tryptone, and pH. The optimum mediumcompositions were as follows: soybean peptone5g/L, LSO7.91g/L, tryptone3.66g/L,yeast extract10g/L, and Twain-801mL, of L-cysteine hydrochloride0.5g/L, inorga-nic salt solution40mL, pH7.04，Final dry weight of biomass was4.722g/L. Theregression model of the dried cells weight was listed as follows:
Y=19.327x_1+15.896x_2+17.720x_3-1.008x_1x_1-0.515x_1x_2-0.212x_1x_3-2.103x_2x_2+0.509x_2x_3-1.271x_3x_3-163.217
The LSO was extracted in hot water and it was isolated by DEAE-52, SephadexG-15and Sephadex G-25, which contained three components: LSO1, LSO2, andLSO3-2. The results of UV band scanning analysis showed that the protein had beenbasically removed from the LSO dissolvent after being purified. High performanceliquid chromatography analysis showed that the sugar chain of LSO was consist ofD-fructose. The analysis of infrared spectra fingerprint characteristic absorption peakshowed that the three components of LSO contain carboxyl, LSO1and LSO2monosaccharide residues was bonded by the α-glycosidic and consited by pyran ringform.
以莲子作为原料提取低聚糖，研究莲子低聚糖对青春双歧杆菌的体外增殖作用。结果表明：与葡萄糖(GLU)、低聚木糖(XOS)和低聚果糖(FOS)相比，莲子低聚糖(LSO)对青春型双歧杆菌增殖效果极为显著(P<0.01)。培养基中莲子低聚糖最佳添加浓度为7g/L。菌体生长曲线的研究表明：在莲子低聚糖培养基中生长的双歧杆菌停滞期较葡萄糖培养基更短，最终菌体浓度较高，说明莲子低聚糖是一种有效的双歧杆菌增殖因子。
以葡萄糖、低聚木糖和低聚果糖为对照，研究莲子低聚糖对双歧杆菌耐模拟胃肠道逆环境特性的影响。结果表明：pH2.0环境下保持1.5h和pH2.0、pH3.0环境下保持3h，莲子低聚糖能显著提高双歧杆菌的酸耐受性(P<0.05)。1%牛胆盐环境下，莲子低聚糖相较于低聚木糖和低聚果糖能显著提高双歧杆菌的牛胆盐耐受性(P<0.05)。在模拟胃肠液环境中，莲子低聚糖具有提高双歧杆菌模拟胃肠液耐受性的能力，此能力与葡萄糖、低聚木糖和低聚果糖对比无差异(P>0.05)。说明莲子低聚糖能有效提高双歧杆菌在模拟胃肠道逆环境中的耐受能力，提示莲子低聚糖可能具有较好的耐消化特性。
采用部分因子和中心组合试验设计法结合响应面分析法优化双歧杆菌培养基配方，结果表明：影响培养双歧杆菌菌体干重的培养基因子为莲子低聚糖、胰蛋白胨和pH。最佳培养基配方为：大豆蛋白胨5g/L，莲子低聚糖7.91g/L，胰蛋白胨3.66g/L，酵母粉10g/L，吐温-801mL，L-半胱氨酸盐酸盐0.5g/L，无机盐溶液40mL，pH7.04。配方优化后的培养基可获得菌体干重为4.722g/L，菌体干重的回归模型为：
Y=19.327x_1+15.896x_2+17.720x_3-1.008x_1x_1-0.515x_1x_2-0.212x_1x_3-2.103x_2x_2+0.509x_2x_3-1.271x_3x_3-163.217
莲子低聚糖经DEAE纤维素-52、葡聚糖凝胶G-15和G-25分离后，得到三种组分：LSO1、LSO2和LSO3-2。紫外全波段扫描分析结果表明，低聚糖溶液中的蛋白质已基本除尽。高效液相色谱分析结果表明，LSO1、LSO2和LSO3-2的糖链都由D-果糖组成。红外光谱指纹区特征吸收峰分析结果表明，莲子低聚糖分离出的三个低聚糖组分都含有羧基，其中LSO1和LSO2组分的单糖基为α-糖苷键构型，以吡喃环形式存在。
Lotus-Seed (Nelumbo nucifera Gaertn.) is one of the special economical resourcein Fujian Province, which contains rich nutrition and special health value. In thispaper, oligosaccharides were extracted as the material from lotus-seed, theproliferative effects of Bifidobacterium in vitro, the effects of Bifidobacteriumadolescence with acquired tolerance to simulated human gastrointestinal tract, theRSM was used to optimize Bifidobacterium medium components, the separatedtechnology and the structure information were studied. The application of oligo-saccharides is expanded, the basis data were offered for the lotus-seed’s functionresearch.
The proliferative effects of lotus-seed oligosaccharides on Bifidobacterium ado-lescentis in vitro were studied. The results showed that: the proliferative effects ofLSO was considerable significant difference with GLU, XOS and FOS (P<0.01). Thebest concentration of LSO in medium was7g/L. The retardate time of Bifidobacter-ium adolescence in LSO medium was shorter than GLU medium, which can be shownthat LSO was an effective Bifidobacterium factor proliferation.
The simulated gastric tolerance of Bifidobacterium adolescence with LSO wasassayed and compared with GLU, XOS and FOS. The results showed that: Bifido-bacterium adolescence was incubated at pH2.0for1.5h or pH2.0and pH3.0for3h,the acid tolerance of Bifidobacterium adolescence can be improved signifycantly byLSO in this environment (P<0.05). The bile tolerance of Bifidobacterium adolescencecan be improved significantly by LSO in the1%bovine bile salt and compared withXOS and FOS (P<0.05). The simulated gastric juices tolerance of Bifidobacteriumadolescence with LSO was not significant difference with the others by in thesimulated digestive juice (P>0.05). The results indicated that the ability ofBifidobacterium adolescence in simulated human gastrointestinal tract can beenhanced by LSO, suggesting that the resistance to digestion of LSO may beperformed.
Fractional factorial and central composite experimental design combined with response surface methodology was used to optimize the composition of Bifido-bacterium adolescence medium, the results show that: the important factors of theBifidobacterium medium which made influence for the dry weight of Bifidobacteriumadolescence was lotus-seed oligosaccharides, tryptone, and pH. The optimum mediumcompositions were as follows: soybean peptone5g/L, LSO7.91g/L, tryptone3.66g/L,yeast extract10g/L, and Twain-801mL, of L-cysteine hydrochloride0.5g/L, inorga-nic salt solution40mL, pH7.04，Final dry weight of biomass was4.722g/L. Theregression model of the dried cells weight was listed as follows:
Y=19.327x_1+15.896x_2+17.720x_3-1.008x_1x_1-0.515x_1x_2-0.212x_1x_3-2.103x_2x_2+0.509x_2x_3-1.271x_3x_3-163.217
The LSO was extracted in hot water and it was isolated by DEAE-52, SephadexG-15and Sephadex G-25, which contained three components: LSO1, LSO2, andLSO3-2. The results of UV band scanning analysis showed that the protein had beenbasically removed from the LSO dissolvent after being purified. High performanceliquid chromatography analysis showed that the sugar chain of LSO was consist ofD-fructose. The analysis of infrared spectra fingerprint characteristic absorption peakshowed that the three components of LSO contain carboxyl, LSO1and LSO2monosaccharide residues was bonded by the α-glycosidic and consited by pyran ringform.
引文
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