【作者】 阿荣；

【导师】 张美莉；

【作者基本信息】 内蒙古农业大学 ， 农产品加工及贮藏工程， 2019， 博士

【摘要】 醪糟是我国的传统发酵食品,目前醪糟的种类日益丰富,满足不同人群的需求,并且由于醪糟酒精度低、营养丰富、风味独特等特点,极大地促进了醪糟行业的发展。燕麦醪糟中含有多糖等生物活性成分,免疫调节活性被公认为是多糖最重要的生物活性。本文系统研究了燕麦醪糟制作工艺的优化、多糖提取、脱蛋白工艺优化;结合两种多糖分离方法DEAE Sepharose-FF阴离子交换柱和Superdex G-100凝胶柱进行多糖分离纯化,采用高效凝胶渗透色谱法测定分子量,采用气相色谱法测定单糖组成,FT-IR对多糖进行红外光谱扫描,通过一维、二维NMR解析了燕麦醪糟多糖的结构,同时评价了燕麦醪糟多糖的体外抗氧化、对结肠癌细胞Lovo细胞的生长影响以及体外免疫调节等作用。主要研究结果如下:1、利用不同粒度的燕麦、黄米进行发酵制成醪糟产品,通过对其总糖、还原糖、多酚含量、总抗氧化性、pH值、感官评定的测定得出80 目的燕麦、黄米发酵醪糟为最佳。通过单因素实验确定最佳的料液比为1:3、燕麦与黄米添加比例为3:7、加曲量为8%。2、在单因素实验的基础上,通过正交试验设计,确立了燕麦醪糟多糖的最佳醇沉工艺参数:乙醇浓度为85%、醇沉pH值为8、醇沉时间为20 h,此条件下多糖的提取量为 11.09 g/L。3、在单因素实验的基础上,通过正交试验设计,确立了燕麦醪糟粗多糖的Sevage法脱蛋白最佳工艺条件:Sevage试剂与多糖溶液的体积比为3:2,氯仿与正丁醇体积比为5:1,振摇时间为20 min,除蛋白次数2次。4、采用DEAE-Sepharose FF色谱柱和Superdex G-100凝胶对燕麦粗多糖进行分离纯化,其洗脱最佳流速为1.0mL/min、最佳洗脱浓度梯度为0→0.2mol/L→0.4 mol/L→>0.6 mol/L NaCl缓冲液进行冲洗,上样最佳浓度为10 mg/mL。在最终扩大缓冲液浓度至2.0 mol/LNaCl后得到三种白色的燕麦醪糟多糖组分LCPS-1、LCPS-2、LCPS-3;经过Superdex G-100凝胶再次纯化后只有LCPS-2的HPGPC洗脱峰呈单一、对称正态分布,并且测得多糖组分LCPS-2是分子量为49.99 KDa的高纯度多糖组分。通过单糖组成分析,阿拉伯糖、木糖、半乳糖是构成LCPS-2的主要组分,并含有少量葡萄糖和甘露糖。NMR 证明,LCPS-2 中含→5)-α-L-Araf-(l→α-L-Araf-(1→3,6)-β-D-Galp-(1→的多糖片段。5、燕麦醪糟多糖组分的体外抗氧化实验表明,抗氧化性强弱顺序为:LCPS-2>LCPS-1>粗多糖。在实验范围内,随着燕麦醪糟多糖浓度的增加,DPPH自由基清除能力、羟基自由基清除能力及总抗氧化力逐渐增强,其中LCPS-2对DPPH自由基清除能力最强。当LCPS-2浓度达到600μg/mL时,对DPPH自由基、羟基自由基和总抗氧化力分别达到90%、31%和20.6 U/mL。选择抗氧化能力最强的LCPS-2在体外与结肠癌细胞Lovo细胞共同培养后,发现LCPS-2对于Lovo细胞生长没有抑制作用。6、LCPS-2的免疫活性评价结果表明,LCPS-2在50～400 μg/mL范围内能促进小鼠脾淋巴细胞增殖,实验范围内均能增强对中性红吞噬能力,提高NO分泌含量(P<0.01)。LCPS-2各浓度组对小鼠脾淋巴细胞培养上清中IL-2、IFN-y水平、NK细胞杀伤活性均高于空白对照组(P<0.05);LCPS-2浓度为200 μg/mL时,小鼠脾淋巴细胞上清液中的CD3+亚群水平明显升高(P<0.05),CD4+、CD8+亚群水平升高不显著。更多还原

【Abstract】 Lao-chao is a traditional fermented food which has a long history in China and was popular among the folk.At present,the varieties of lao-chao increased to satisfy the needs of different groups of people.Because the low alcohol content,rich nutrition and unique flavor,it has greatly promoted the industrial development of the lao-chao.However,there are few studies focus on bioactivity of lao-chao.This paper studied optimization of manufacturing oat lao-chao,extraction of polysaccharide and removal of its protein,separation and purification of polysaccharide.The separation and purification process based on anion exchange chromatography DEAE-sepharose FF and superdex G-100 gel filtration,molecule size of polysaccharide was determined by high performance gel premeatioin chromatography(HPGPC).The monosaccharide composition analyzed by gas chromatography.The structure of polysaccharide was analyzed by infrared scanning and 1DNMR and 2DHMR.We evaluated the oxidation resistance of polysaccharide and antitumor activity on cancer cell and immunoregulation activities of polysaccharide in vitro.The main results of our study are as follows:1.Lao-chao was fermented with different particle sizes of oats and prosco millet,by measuring total sugar,reducing sugar,polyphenol content,total antioxidant activity,pH value and sensory evaluation,80 mesh’s products were best.The optimum ratio of solid-liquid was 1:3,adding portion of prosco oat and millet was 3:7,and the ratio of starter was 8%.2.Based on the single factor experiment,the optimal alcohol sedimentation process parameters of lao-chao polysaccharides were established by orthogonal experimental design:ethanol concentration was 75%,alcohol precipitation pH was 8,and alcohol precipitation time was 20h.The results of orthogonal test on the basis of single factor showed that the optimal optimization conditions were as follows:ethanol concentration was 85%,alcohol precipitation pH was 8,and precipitation time was 20 h.Under this condition,the extraction amount of polysaccharide was 11.09 g/L.3.The results of the single factor experiment showed that the volume ratio of the best adding portion of polysaccharide solution and sevage agent was 1:1;the ratio of the best chloroform to n-Butanol was 5:1;the best shaking time was 15min;the times of getting rid of protein was 3 times.After the final four-factor and three-level orthogonal experiments,the optimal process conditions were obtained:portion of polysaccharide solution and sevage agent was 3:2,the volume ratio of chloroform to n-Butanol was 5:1,the shaking time was 20minutes.The times of protein removals was 2 times.4.The crude polysaccharide of oat lao-chao was separated and purified by DEAE-Sepharose FF column and superdex G-100 gel purification system.The optimum elution rate was 1.0mL/min,and the optimal elution concentration gradient of NaCl buffer was 0→0.2 mol/L→0.4 mol/L→0.6 mol/L,and the optimal injecting concentration of polysaccharide is 10mg/mL.After the final expansion of the NaCl buffer concentration to 2.0mol/L,three white polysaccharide components LCPS-1,LCPS-2 and LCPS-3 were obtained;After purification by the superdex G-100 polysaccharide gel purification system,only LCPS-2 was obtained.The elution peak showed a single,symmetric normal distribution by HPGPC,a high-purity polysaccharide component was obtained with molecular weight of 49.99 KDa.The monosaccarides compositoin of LCPS-2 was analyzed by GC-MS,arabinose,xylose and galactose are the main components of LCPS-2,a small amount of polysaccharide were glucose and mannose;β-glycosidic bond configuration contained in sugar residue.FT-IR and NMR,LCPS-2 contains fragment of→5)-α-L-Araf-(1→α-L-Araf-(1→3,6)-β-D-Galp-(1→.5.In vitro anti oxidant activity of oat lao-chao polysaccharides showed that the strong and weak order of antioxidant strength was LCPS-2>LCPS-1>crude polysaccharide.Within the experimental range,DPPH free radical scavenging ability,hydroxyl radical scavenging ability and total antioxidant capacity gradually increased during the increasing concentration of polysaccharide.Among polysaccharides of oat lao-chao LCPS-2 had the strongest ability to scavenge DPPH free radicals.When the concentration reached 600μg/mL,the DPPH free radicals,hydroxyl radicals and total antioxidant capacity reached 90%,31%and 20.6 U/mL,respectively.After the selection of LCPS-2,which has the strongest antioxidant capacity,we co-cultured colon cancer cell line Lovo in vitro with LCPS-2,it was found that LCPS-2 had no inhibitory effect on Lovo cell growth.6.Immunomodulatory activity of lao-chao polysaccharides:The immunological activity of LCPS-2 polysaccharide fraction was evaluated by in vitro cellular immunoassay.The results showed that purified polysaccharide LCPS-2 could promote the proliferation of mouse spleen lymphocytes in the range of 50～400 μg/mL.The ability of NO phago cytosis could be enhanced in the experimental range,and the content of NO was significantly increased(P<0.01).The levels of the IFN-y and IL-2 in supertanant of the mouse spleen lymphocyte LCPS-2 group were higher than those in the blank control group(P<0.05)and NK cell killing activity in the supernatant of mouse spleen lymphocyte were also higher than those in the blank control group(P<0.05).The levels of CD3+ subset group was increased significantly when concentration of LCPS-2 was 200 μg/mL(P<0.05),the levels of CD4+and CD8+ subset groups increased not significantly.更多还原