CAS OpenIR
黄秋葵嫩果干燥、多糖规模化制备及其性能研究
王华
Thesis Advisor赵兵
2020-06-01
Degree Grantor中国科学院大学
Degree Name博士
Degree Discipline生物化工
Keyword黄秋葵,干燥,多糖,分离纯化,生物活性
Abstract

黄秋葵(Abelmoschus esculentus(L.)Moench)为锦葵科秋葵属一年生草本植物,其嫩果含果胶及多糖等,具有多种保健功能,开发利用价值高。黄秋葵已在国内多个地区种植,产量大、市场价值高,但作为新鲜蔬菜鲜食需求量有限,采收期集中、鲜果难储存、易于纤维化造成资源浪费,传统干燥技术造成营养及功能成分流失,缺乏适宜于规模化生产的黄秋葵提取加工技术,缺乏以生物活性为基础的新产品等,这些问题均成为产业发展瓶颈。本论文从黄秋葵嫩果干燥、多糖规模化制备、理化性质、生物活性等方面进行系统研究,以期为黄秋葵资源髙值化利用和产业发展提供支撑。论文的主要研究内容和结果如下:1.黄秋葵嫩果干燥研究采用实验室自主研制的真空脉动超声干燥设备,分析了黄秋葵嫩果在不同干燥过程中的干燥时间、总能耗以及多糖等功能成分的含量,研究了不同预处理方式以及不同干燥方法对黄秋葵嫩果干燥速率及品质的影响。结果表明:超声联合真空干燥技术(ultrasound pulse with vacuum drying,UCVD)比真空冷冻干燥技术的干燥时间和总能耗分别减少53.33%和21.80%,UCVD比热风干燥技术的干燥时间和总能耗分别减少2.78%和56.64%。UCVD处理的黄秋葵的粗多糖得率为17.34%,显著高于其他干燥样品。干燥产品较好地保留了其他生物活性物质含量和复水潜力。通过研究有效解决了传统超声预处理营养有效成分流失,超声需要溶剂介质传播和物料预处理之间的难题,同时也解决了后续多糖提取所需原料供给问题。2.黄秋葵多糖规模化制备研究通过单因素实验和正交优化试验,确定了黄秋葵多糖提取工艺的最佳条件:料液比为1:20、提取温度为85℃、提取时间为2 h、提取2次。重复5次的验证试验得到黄秋葵多糖平均得率为16.78%,RSD为1.16%。运用过程工程的方法和理论,对比分析了实验室放大、中试及大规模工业化试生产三个不同工程放大阶段中黄秋葵多糖性质,研究结果表明:黄秋葵多糖得率仅略低于实验室优化条件下的多糖得率,Rha/GalA比值与红外图谱结果显示在不同操作单元过程中黄秋葵多糖是酸性多糖,并具有RG-I(聚鼠李半乳糖醛酸Ⅰ)结构。在任一放大阶段,随着热水搅拌提取、真空浓缩、真空干燥单元操作顺序进行,多糖分子量呈现降低趋势。比较实验室放大实验、中试生产、工业化生产中的黄秋葵多糖性质变化,中试试验和规模化生产的多分散性系数均显示出真空浓缩和真空干燥两个单元操作样品的分子量分布变宽,可能分子结构发生变化。3. 黄秋葵多糖制备分离纯化、理化性质与结构研究比较了分级醇沉、高速逆流色谱(High-speed Countercurrent Chromatography,HSCCC)和膜过滤等三种方法得到的黄秋葵多糖。分级醇沉得到的四个样品是OMPE-1,OMPE-2,OMPE-3,OMPE-4,重均分子量(Mw)分别为2,277、2,970、59.63、3.10 kDa;HSCCC得到三个样品是OMPH-1,OMPH-2,OMPH-3,Mw分别是2.51、1.94、2,586 kDa;膜过滤得到的三个样品是OMPMF,OMPUF,OMPNF,Mw分别为2,804、1.39、0.69 kDa。三种方法均分离得到Mw在2,500-3,000 kDa之间,多糖粘度随其分子量的增加而增加,且PDI在2.0左右的黄秋葵多糖组分,即OMPE-2、OMPMF和OMPH-3。三种多糖的单糖组成均有葡萄糖、半乳糖、半乳糖醛酸、阿拉伯糖、葡萄糖醛酸、甘露糖、鼠李糖。OMPE-2、OMPMF和OMPH-3的Rha/GalA比值分别为71.36%、64.51%和72.31%,且红外图谱结果显示在1146 cm-1处均具有糖醛酸的特征吸收峰,说明其均具有RG-I结构。利用原子力显微镜研究了黄秋葵多糖在溶液中的空间显微结构,结果表明:黄秋葵多糖呈不规则球状排列,且在高浓度溶液中高度聚集,使其具有特有的高粘度特性。4. 黄秋葵多糖生物活性评价 从体外抗氧化活性、模拟胃肠液环境下抗氧化活性、肝细胞毒性试验以及动物实验等方面,对黄秋葵多糖的生物活性进行评价。黄秋葵多糖样品体外抗氧化活性评价中,黄秋葵多糖样品的ABTS•+清除活性从强到弱依次是OMPE-4>OMPMF>OMPE-2>OMPUF>OMPH-3>OMPE-3>OMPE-1,黄秋葵多糖样品的FRAP活性从强到弱依次是OMPMF>OMPE-2>OMPUF>OMPE-4>OMPH-3>OMPE-3>OMPE-1,其中多糖组分OMPMF和OMPE-2具有相似的单糖组成,均具有半乳糖、半乳糖醛酸、葡萄糖醛酸、甘露糖和鼠李糖。模拟肠液环境下,FRAP活性在前100 min内,随着时间的变化呈现下降的趋势,在100 min之后变化趋缓。正常人肝细胞L02的24 h毒性实验结果表明,黄秋葵多糖不同样品浓度从0.10 mg/mL提高到1.00 mg/mL时,L02细胞存活率均保持在90%左右,即使浓度增到2.00 mg/mL时细胞存活率也保持在80.00%以上,因此,黄秋葵多糖对L02细胞没有显著毒性(P>0.05)。黄秋葵水提物低、中、高不同剂量组的实验小鼠抗疲劳实验中,血尿氮素浓度分别下降了18.26%、12.84%、13.56%;肝糖原含量分别提高了23.61%、2.89%、11.56%;血液乳酸清除率分别为64.64%、90.67%、86.89%。实验结果表明:黄秋葵水提物具有显著的缓解疲劳的功能。;Okra (Abelmoschus esculentus (L.) Moench), an annual herb of mallow, which contains pectin and polysaccharide, has many health functions and high value of development and utilization. Okra has been planted in many areas in China, with large output and high market value. However, as a fresh vegetable, the demand for fresh food is limited, the harvesting period is centralized, the fresh fruit is difficult to store, and it is easy to fibrosis, which leads to waste of resources. These have become the bottleneck of industrial development, such as the loss of nutrition and functional components caused by traditional drying technology, the lack of extraction and processing technology suitable for large-scale production of okra, the lack of new product development based on biological activity. In order to provide basis for high-value utilization of resources and industrial development, the drying method of okra pods, large-scale preparation of okra polysaccharide, physical and chemical properties and biological activities were systematically studied in this paper. The main contents and results are as follows:1. Drying method of okra pods.The effects of different pretreatment methods and drying methods on the drying rate and quality of okra pods were systematically studied with the vacuum pulse ultrasonic drying equipment (CN 104677066b) developed by ourselves. The drying time, total energy consumption and the content of polysaccharide and other functional components of okra were analyzed. The drying time and total energy consumption of ultrasound pulse with vacuum drying (UCVD) technology, which was developed independently by the experiment, were 53.33% and 21.80% shorter than vacuum freeze drying technology, and 2.78% and 56.64% shorter than hot air drying technology, respectively. The crude polysaccharide extraction rate of okra treated by UCVD was 17.34%, which was significantly higher than other samples. The dried product also retains the content of other bioactive substances and rehydration potential. UCVD effectively solves the problems of the loss of nutrients in traditional ultrasonic pretreatment, the transmission of solvent media and material pretreatment, and the supply of raw materials for subsequent polysaccharide extraction.2. Scale up test of preparation process of okra polysaccharide.The hot water stiring extraction technology of okra polysaccharide was studied. The optimum extraction conditions of okra polysaccharides were determined by single factor experiment and orthogonal experiment: the ratio of solid to liquid was 1:20, the extraction temperature was 85ºC, the extraction time was 2 h, the extraction was for 2 times. The average yield of okra polysaccharide was 16.78% and RSD was 1.16% in the repeated validation test. The properties of okra polysaccharide in laboratory scale-up, pilot plant scale-up and large-scale industrial scale scale-up were compared using the method and theory of process engineering. The yield of okra polysaccharide was slightly lower than that under the optimized conditions in the laboratory. The results of Rha/GalA ratio and infrared spectrum showed that okra polysaccharide was acid polysaccharide in different operation units, and had poly rhamnogalacturonic acid I (RG-I) structure. In the same scale-up stage of the project, the molecular weight of polysaccharide decreased with the operation sequence of hot water agitation extraction, vacuum concentration and vacuum drying unit. Compared with the changes of the properties of okra polysaccharide in laboratory scale-up experiment, pilot production and industrial production, the polydispersity coefficient of the pilot test and large-scale production shows that the molecular weight distribution of the two units of vacuum concentration and vacuum drying is wider, and the molecular structure may change.3. Preparation, properties and structure of okra polysaccharide.The separation and purification of okra polysaccharide were studied. The separation of okra polysaccharide by alcohol precipitation, high speed counter current chromatography (HSCCC) and membrane filtration were compared.Four components of okra polysaccharide (OMPE-1, OMPE-2, OMPE-3, OMPE-4) were separated by alcohol precipitation method. Their molecular weight (Mw) of OMPE-1, OMPE-2, OMPE-3, OMPE-4 were 2,277, 2,970, 59.63 and 3.10 kDa respectively. Three samples of okra polysaccharides (OMPH-1, OMPH-2, OMPH-3) were obtained by HSCCC. The Mw of OMPH-1, OMPH-2, OMPH-3 were 2.51, 1.94 and 2,586 kDa respectively. The Mw of three samples (OMPMF, OMPUF OMPNF) separated by membrane filtration were 2,804, 1.39 and 0.69 kDa. The polydispersity index (PDI) of OMPE-2, OMPMF, and OMPH-3 were 1.91, 1.98 and 2.06, respectively. The molecular weight distribution of polysaccharides was similar. The monosaccharide composition of three samples (OMPE-2, OMPMF and OMPH-3) contained glucose, galactose, galacturonic acid, arabinose, glucuronic acid, mannose and rhamnose. The results of Rha/GalA ratio (71.36%, 64.51% and 72.31% respectively) and infrared spectrum showed that they all had RG-I structure.The space microstructure of okra polysaccharide in solution was studied by AFM for the first time. The results showed that the okra polysaccharide was in irregular spherical arrangement and highly concentrated in high concentration solution, which made it have special high viscosity characteristics.4. Biological activity of okra polysaccharide. The biological activity of okra polysaccharide was evaluated from the aspects of antioxidant activity in vitro, antioxidant activity in simulated gastrointestinal fluid environment, hepatotoxicity test and animal experiment. The antioxidative activity of okra polysaccharide in vitro and the alleviation of physical fatigue were studied. The ABTS•+scavenging activity in order were OMPE-4 >OMPMF > OMPE-2 > OMPUF > OMPH-3 > OMPE-3 > OMPE-1 and the FRAP activities in the evaluation of antioxidant activity in vitro were OMPMF > OMPE-2 > OMPUF > OMPE-4 > OMPH-3 > OMPE-3 > OMPE-1. The polysaccharide component OMPMF and OMPE-2 have similar monosaccharide composition, all of which have galactose, galacturonic acid, glucuronic acid, mannose and rhamnose. The FRAP activity of okra polysaccharide decreased with time in the first 100 minutes. The results of 24-h toxicity experiments of L02 showed that when the concentration of okra polysaccharide was increased from 0.10 to 1.00 mg/mL, the survival rate of L02 cells remained at about 90%, even when the concentration was increased to 2.00 mg/mL, the survival rate of L02 cells also remained at over 80.00%, so okra polysaccharide had no significant toxicity to L02 cells (P > 0.05). In the blood urine nitrogen test of mice, the water extract of okra has obvious function of relieving fatigue. The concentrations of blood urine nitrogen in the low, medium, and high doses of okra extract decreased by 18.26 %, 12.84 %, and 13.56 % respectively. In mice liver glycogen test, the contents of liver glycogen in different dose groups increased by 23.61 %, 2.89 % and 11.56 % respectively. In the blood lactic acid level test of mice before and after swimming, the removal rate of blood lactic acid in different doses were 64.64 %, 90.67 % and 86.89 % respectively. 

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/49663
Collection中国科学院过程工程研究所
Recommended Citation
GB/T 7714
王华. 黄秋葵嫩果干燥、多糖规模化制备及其性能研究[D]. 中国科学院大学,2020.
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