牛初乳中功能寡糖的分离纯化技术研究

发布时间：2018-04-15 05:00

本文选题：牛初乳 + 功能乳寡糖　；参考：《华东理工大学》2017年硕士论文

【摘要】：牛初乳中含有种类丰富且具有生理活性的功能寡糖,在食品和保健品领域有潜在的应用前景。目前对这些寡糖的研究尚不充分,对于结构复杂、种类较多的混合寡糖缺乏有效的分离方法,极大地限制了牛初乳中功能寡糖的开发和应用。本论文旨在利用膜分离耦合色谱分离技术对牛初乳中的功能寡糖特别是唾液酸化的混合寡糖进行分离纯化,优化并得到最佳的分离工艺参数,获得高纯度的唾液酸乳糖,对牛初乳的高值化利用提供依据。首先,本研究建立了一种快速、准确的分析测定牛初乳中糖类物质的方法;并通过乳糖、半乳糖、3'-唾液酸乳糖、6'-唾液酸乳糖四种牛乳中含量较多的糖类标准品对该方法进行验证。结果表明:乳糖、半乳糖、3'-唾液酸乳糖、6'-唾液酸乳糖,分别在0.03-5.00 mg/mL、0.02-5.00 mg/mL、0.05-2.00 mg/mL、0.05-2.00 mg/mL 浓度范围内,该方法具有良好的线性关系(R20.99)和重复性(峰面积的RSD4%,保留时间RSD2%);四种标准品的回收率均在100%-115%之间,LOD、LOQ分别为9 μg/mL、30 μg/mL,7 μg/mL、20μg/mL,125μg/mL、200μg/mL,350μg/mL、450μg/mL。基于建立的检测方法对牛初乳中主要寡糖组分进行了分析测定,发现牛初乳中大约有27种不同的功能寡糖,其含量大约为1.93 g/L。其次,根据牛初乳组成成分的物理化学特性,选择了截留分子量为10 kDa的超滤膜和截留分子量为280 Da的纳滤膜对牛初乳中的寡糖进行初步分离纯化。结果表明:超滤过程中压力和温度对膜通量具有明显的影响,但对蛋白质截留率和总糖的回收率无明显作用。在最适的操作压力和操作温度(5 bar,40℃)条件下蛋白质的截留率为96.0%,总糖的回收率为98.3%。纳滤分离过程中膜通量随着压力和温度的增大明显的变大,单糖和乳糖的截留率随着压力的增大先升高后降低,随着温度的升高不断的降低;而压力和温度的变化对寡糖的截留率无明显的影响。在最适的操作压力和操作温度(8 bar,45℃)条件下,单糖和乳糖的截留率为41.9%,寡糖的截留率为97.0%。最后,对膜分离得到的寡糖粗提液的成分进行测定,发现乳糖和唾液酸乳糖含量最高。根据乳糖和唾液酸乳糖的性质差异及色谱分离技术的原理,选择D315、QY-P001、QY-Z001、PCR642Ca、CGC100X8五种树脂作为研究对象,综合乳糖和唾液酸乳糖的分离度、回收率和保留时间等因素,筛选合适的树脂。结果表明:QY-Z001型阳离子交换树脂是最适合分离唾液酸乳糖和乳糖的树脂,且在该树脂上乳糖为强保留组分,唾液酸乳糖为弱保留组分。以QY-Z001树脂作为固定相,去离子水作为流动相,研究了径高比、柱温、洗脱速度、进样浓度等因素对乳糖和唾液酸乳糖分离效果的影响。结果表明:色谱柱径高比为1:35、温度为55℃、流速1.0 mL/min、寡糖混合物浓度为100 mg/mL的条件下,寡糖混合物中唾液酸乳糖和乳糖的分离效果最好,分离度可以达到0.96;色谱分离得到的样品中唾液酸乳糖和乳糖的纯度分别为84.6%、99.3%,回收率分别为76.9%、100.0%。树脂QY-Z001适合牛初乳寡糖混合物中唾液酸乳糖和乳糖的分离,且具有良好的分离效果。研究结果为牛初乳中功能寡糖的分离纯化提供了理论依据。
[Abstract]:Bovine colostrum contains a variety of functional oligosaccharides with physiological activity, have potential applications in food and health care products. The study of these oligosaccharides is not sufficient, for the complex structure, many types of mixed oligosaccharides lack effective separation methods, which greatly limits the development and application of functional oligosaccharides in bovine colostrum this paper aims to use membrane separation. The coupling function of chromatographic separation of bovine colostrums oligosaccharides in particular hybrid oligosaccharide sialyl were purified and separated, optimize the best technology parameter and obtain high purity sialyllactose, bovine colostrum high value utilization is provided. Firstly, this study established a rapid the analysis method, accurate determination of carbohydrates in bovine colostrum; and by lactose, galactose, 3'- sialyllactose, 6'- sialyllactose four milk content of more sugar The standard to verify the method. The results showed that: lactose, galactose, 3'- sialyllactose, 6'- sialyllactose, respectively, in 0.03-5.00 mg/mL, 0.02-5.00 mg/mL, 0.05-2.00 mg/mL, 0.05-2.00 mg/mL concentration range, the method has a good linear relationship (R20.99) and repeatability (peak area of RSD4%, retention time RSD2%); four standard recovery rate was between 100%-115%, LOD and LOQ were 9 g/mL, 30 g/mL, 7 g/mL, 20 g/mL, 125 g/mL, 200 g/mL, 350 g/mL, 450 g/mL. based on the detection method of primary bovine milk oligosaccharides group by analysis, found that there are about 27 different species of functional oligosaccharides in bovine colostrum, the content of about 1.93 g/L. second, according to the physical and chemical properties of bovine colostrum composition, the molecular weight cut-off of 10 kDa membrane with a MWCO of 280 Da nanofiltration membrane in bovine colostrum Oligosaccharides were purified. The results showed that the ultrafiltration pressure and temperature has obvious influence on the membrane flux, but the rate has no significant effect on the recovery of protein retention rate and total sugar. The operating pressure and the optimum temperature (5 bar, 40 C) under the condition of protein retention rate was 96%. The total sugar recovery rate of 98.3%. in the process of nanofiltration membrane flux with temperature and pressure increased significantly larger, monosaccharide and lactose retention rate with the increase of pressure increased first and then decreased, with the increase of temperature decreasing; and the pressure and temperature of the oligosaccharides retention rate had no significant effect. In the operating pressure and temperature optimum (8 bar, 45 DEG C) condition, monosaccharide and lactose retention rate of 41.9%, oligosaccharides retention rate is 97.0%. finally, the membrane separation obtained oligosaccharide crude extract components were measured and found that lactose and sialic acid The highest content of lactose and lactose. According to the differences between the sialyllactose and the chromatographic separation technology principle, select D315, QY-P001, QY-Z001, PCR642Ca, CGC100X8 five resin as the research object, synthesis of lactose and sialyllactose separation factors, the recovery rate and retention time, selecting the appropriate resin. The results showed that: QY-Z001 cation exchange resin is the most suitable resin for separation of sialyllactose and lactose, and the resin for lactose retention components, sialyllactose for weak retention components. Using QY-Z001 resin as stationary phase, deionized water as mobile phase, the ratio of diameter to height, column temperature, elution speed, influence the separation effect the sample concentration of lactose and sialyllactose. The results showed that the column ratio is 1:35, the temperature is 55 DEG C, the flow rate of 1 mL/min, the concentration of a mixture of 100 mg/mL, oligosaccharide mixture in saliva The best separation effect of acid lactose and lactose, the separation degree can reach 0.96; the purity of chromatographic separation of sialyllactose and lactose samples obtained were 84.6%, 99.3%, the recovery rate was 76.9%, 100.0%. separation resin QY-Z001 for sialyllactose and lactose in bovine colostrum oligosaccharide mixture, and has a good separation effect. The research results provide a theoretical basis for the separation function of bovine colostrum in the purification of oligosaccharides.

【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS252.1

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