壳聚糖澄清猕猴桃果汁及其澄清机理的探讨
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摘要
我国是猕猴桃的原产国,品种资源丰富。猕猴桃果实营养丰富,可防治坏血病、冠心病等疾病,Vc含量比一般水果高数十倍, 具很高的开发价值。猕猴桃属浆果,
采收时正值高温季节,果实采后极易腐烂,严重影响猕猴桃种植业发展。故研究猕猴桃加工技术,对充分开发利用我国的猕猴桃资源,提高农产品经济价值有重要的意义。猕猴桃果实中含有较高的蛋白质,与其它水果相比,是一种难以加工的水果品种,在猕猴桃清汁生产和贮藏过程中易引起混浊和二次沉淀。本课题从实际情况出发,研究了壳聚糖澄清猕猴桃果汁的生产工艺及其澄清机理。主要的研究内容和结果如下:
首先由不同粒度的甲壳素制备壳聚糖,结果表明粒度小的甲壳素制取的壳聚糖脱乙酰度比粒度大的甲壳素制取的大;凝胶过滤色谱测定不同壳聚糖相对分子质量分布得出:脱乙酰反应10小时所得壳聚糖的相对分子质量较反应8小时的小,脱乙酰反应14小时时,多糖主链的糖苷键已经发生断裂,产生了相对分子质量更小的多糖组分。
通过单因素实验和拟因子正交实验设计找出了壳聚糖澄清猕猴桃压榨过滤后果汁(原果汁)的最优条件为:用脱乙酰度80%的壳聚糖,加入量0.4g/L,加入壳聚糖后原果汁于50℃保温30分钟,pH为自然pH值,壳聚糖澄清原果汁后离心20分钟;用此条件制得的清汁透光率达到98%。贮藏实验结果表明低温有利于清汁的贮藏稳定。
离子交换层析色谱分离壳聚糖的研究结果表明:离子强度线性梯度洗脱时洗脱体积的增加有利于壳聚糖的分离;增加离子交换柱的床体积,分离制备了澄清原果汁效果最好的脱乙酰度80%壳聚糖样品(壳聚糖k),分离得到三个组分(a、b、c),收集、透析脱盐,脱盐率分别达到99.7%、99.5%、99.6%;红外图谱得壳聚糖k、a、b、c的脱乙酰度分别为82%,56%,73%,88%,粘度法测定得壳聚糖k、a、b、c的相对分子质量:1140000、1350000、740000、370000。
分别用壳聚糖k、a、b、c澄清原果汁,对澄清前后果汁中的化学成分变化和澄清后清汁透光率、色泽的研究结果表明:壳聚糖k、a、b、c都可以有效的澄清原果汁,澄清后清汁透光率都达到95%以上;壳聚糖k澄清后的清汁色泽浅,壳聚糖c澄清后的清汁色泽较深;分别用壳聚糖k、a、b、c澄清原果汁,澄清前后果汁中可溶性固形物和Vc含量相同,不影响猕猴桃果汁的营养价值;澄清后,原果汁中的蛋白质含量分别降低了67.35%、69.00%、72.70%、68.38%,总酚含量分别降低了6.49%、11.90%、16.44%、14.62%。贮藏实验表明:壳聚糖b澄清原果汁所得清汁的贮藏稳定性较好。
原果汁中多酚提取物的紫外-可见扫描结果表明原果汁中的多酚有可能是黄酮和黄烷醇类多酚。不同大孔吸附树脂对果汁中多酚物质的吸附解吸研究表明, DA201-A吸附
树脂对果汁中多酚物质的吸附性能较好,对多酚的吸附率达到78.63%;高效液相色谱对果汁中多酚的分析结果表明:分别用壳聚糖k、a、b、c澄清原果汁后,所得清汁中的多酚种类不同。HPLC-ESI/MS分析结果表明:吸附树脂分离原果汁中多酚物质所得乙醇洗脱组分又经C-18小柱分离的甲醇洗脱组分中的多酚样品中含有槲皮素-3-鼠李糖、山奈素-3-鼠李糖苷、山奈素-3-芸香糖苷、表没食子儿茶素、原花青素的二聚体、槲皮素-3-葡萄糖、表儿茶素没食子酸酯、原花青素的二聚单酯、山奈素-3-葡萄糖苷和芦丁。
壳聚糖k澄清原果汁后所得絮凝物溶于不同pH缓冲液中蛋白质的溶出量不同,pH4.5-7.5,蛋白质溶出量由低到高变化很大,pH 4.0以下蛋白质未能溶出,pH7.5以上蛋白质溶出量随pH提高增加不明显。凝胶过滤色谱测定自絮凝物中溶出的蛋白质相对分子质量分布的实验结果表明:絮凝物(分别用壳聚糖k 、a、b、c澄清原果汁后所得)分别用不同缓冲液复溶其中蛋白质时,蛋白质的Sepharose CL-6B凝胶过滤色谱图不同。
光学显微镜观察壳聚糖k澄清原果汁前后果汁中粒子状态的结果表明:原果汁中含有圆形粒子,粒子为多分散态;壳聚糖加入原果汁后,分散的少数粒子先聚集在一起,随时间延长,聚集的粒子数目增多,逐渐形成一个核,周围的粒子以这个核为中心以堆积方式聚集在一起,絮凝物体积不断增大。原果汁中粒子的平均粒径为38.322μm,壳聚糖k加入原果汁中保温10min后粒子的平均粒径为44.143μm,保温20min后粒子的平均粒径为62.995μm。壳聚糖k的加入可促使果汁中物质之间的相互聚集,形成大的颗粒,从而絮凝原果汁中的某些物质,达到澄清果汁的目的。
扫描电子显微镜观察分别用壳聚糖k、a、b、c澄清原果汁后所得到絮凝物超微结构表明:分别用壳聚糖k、a、b、c澄清原果汁后,得到絮凝物的结构和形态是不同的;经过壳聚糖k、a澄清处理后,絮凝物中颗粒之间的聚集较对照样紧密, 壳聚糖b、c澄清原果汁后所得絮凝物具有网络结构。
用果胶-蛋白质-丹宁酸混合体系作为猕猴桃原果汁的模拟体系研究结果表明:1、果胶溶液中果胶分子聚集物呈中空球形均匀分散;2、果胶/牛血清白蛋白混合溶液中的球形粒子有两种:粒子中央光线可透过,照片上呈环形;粒子的中央包含有单个或多个球形粒子的聚集体,认为粒子的中央是蛋白质,外层是果胶;3、果胶的存在抑制或减弱了蛋白质-丹宁酸之间的相互作用,果胶起到防止蛋白质-多酚产生沉淀的作用,增强了溶液的稳定性;4、壳聚糖加入果胶-蛋白质-丹宁酸混合溶液中时,可?
Kiwifruit is affluent in variety and resource in China where is its original habitat. Kiwifruit is abundant in Vitamin C, the concentration of which is ten times more than other fruits. This gives kiwifruit the properties to prevent scurvy, hypertension, coronary heart disease etc. Kiwifruit belongs to bacca. It is sensitive to ethylene and easily decomposes after picking, which affect the plantation development of kiwifruit weightily. Therefore, studying the processing technique of kiwifruit is important to exploit it as a resource and improve its economic value. Kiwifruit is high in protein compared with many other fruits and this causes haze problems in clear juice processing and storage. The objective of this dissertation was to develop a technology for processing clear kiwifruit juice using chitosan and to investigate the mechanism involved in the clarification treatment.
Chitosan was prepared from chitin with different granularity. The reactive trends were similar under the reaction condition of same temperature, same lye concentration and different reaction times. The degree of deacetylation (DD) of chitosan increased quickly in the first several hours of the reaction, and then remained stationary. DD of chitosan made from chitin with small granularity was higher than that with big granularity. Gel permeation chromatography analysis showed that the molecular weight of chitosan prepared at 10 hours reaction time was smaller than that at 8 hours reaction time and the main chain of chitosan had been ruptured at 14 hours reaction time.
According to the results of single and orthogonal tests, the procedure for chitosan clarification kiwifruit pressing juice was as follows: selected kiwifruits were washed, sterilized , rinsed and pressed; the juice was filtered then added into 0.4g/L chitosan with 80% degree of deacetylation and kept for 30min at 50℃ constant temperature; and then the juice was centrifuged. Percent transmittance of clear kiwifruit juice prepared under this condition was 98%. The result of the storage experiment showed that low temperature was propitious to storage stability of clear juice.
The effects of factors such as the eluate volume on the separation of chitosan by ion- exchange chromatography were also studied. The results demonstrated that increase in eluate volume were favorable to isolation with a continuous gradient. Chitosan with 80% degree of deacetylation (chitosan k) was separated. Three fractions were collected, concentrated under vacuum and lyophilized. The DD of chitosan k, a, b and c were 82%,56%,73% and 88%, respectively, as calculated from IR spectra. Their molecular weight daltons were, respectively, 1140000,1350000,740000 and 370000.
Chemical change, percent transmittance and color of kiwifruit juice before and after chitosan k, a, b and c fining treatments were studied. The results showed that chitosan k, a, b and c could clarify pressed kiwifruit juice effectively. Percent transmittance of the clear juice after fining was higher than 95%. Protein concentration in juice before and after chitosan k, a, b and c treatments reduced by 67.35%,69.00%,72.70%,68.38% and polyphenoly concentration reduced by 6.49%、11.90%、16.44%、14.62%,respectively. The concentration of Vitamin C in juice and soluble solids content of juice were the same before and after chitosan k, a, b and c clarification treatments. Clear juice treated with chitosan k was lighter colored than that clarified with chitosan c. During storage kiwifruit juice fined with chitosan b was more stable than that clarified using chitosan k, a and c.
Results from ultra-visual scanning study of kiwifruit juice before and after chitosan k
clarification treatment showed that polyphenol in natural juice maybe flavanol and flavonoid. Several macroporous adsorption resins were studied for selecting the best one to separate polyphenol. The result showed the adsorption properties of DA201-A macroporous resin was better than that of DA201-B and DA201-C. 78.6% of the polyphenol in the juice was adsorbed, and the yield o
采收时正值高温季节,果实采后极易腐烂,严重影响猕猴桃种植业发展。故研究猕猴桃加工技术,对充分开发利用我国的猕猴桃资源,提高农产品经济价值有重要的意义。猕猴桃果实中含有较高的蛋白质,与其它水果相比,是一种难以加工的水果品种,在猕猴桃清汁生产和贮藏过程中易引起混浊和二次沉淀。本课题从实际情况出发,研究了壳聚糖澄清猕猴桃果汁的生产工艺及其澄清机理。主要的研究内容和结果如下:
首先由不同粒度的甲壳素制备壳聚糖,结果表明粒度小的甲壳素制取的壳聚糖脱乙酰度比粒度大的甲壳素制取的大;凝胶过滤色谱测定不同壳聚糖相对分子质量分布得出:脱乙酰反应10小时所得壳聚糖的相对分子质量较反应8小时的小,脱乙酰反应14小时时,多糖主链的糖苷键已经发生断裂,产生了相对分子质量更小的多糖组分。
通过单因素实验和拟因子正交实验设计找出了壳聚糖澄清猕猴桃压榨过滤后果汁(原果汁)的最优条件为:用脱乙酰度80%的壳聚糖,加入量0.4g/L,加入壳聚糖后原果汁于50℃保温30分钟,pH为自然pH值,壳聚糖澄清原果汁后离心20分钟;用此条件制得的清汁透光率达到98%。贮藏实验结果表明低温有利于清汁的贮藏稳定。
离子交换层析色谱分离壳聚糖的研究结果表明:离子强度线性梯度洗脱时洗脱体积的增加有利于壳聚糖的分离;增加离子交换柱的床体积,分离制备了澄清原果汁效果最好的脱乙酰度80%壳聚糖样品(壳聚糖k),分离得到三个组分(a、b、c),收集、透析脱盐,脱盐率分别达到99.7%、99.5%、99.6%;红外图谱得壳聚糖k、a、b、c的脱乙酰度分别为82%,56%,73%,88%,粘度法测定得壳聚糖k、a、b、c的相对分子质量:1140000、1350000、740000、370000。
分别用壳聚糖k、a、b、c澄清原果汁,对澄清前后果汁中的化学成分变化和澄清后清汁透光率、色泽的研究结果表明:壳聚糖k、a、b、c都可以有效的澄清原果汁,澄清后清汁透光率都达到95%以上;壳聚糖k澄清后的清汁色泽浅,壳聚糖c澄清后的清汁色泽较深;分别用壳聚糖k、a、b、c澄清原果汁,澄清前后果汁中可溶性固形物和Vc含量相同,不影响猕猴桃果汁的营养价值;澄清后,原果汁中的蛋白质含量分别降低了67.35%、69.00%、72.70%、68.38%,总酚含量分别降低了6.49%、11.90%、16.44%、14.62%。贮藏实验表明:壳聚糖b澄清原果汁所得清汁的贮藏稳定性较好。
原果汁中多酚提取物的紫外-可见扫描结果表明原果汁中的多酚有可能是黄酮和黄烷醇类多酚。不同大孔吸附树脂对果汁中多酚物质的吸附解吸研究表明, DA201-A吸附
树脂对果汁中多酚物质的吸附性能较好,对多酚的吸附率达到78.63%;高效液相色谱对果汁中多酚的分析结果表明:分别用壳聚糖k、a、b、c澄清原果汁后,所得清汁中的多酚种类不同。HPLC-ESI/MS分析结果表明:吸附树脂分离原果汁中多酚物质所得乙醇洗脱组分又经C-18小柱分离的甲醇洗脱组分中的多酚样品中含有槲皮素-3-鼠李糖、山奈素-3-鼠李糖苷、山奈素-3-芸香糖苷、表没食子儿茶素、原花青素的二聚体、槲皮素-3-葡萄糖、表儿茶素没食子酸酯、原花青素的二聚单酯、山奈素-3-葡萄糖苷和芦丁。
壳聚糖k澄清原果汁后所得絮凝物溶于不同pH缓冲液中蛋白质的溶出量不同,pH4.5-7.5,蛋白质溶出量由低到高变化很大,pH 4.0以下蛋白质未能溶出,pH7.5以上蛋白质溶出量随pH提高增加不明显。凝胶过滤色谱测定自絮凝物中溶出的蛋白质相对分子质量分布的实验结果表明:絮凝物(分别用壳聚糖k 、a、b、c澄清原果汁后所得)分别用不同缓冲液复溶其中蛋白质时,蛋白质的Sepharose CL-6B凝胶过滤色谱图不同。
光学显微镜观察壳聚糖k澄清原果汁前后果汁中粒子状态的结果表明:原果汁中含有圆形粒子,粒子为多分散态;壳聚糖加入原果汁后,分散的少数粒子先聚集在一起,随时间延长,聚集的粒子数目增多,逐渐形成一个核,周围的粒子以这个核为中心以堆积方式聚集在一起,絮凝物体积不断增大。原果汁中粒子的平均粒径为38.322μm,壳聚糖k加入原果汁中保温10min后粒子的平均粒径为44.143μm,保温20min后粒子的平均粒径为62.995μm。壳聚糖k的加入可促使果汁中物质之间的相互聚集,形成大的颗粒,从而絮凝原果汁中的某些物质,达到澄清果汁的目的。
扫描电子显微镜观察分别用壳聚糖k、a、b、c澄清原果汁后所得到絮凝物超微结构表明:分别用壳聚糖k、a、b、c澄清原果汁后,得到絮凝物的结构和形态是不同的;经过壳聚糖k、a澄清处理后,絮凝物中颗粒之间的聚集较对照样紧密, 壳聚糖b、c澄清原果汁后所得絮凝物具有网络结构。
用果胶-蛋白质-丹宁酸混合体系作为猕猴桃原果汁的模拟体系研究结果表明:1、果胶溶液中果胶分子聚集物呈中空球形均匀分散;2、果胶/牛血清白蛋白混合溶液中的球形粒子有两种:粒子中央光线可透过,照片上呈环形;粒子的中央包含有单个或多个球形粒子的聚集体,认为粒子的中央是蛋白质,外层是果胶;3、果胶的存在抑制或减弱了蛋白质-丹宁酸之间的相互作用,果胶起到防止蛋白质-多酚产生沉淀的作用,增强了溶液的稳定性;4、壳聚糖加入果胶-蛋白质-丹宁酸混合溶液中时,可?
Kiwifruit is affluent in variety and resource in China where is its original habitat. Kiwifruit is abundant in Vitamin C, the concentration of which is ten times more than other fruits. This gives kiwifruit the properties to prevent scurvy, hypertension, coronary heart disease etc. Kiwifruit belongs to bacca. It is sensitive to ethylene and easily decomposes after picking, which affect the plantation development of kiwifruit weightily. Therefore, studying the processing technique of kiwifruit is important to exploit it as a resource and improve its economic value. Kiwifruit is high in protein compared with many other fruits and this causes haze problems in clear juice processing and storage. The objective of this dissertation was to develop a technology for processing clear kiwifruit juice using chitosan and to investigate the mechanism involved in the clarification treatment.
Chitosan was prepared from chitin with different granularity. The reactive trends were similar under the reaction condition of same temperature, same lye concentration and different reaction times. The degree of deacetylation (DD) of chitosan increased quickly in the first several hours of the reaction, and then remained stationary. DD of chitosan made from chitin with small granularity was higher than that with big granularity. Gel permeation chromatography analysis showed that the molecular weight of chitosan prepared at 10 hours reaction time was smaller than that at 8 hours reaction time and the main chain of chitosan had been ruptured at 14 hours reaction time.
According to the results of single and orthogonal tests, the procedure for chitosan clarification kiwifruit pressing juice was as follows: selected kiwifruits were washed, sterilized , rinsed and pressed; the juice was filtered then added into 0.4g/L chitosan with 80% degree of deacetylation and kept for 30min at 50℃ constant temperature; and then the juice was centrifuged. Percent transmittance of clear kiwifruit juice prepared under this condition was 98%. The result of the storage experiment showed that low temperature was propitious to storage stability of clear juice.
The effects of factors such as the eluate volume on the separation of chitosan by ion- exchange chromatography were also studied. The results demonstrated that increase in eluate volume were favorable to isolation with a continuous gradient. Chitosan with 80% degree of deacetylation (chitosan k) was separated. Three fractions were collected, concentrated under vacuum and lyophilized. The DD of chitosan k, a, b and c were 82%,56%,73% and 88%, respectively, as calculated from IR spectra. Their molecular weight daltons were, respectively, 1140000,1350000,740000 and 370000.
Chemical change, percent transmittance and color of kiwifruit juice before and after chitosan k, a, b and c fining treatments were studied. The results showed that chitosan k, a, b and c could clarify pressed kiwifruit juice effectively. Percent transmittance of the clear juice after fining was higher than 95%. Protein concentration in juice before and after chitosan k, a, b and c treatments reduced by 67.35%,69.00%,72.70%,68.38% and polyphenoly concentration reduced by 6.49%、11.90%、16.44%、14.62%,respectively. The concentration of Vitamin C in juice and soluble solids content of juice were the same before and after chitosan k, a, b and c clarification treatments. Clear juice treated with chitosan k was lighter colored than that clarified with chitosan c. During storage kiwifruit juice fined with chitosan b was more stable than that clarified using chitosan k, a and c.
Results from ultra-visual scanning study of kiwifruit juice before and after chitosan k
clarification treatment showed that polyphenol in natural juice maybe flavanol and flavonoid. Several macroporous adsorption resins were studied for selecting the best one to separate polyphenol. The result showed the adsorption properties of DA201-A macroporous resin was better than that of DA201-B and DA201-C. 78.6% of the polyphenol in the juice was adsorbed, and the yield o
引文
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