甘薯淀粉生产废液中糖蛋白的提取及其生物活性和结构的研究
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摘要
我国是甘薯生产世界第一大国,甘薯在加工中主要用于甘薯淀粉的生产。而甘薯淀粉生产中产生大量的废水,废水中又富含多糖和糖蛋白类物质,这些有机物是造成环境污染的主要原因。同时,日本科学家发现甘薯中存在的糖蛋白具有很好的调节免疫力和抗肿瘤的功能。如果能把甘薯生产淀粉的废水中的糖蛋白提取出来不但可以减少甘薯生产淀粉废水的环境污染,而且可以得到一种具有高附加值的生物活性物质,具有很大的理论意义和实用价值。
本课题采用超滤技术从甘薯淀粉生产废水中提取甘薯糖蛋白,并对其生物活性和结构进行了研究,主要研究内容如下:
确定了从甘薯淀粉生产废水中提取甘薯糖蛋白的工艺路线:甘薯淀粉生产的废水经过两次离心(1,900和3,000r/min)和过滤(400目滤布)后,通过无机陶瓷膜超滤进行初级浓缩,然后再经过真空浓缩,加入70%(终浓度)的乙醇得到沉淀,沉淀经过冷冻干燥得到甘薯粗糖蛋白样品。
采用中空纤维聚砜超滤设备浓缩甘薯淀粉生产废水的最佳工艺条件、糖蛋白的截留率和粗糖蛋白的提取率分别为:压力0.17MPa,pH=6.5,温度20℃。将100L料液进行浓缩,体积浓缩倍数为7.7倍,糖蛋白浓缩倍数5.25倍,糖蛋白的截留率为68.25%,粗糖蛋白的提取率为0.67%(占甘薯鲜重)。采用无机陶瓷超滤设备浓缩甘薯淀粉生产废水的最佳工艺条件、糖蛋白的截留率和粗糖蛋白的提取率分别为:压力0.35MPa,pH=6.5,温度20℃,u=2m/s。将100L料液进行浓缩,体积浓缩倍数为8.3倍,糖蛋白浓缩倍数为7.5倍,糖蛋白的截留率达到91%,粗糖蛋白的提取率为0.88%(占甘薯鲜重)。
两种超滤膜各有特点,虽然中空纤维聚砜超滤设备成本低,处理量大,但是和无机陶瓷膜超滤设备相比膜污染严重,膜清洗困难,糖蛋白损失多,所以相比较而言无机陶瓷膜超滤设备更适合于从甘薯淀粉生产废水提取糖蛋白。
建立了采用无机陶瓷膜超滤设备从甘薯淀粉生产废水中提取糖蛋白的数学模型:
采用该数学模型和相应的数学软件,当超滤操作条件改变时,可以预测超滤通量的变化;同时当通量一定时,采用该模型可以选择合适的操作条件。通过甘薯粗糖蛋白的急性毒性试验,结果证明甘薯粗糖蛋白并不存在急性毒性。研究了甘薯粗糖蛋白的调节免疫力生物活性,小鼠碳阔清试验证明甘薯粗糖蛋白具
China is the biggest country of Sweet Potato production, most of Sweet Potato wasmanufactured into starch and a great deal of wastewater was produced during the SweetPotato starch production in China. The wastewater will pollute environment because thereare a large number of organics in it. Most of organics of wastewater are glycoprotein, whichhave excellent immunodulating and antitumor biological activity found by Japanesescientists. If the glycoprotein can be extracted from the waste water, not only the pollutingproblem can be resolved, but also can obtain a kind of biological activity product highappending value.
Ultrafiltration was used for extracting glycoprotein from wastewater of Sweet Potatostarch production and the structure and biological activities of the glycoprotein were studiedin this paper. Major content of this paper as following:
Technical route of extracting glycoprotein from wastewater of Sweet Potato starchproduction were confirmed as: wastewater was centrifuged twice (1,900 and 3,000rpm) andfiltrated with filtrating fabric (400 mesh), then concentrated with ultrafiltrator and vacuumconcentrator, then precipitated with 70% alcohol (final concentration), then freezed dryinginto powder.
Optimum technical parameters and the retentive ratio and extracting ratio ofglycoprotein using hollow fabric ultrafiltrator was studied: pressure 0.17MPa, pH 6.5,temperature 20℃;volume concentration times after concentrating 100L wastewater was 7.7,glycoprotein concentration times was 5.25, retentive ratio of glycoprotein was 68.25%,extracting ratio of crude glycoprotein was 0.67%.
Optimum technical parameters and the retentive ratio and extracting ratio ofglycoprotein using ceramic ultrafiltrator was studied: pressure 0.35MPa, pH 6.5,temperature 20℃, cross-flow velocity 2m/s;Volume concentration times after concentrating100L wastewater was 8.3, glycoprotein concentration time was 7.5, retentive ratio ofglycoprotein was 91.0%, extracting ration of crude glycoprotein was 0.88%.
Although hollow ultrafiltrator can deal with large number of wastewater and use at alow cost, its membrane was polluted badly and difficult to clean, and lost of glycoproteinwas seriously compared with ceramic ultrafiltrator. So ceramic ultrafiltrator was moresuitable for concentrating glycoprotein from wastewater of Sweet Potato starch productionthan hollow fabric ultrafiltrator.
A mathematic model of concentrating glycoprotein from wastewater of Sweet Potatostarch production using ceramic ultrafiltrator was described as following:26232. 43510.37780.9342390.91290.184910000ucPucJ =+ ? ?P?×+?Using this model and corresponding software, flux change can be predicted andoperating conditions also can be selected when flux confirmed.Glycoprotein of Sweet Potato has no acute toxicity certified by mice acute toxicityexperiment.Glycoprotein of Sweet Potato has excellent immunomodulating activity certified bycarbon clearance ratio of mice. Glycoprotein has the highest clearance index (4.52) at thedose of 50mg /kg.d. When used together with Cycolphosphamide, glycoprotein also canreduce damage of body by Cycolphosphamide.Glycoprotein of Sweet Potato has excellent antitumor activity certified by mice againstSarcoma 180 implanted tumor experiment. Glycoprotein has the highest tumor inhibitionrate (62%) at the dose of 50mg /kg.d. Glycoprotein also can enhance antitumor effect whenused together with Cycolphosphamide.Antitumor mechanism of glycoprotein of Sweet Potato was studied. Glycoproteinexerted its antitumor effect possibly through enhancing immunity of body, which proved bythe effects of proliferation of active splenocyte by ConA and S180 inhibition in vitroexperiment.Glycoprotein was separated and purified for screening high biological activitycomponents. Combining sulfate acid phase precipitation, ion-exchange, gel filtration,affinity chromatogram with the determining the effects of proliferation of active splenocyteby ConA and S180 inhibition in vitro experiment, Sp-3241 was got. Its purity was certified bygel filtration, PAGE and HPLC. The simulating index of no-active spleen lymphocyte invitro of the purified fraction SP-3241 was 1.727, which was enhanced 40.75% comparedwith index of crude glycoprotein, and the simulating index of active spleen lymphocyte invitro was to 1.965,which was enhanced 49.43% compared with index of crude glycoprotein.The structure of SP-3241 was studied, the result describe as following: SP-3241 is akind of glycoprotein;protein content is 91.60%;sugar content is 6.43%;it is rich in Asp(13.59%), Glu (7.75%) and Leu (7.27%);there were 4 kinds of sugar in the glycoproteinwith a ratio of glucose: galactose: rhamnose: arabinose 10.2:1.25:1:2.5;the relativemolecule weight of the glycoprotein determined by gel separation chromatogram and HPLCis 32359 and 32606Da respectively;sugar and protein is linked with O-link band and sugarlinked with Thr. There were 2 kinds of glycans in the glycoprotein (glycan-1 and glycan-2).
Relative molecule weight of glycan-1 was 1725Da and there were 3 kinds of sugar in theglycan-1 with a ratio of rhamnose: glucose: galactose 1:10:1.2. Relative molecule weight ofglycan-1 was 468Da and there were 2 kinds of sugar in the glycan-2 with a ratio ofarabinose: glucose 1:2.05.Relationship between structure and biological activity was studied. The result was:there was no immunomodulating activity when using protein or sugar chain along;there wasimmunomodulating activity only when protein and sugar chains being linking together.
本课题采用超滤技术从甘薯淀粉生产废水中提取甘薯糖蛋白,并对其生物活性和结构进行了研究,主要研究内容如下:
确定了从甘薯淀粉生产废水中提取甘薯糖蛋白的工艺路线:甘薯淀粉生产的废水经过两次离心(1,900和3,000r/min)和过滤(400目滤布)后,通过无机陶瓷膜超滤进行初级浓缩,然后再经过真空浓缩,加入70%(终浓度)的乙醇得到沉淀,沉淀经过冷冻干燥得到甘薯粗糖蛋白样品。
采用中空纤维聚砜超滤设备浓缩甘薯淀粉生产废水的最佳工艺条件、糖蛋白的截留率和粗糖蛋白的提取率分别为:压力0.17MPa,pH=6.5,温度20℃。将100L料液进行浓缩,体积浓缩倍数为7.7倍,糖蛋白浓缩倍数5.25倍,糖蛋白的截留率为68.25%,粗糖蛋白的提取率为0.67%(占甘薯鲜重)。采用无机陶瓷超滤设备浓缩甘薯淀粉生产废水的最佳工艺条件、糖蛋白的截留率和粗糖蛋白的提取率分别为:压力0.35MPa,pH=6.5,温度20℃,u=2m/s。将100L料液进行浓缩,体积浓缩倍数为8.3倍,糖蛋白浓缩倍数为7.5倍,糖蛋白的截留率达到91%,粗糖蛋白的提取率为0.88%(占甘薯鲜重)。
两种超滤膜各有特点,虽然中空纤维聚砜超滤设备成本低,处理量大,但是和无机陶瓷膜超滤设备相比膜污染严重,膜清洗困难,糖蛋白损失多,所以相比较而言无机陶瓷膜超滤设备更适合于从甘薯淀粉生产废水提取糖蛋白。
建立了采用无机陶瓷膜超滤设备从甘薯淀粉生产废水中提取糖蛋白的数学模型:
采用该数学模型和相应的数学软件,当超滤操作条件改变时,可以预测超滤通量的变化;同时当通量一定时,采用该模型可以选择合适的操作条件。通过甘薯粗糖蛋白的急性毒性试验,结果证明甘薯粗糖蛋白并不存在急性毒性。研究了甘薯粗糖蛋白的调节免疫力生物活性,小鼠碳阔清试验证明甘薯粗糖蛋白具
China is the biggest country of Sweet Potato production, most of Sweet Potato wasmanufactured into starch and a great deal of wastewater was produced during the SweetPotato starch production in China. The wastewater will pollute environment because thereare a large number of organics in it. Most of organics of wastewater are glycoprotein, whichhave excellent immunodulating and antitumor biological activity found by Japanesescientists. If the glycoprotein can be extracted from the waste water, not only the pollutingproblem can be resolved, but also can obtain a kind of biological activity product highappending value.
Ultrafiltration was used for extracting glycoprotein from wastewater of Sweet Potatostarch production and the structure and biological activities of the glycoprotein were studiedin this paper. Major content of this paper as following:
Technical route of extracting glycoprotein from wastewater of Sweet Potato starchproduction were confirmed as: wastewater was centrifuged twice (1,900 and 3,000rpm) andfiltrated with filtrating fabric (400 mesh), then concentrated with ultrafiltrator and vacuumconcentrator, then precipitated with 70% alcohol (final concentration), then freezed dryinginto powder.
Optimum technical parameters and the retentive ratio and extracting ratio ofglycoprotein using hollow fabric ultrafiltrator was studied: pressure 0.17MPa, pH 6.5,temperature 20℃;volume concentration times after concentrating 100L wastewater was 7.7,glycoprotein concentration times was 5.25, retentive ratio of glycoprotein was 68.25%,extracting ratio of crude glycoprotein was 0.67%.
Optimum technical parameters and the retentive ratio and extracting ratio ofglycoprotein using ceramic ultrafiltrator was studied: pressure 0.35MPa, pH 6.5,temperature 20℃, cross-flow velocity 2m/s;Volume concentration times after concentrating100L wastewater was 8.3, glycoprotein concentration time was 7.5, retentive ratio ofglycoprotein was 91.0%, extracting ration of crude glycoprotein was 0.88%.
Although hollow ultrafiltrator can deal with large number of wastewater and use at alow cost, its membrane was polluted badly and difficult to clean, and lost of glycoproteinwas seriously compared with ceramic ultrafiltrator. So ceramic ultrafiltrator was moresuitable for concentrating glycoprotein from wastewater of Sweet Potato starch productionthan hollow fabric ultrafiltrator.
A mathematic model of concentrating glycoprotein from wastewater of Sweet Potatostarch production using ceramic ultrafiltrator was described as following:26232. 43510.37780.9342390.91290.184910000ucPucJ =+ ? ?P?×+?Using this model and corresponding software, flux change can be predicted andoperating conditions also can be selected when flux confirmed.Glycoprotein of Sweet Potato has no acute toxicity certified by mice acute toxicityexperiment.Glycoprotein of Sweet Potato has excellent immunomodulating activity certified bycarbon clearance ratio of mice. Glycoprotein has the highest clearance index (4.52) at thedose of 50mg /kg.d. When used together with Cycolphosphamide, glycoprotein also canreduce damage of body by Cycolphosphamide.Glycoprotein of Sweet Potato has excellent antitumor activity certified by mice againstSarcoma 180 implanted tumor experiment. Glycoprotein has the highest tumor inhibitionrate (62%) at the dose of 50mg /kg.d. Glycoprotein also can enhance antitumor effect whenused together with Cycolphosphamide.Antitumor mechanism of glycoprotein of Sweet Potato was studied. Glycoproteinexerted its antitumor effect possibly through enhancing immunity of body, which proved bythe effects of proliferation of active splenocyte by ConA and S180 inhibition in vitroexperiment.Glycoprotein was separated and purified for screening high biological activitycomponents. Combining sulfate acid phase precipitation, ion-exchange, gel filtration,affinity chromatogram with the determining the effects of proliferation of active splenocyteby ConA and S180 inhibition in vitro experiment, Sp-3241 was got. Its purity was certified bygel filtration, PAGE and HPLC. The simulating index of no-active spleen lymphocyte invitro of the purified fraction SP-3241 was 1.727, which was enhanced 40.75% comparedwith index of crude glycoprotein, and the simulating index of active spleen lymphocyte invitro was to 1.965,which was enhanced 49.43% compared with index of crude glycoprotein.The structure of SP-3241 was studied, the result describe as following: SP-3241 is akind of glycoprotein;protein content is 91.60%;sugar content is 6.43%;it is rich in Asp(13.59%), Glu (7.75%) and Leu (7.27%);there were 4 kinds of sugar in the glycoproteinwith a ratio of glucose: galactose: rhamnose: arabinose 10.2:1.25:1:2.5;the relativemolecule weight of the glycoprotein determined by gel separation chromatogram and HPLCis 32359 and 32606Da respectively;sugar and protein is linked with O-link band and sugarlinked with Thr. There were 2 kinds of glycans in the glycoprotein (glycan-1 and glycan-2).
Relative molecule weight of glycan-1 was 1725Da and there were 3 kinds of sugar in theglycan-1 with a ratio of rhamnose: glucose: galactose 1:10:1.2. Relative molecule weight ofglycan-1 was 468Da and there were 2 kinds of sugar in the glycan-2 with a ratio ofarabinose: glucose 1:2.05.Relationship between structure and biological activity was studied. The result was:there was no immunomodulating activity when using protein or sugar chain along;there wasimmunomodulating activity only when protein and sugar chains being linking together.
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88. Colangelo J, Orlando R, On-target exoglycosidase digestions/MALDI-MS for determining the primary structures of carbohydrate chains, Anal Chem, 1999,71:1479~1482.
89. Geyer H, Schmitt S, Wuhrer M, Geyer R, Structural analysis of glycoconjugates by on-target enzymatic digestion and MALDI-TOF-MS, Anal Chem, 1999,71:476~482.
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