风味肽的表达与分离纯化

论文编号:SP010  论文字数:30504,页数:54

摘  要
牛肉风味肽(BMP)是一种最初发现于牛肉中带有牛肉香味的八肽，因其独特的风味有望成为食品工业一种新型风味强化剂。本文主要对BMP的诱导表达条件、稳定性、预处理条件及分离纯化方法进行了如下研究：
优化了毕赤酵母工程菌的生长及发酵条件：种子培养温度为28℃，诱导期采用BSM为诱导培养基，诱导温度28℃，pH值3.0，转速190r/min，每24小时分批补加终浓度为0.5%(V:V)的甲醇诱导，1%酪蛋白水解物(CA)为氮源，最佳诱导时间为96小时，BMP产量最高可达623mg/L。
BMP的稳定性试验：90℃分别热处理5min、10min、15min、20min，BMP仍具有很好的稳定性；在pH3.0-8.0下室温放置48小时，均呈现较好的稳定性；抗蛋白酶水解试验证明，BMP具有很好的抗胃蛋白酶能力，而抗中性、碱性蛋白酶能力较弱。
发酵液预处理试验结果表明，因杂质同样具有较好的热稳定性，使得热处理法不适于前期的除杂质；活性炭吸附最佳pH值为7.0-8.0之间；乙醇沉淀除杂质最佳pH为7.0。
采用大孔树脂吸附法、离子交换法和凝胶过滤法对BMP进行粗分离，并利用反相高效液相色谱(RP-HPLC)进行定性和定量检测。经比较发现，葡聚糖G-25凝胶过滤法最终的得率、纯度及纯化倍数均高于其它两种方法。
利用RP-HPLC，采用二元梯度洗脱方法，通过两次制得的BMP纯品纯度达到95.26%。
关键词：牛肉风味肽；毕赤酵母；诱导表达；稳定性；分离纯化

ABSTRACT
Beefy Meaty Peptide(BMP)，an thermostable octapeptide found in beef with beefy flavor，has a good potential for application in food industry as a new kind of flavor enhancer.In this paper, the inducing condition,stability,pretreatment and isolation methods of BMP were studied.
The optimized inducing condition was as follows: BSM was chosen as the inducing medium, added 0.5%(V:V) methanol,the nitrogen source (1%CA) per 24 hours,then incubated at 28℃, pH 3.0, rotating at 190rpm. The optimum inducing time was 96 hours.The secreted peptide quantity under the conditions in the flask fermentation had been obviously improved. The expressed peptide yield reached 623mg/L .
The stability of BMP in recombined Pichia pastoris yeast fermentation was studied.The results showed that the target peptide was still stable after heated at 90℃ within 5min, 10min, 15min, 20min,respectively.Also,it kept stable between pH3.0 and pH8.0 within 48 hours in room temperature.Besides,BMP can withstand pepsin, while was relative instable afer hydrolyzed by Neutrase and Alcalase.
It was revealed that heat-solution was not satisfying during the pretreatment, as the impurities were also thermostable. However, the active carbon adsorption was more effective at pH7.0-8.0,while the ethanol precipitation was better at pH 7.0.
Macroporus adsorption resin, ion-exchange and gel-filtration chromatography were performanced according to the standard protocols. The content of BMP was detected by RP-HPLC. It was found that the purification effect of the Sephadex G-25 gel filtration chromatography was better.
Finaly, BMP was gathered and analyzed by RP-HPLC, eluted with bi-gradient mobile solvents at a flow rate of 1.0 mL/min and UV detection at 230nm．The purity of BMP was 95.26%.

Key words:BMP, Pichia pastoris yeast, inducing, stability, isolation&purification