聚乙二醇/盐双水相萃取L-苯丙氨酸的研究

研究了聚乙二醇/盐双水相体系的成相行为及L-苯丙氨酸在双水相中的分配规律,其中包括聚乙二醇的分子量、聚乙二醇质量分数、盐的种类及加入量、L-苯丙氨酸初始浓度和pH对萃取分离的影响。当聚乙二醇1000的质量分数为27%,磷酸氢二钾的质量浓度为0.15g/mL,L-苯丙氨酸的质量浓度为10g/L,体系的pH为8.5时,L-苯丙氨酸的萃取率最高为99.5%,分配系数最大为186.5。     

NO熏蒸处理对冷藏枇杷果实木质化劣变的影响

为了探讨外源一氧化氮（NO）熏蒸处理对冷藏枇杷果实木质素代谢的影响,以‘解放钟’枇杷果实为试材,采用不同水平NO（0、5、15、25、35、45 μL/L）熏蒸处理,于5 ℃条件下贮藏,测定冷藏过程中枇杷果实的硬度、出汁率、可溶性总糖、可滴定酸、木质素含量及木质素代谢相关酶活性的变化。结果表明：NO熏蒸处理可延缓可溶性总糖及可滴定酸含量的下降,并抑制硬度的上升及出汁率的降低,较好地保持果实的商品品质。NO熏蒸处理显著抑制了苯丙氨酸解氨酶、肉桂醇脱氢酶活性的上升,延缓了可溶性总糖含量的降低,且抑制了木质素含量的上升,从而延缓了冷藏枇杷果实木质化劣变进程,以15 μL/L和25 μL/L NO处理效果较为明显。     

5-氨基乙酰丙酸对苹果幼果多酚含量、苯丙氨酸解氨酶活性及其基因表达的影响

探究5-氨基乙酰丙酸（aminolevulinic acid,ALA）处理对苹果幼果酚类物质、苯丙氨酸解氨酶（phenylalanin ammonia-lyase,PAL）活性及其基因表达量的影响。在苹果疏果期前,利用不同质量浓度ALA处理苹果幼果,采用紫外分光光度法测定ALA处理后12 d内苹果多酚和PAL活性,利用荧光定量法测定苹果幼果PAL基因表达量。结果表明：ALA处理质量浓度在300 mg/L以内,随着质量浓度提高,苹果多酚含量、PAL酶活性及其基因表达量均升高,到400 mg/L时各项指标均表现出下降趋势;相同质量浓度ALA处理苹果幼果后,幼果的上述3 项指标均随时间延长而明显提高,在第9天时达到最高值,12 d后开始下降。为提高苹果疏果多酚含量,实现疏果的最大利用率,生产中需要选择适宜的ALA处理浓度和时间采集幼果。     

鲜切雷竹笋冷藏过程中木质化机理的研究

研究鲜切雷竹笋在4℃冷藏条件下苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)和多酚氧化酶(PPO)活力及丙二醛(MDA)、多酚、木质素含量的变化规律,探讨其木质化机理。结果显示：在冷藏过程中,PAL、POD活力、MDA含量呈先上升,后下降的趋势;PPO活力在前6d缓慢上升,第15天开始下降;多酚含量在冷藏前6d急剧下降,后缓慢上升,第12天又开始下降;木质素含量不断增加,基部为5%～26%,中部为3%～24%,尖部为2%～22%。结果表明：在冷藏期内,PAL、POD活力大幅度增加,是导致鲜切雷竹笋木质化的关键酶;酚类物质作为木质素合成的前体物质,参与木质素的合成;MDA可能参与鲜切雷竹笋的木质化进程,但需进一步验证;木质素含量从基部向尖部逐渐降低,木质化进程是从基部向尖部推进的。     

胡萝卜中过氧化物酶和苯丙氨酸解氨酶特性及抑制条件的研究

经硫酸铵分级沉淀、Sephadex G-100凝胶过滤层析纯化,得到部分纯化的胡萝卜过氧化物酶(POD)和苯丙氨酸解氨酶(PAL).这两种酶的纯化倍数分别为7.44倍和5.87倍,回收率分别为32.51％和5.06％.酶学性质和动力学性质研究表明,胡萝卜POD和PAL的最适pH分别为5.0和8.4;在不同pH值下POD和PAL的活力及其耐受性变化趋势相似.POD的适宜温度为30℃,当温度40～50℃时,PAL酶活力达到最高点.PAL酶液体积为1.6 mL时,反应速度最快;而POD酶液体积分别为1.0mL和1.6mL时,反应速度出现峰值.当POD和PAL底物浓度分别为44.8×10-6 mol/L和0.02 mol/L时,反应速度达到最大值.Na+、10 mmol/L Ca2+、0.1 mmol/L NL4+、Mg2+可以同时显著地(P＜o.05)抑制胡萝卜POD和PAL酶的活力.胡萝卜POD与愈创木酚的亲合力强,PAL与L-苯丙氨酸的亲合力较小.     

Systematic Modification and Evaluation of Enzyme-Loaded Chitosan Nanoparticles

Polymeric-based nano drug delivery systems have been widely exploited to overcome protein instability during formulation. Presently, a diverse range of polymeric agents can be used, among which polysaccharides, such as chitosan (CS), hyaluronic acid (HA) and cyclodextrins (CDs), are included. Due to its unique biological and physicochemical properties, CS is one of the most used polysaccharides for development of protein delivery systems. However, CS has been described as potentially immunogenic. By envisaging a biosafe cytocompatible and haemocompatible profile, this paper reports the systematic development of a delivery system based on CS and derived with HA and CDs to nanoencapsulate the model human phenylalanine hydroxylase (hPAH) through ionotropic gelation with tripolyphosphate (TPP), while maintaining protein stability and enzyme activity. By merging the combined set of biopolymers, we were able to effectively entrap hPAH within CS nanoparticles with improvements in hPAH stability and the maintenance of functional activity, while simultaneously achieving strict control of the formulation process. Detailed characterization of the developed nanoparticulate systems showed that the lead formulations were internalized by hepatocytes (HepG2 cell line), did not reveal cell toxicity and presented a safe haemocompatible profile.     

Purification and Use of Glycomacropeptide for Nutritional Management of Phenylketonuria

ABSTRACT:  Individuals with phenylketonuria (PKU) cannot metabolize phenylalanine (Phe) and must adhere to a low-Phe diet in which most dietary protein is provided by a Phe-free amino acid formula. Glycomacropeptide (GMP) is the only naturally occurring protein that does not contain Phe, and is of interest as a source of protein for dietary management of PKU. However, commercially available GMP contains too much Phe from residual whey proteins and does not contain adequate levels of all the indispensable amino acids to provide a nutritionally complete protein. The aim of this study was to increase purity of GMP and develop a mass balance calculation for indispensable amino acid supplementation of GMP foods. Cation exchange chromatography, ultrafiltration/diafiltration, and lyophilization were used at the pilot plant scale to decrease Phe. Enough purified GMP (5 kg) was manufactured to provide 15 PKU subjects with a 4-d diet in which the majority of protein was from GMP foods. A mass balance was used to supplement GMP foods so that all indispensable amino acids met or exceeded the daily recommended intake. GMP foods were tested in a human clinical trial as a replacement for the traditional amino acid formula. Nutritionally complete GMP foods created with high purity GMP provide individuals with PKU with more options to manage PKU, which may lead to improved compliance and quality of life.     

冠突散囊菌对燕麦黄酮含量的影响

利用冠突散囊菌（Eurotium cristatum）对燕麦进行固态发酵,以黄酮含量为考察指标,通过单因素试验和正交试验优化燕麦固态发酵条件,并对燕麦发酵过程中黄酮含量与苯丙氨酸解氨酶活力动态变化的相关性进行分析。结果表明,冠突散囊菌固态发酵燕麦的最佳发酵条件为发酵温度30 ℃,基质pH值6.0,接种量0.5%,发酵时间7 d,在此最优发酵条件,燕麦黄酮含量最高,为58.13 mg/g,较优化前提高41.06%;黄酮含量的变化与苯丙氨酸解氨酶活性的变化呈显著正相关。通过冠突散囊菌发酵燕麦提高了燕麦生物活性成分,为燕麦再加工提高附加值提供了有力依据。     

Effects of dietary leucine and phenylalanine on pancreas development,enzyme activity,and relative gene expression in milk-fed Holstein dairy calves

This study aimed to investigate the effect of dietary supplementation with leucine and phenylalanine on pancreas development, enzyme activity, and related gene expression in male Holstein calves. Twenty male Holstein calves [1 d of age, 38 ± 3 kg of body weight (BW)] were randomly assigned to 1 of the following 4 treatment groups with 5 calves in each group: control, leucine supplementation (1.435 g/L of milk), phenylalanine supplementation (0.725 g/L of milk), and leucine and phenylalanine (1.435 + 0.725 g/L of milk). The diets were made isonitrogenous with the inclusion of alanine in each respective treatment. The feeding trial lasted for 8 wk, including 1 wk for adaption and 7 wk for the feeding experiment. Leucine tended to increase the concentration of total pancreatic protein (mg/kg of BW). Phenylalanine increased the concentrations of plasma insulin, cholecystokinin, and pancreatic DNA (mg/g) and the expression of trypsin gene but decreased the pancreatic protein:DNA ratio and tended to decrease the pancreas weight (g/kg of BW). No differences were observed in total pancreatic DNA (mg/pancreas and mg/kg of BW), pancreatic protein (mg/pancreas), or activities of α-amylase, trypsin, and lipase. The relative expression levels of the genes encoding α-amylase and lipase did not differ among the 4 groups. The supplementation of both leucine and phenylalanine showed an interaction on the pancreas weight (g and g/kg of BW) and a tendency of an interaction on the pancreatic protein concentration (mg/g of pancreas and mg/kg of BW) and the plasma glucose concentration. Leucine tended to increase the size of the pancreatic cells, whereas phenylalanine tended to increase the number of pancreatic cells. However, neither AA affected the activities of the pancreatic enzymes of the calves. These results indicate that leucine and phenylalanine supplementation in milk-fed Holstein calves differentially affect pancreatic growth and development.