硫辛酸分子印迹膜及其抗氧化效果

以α-硫辛酸(ALA)为模板分子,N-乙烯基己内酰胺(NVCL)为温敏性功能单体,在制备硫辛酸分子印迹聚合物(ALA-MIPs)的基础上,结合膜技术,选用聚丙烯腈(PAN)和聚偏氟乙烯(PVDF)作为成膜材料,以异相粒子填充膜法制备了ALA-MIPs共混膜,通过扫描电镜考察了分子印迹膜(MIM)的微观形貌,研究了机械性能、亲水性、吸水性、ALA的释放特性。结果表明,PVDF膜具有更好的机械性能和疏水性能,PAN膜有较好的ALA释放量,两者都在20℃下存在温敏性释放。通过测定分子印迹膜在植物油中的POV值,发现PAN-MIM膜有更好的抗氧化效果。     

烯唑醇分子印迹固相萃取膜的制备及其应用

以烯唑醇为模板分子,丙烯酰胺(AM)为功能单体,采用原位聚合法制备出对烯唑醇具有高选择性的分子印迹固相萃取膜;通过紫外光谱试验优化了功能单体,考察了不同功能单体与模板分子的结合能力,同时考察了制备印迹膜时致孔剂的选择和分子印迹膜印迹次数对液体通过性的影响;建立了基于分子印迹固相萃取膜-高效液相色谱法测定粮谷中烯唑醇残留的方法。结果表明,与功能单体α-甲基丙烯酸(MAA)相比,丙烯酰胺(AM)与烯唑醇的结合能力更强,以乙腈为制孔剂制备烯唑醇印迹膜,印迹一次印迹膜较稳定,且液体通透性良好。烯唑醇在0.5~15μg/m L浓度范围内有良好的线性关系(r=0.9987),平均回收率在80.34%~87.03%之间,检出限为2.0μg/g。该方法选择性强、灵敏、可靠,适用于粮谷等复杂基质中烯唑醇的残留检测。     

乳清蛋白可食用膜的酶法改性及机理研究

采用转谷氨酰胺酶(TG)对乳清蛋白的成膜性质进行改性.酶用量为1.6g/L,反应时间120min时,可食用膜的的透湿系数和透氧系数分别降低47%和44%.HPLC分析表明TG处理的蛋白质所含的大分子量组分比例增大;DSC分析表明酶法改性促使可食用膜的变性温度升高;荧光分析显示反应过程中乳清蛋白分子的疏水性增大;表面巯基测定显示酶法改性初期蛋白质结构展开;总巯基测定显示酶法改性除了促使蛋白质分子间形成ε-(λ-谷氨酰基)赖氨酸共价键,同时促进二硫键的形成;扫描电镜显示可食用膜的超微结构更加细致、光滑.结果表明,TG改性有助于改善乳清蛋白可食用膜的阻隔性能.     

阿魏酸-米渣蛋白复合膜的结构及性能

通过测定膜的机械性能、水蒸气透过率、水溶性研究了阿魏酸对米渣蛋白膜性能的影响,通过傅里叶红外光谱、差示扫描量热法、X-射线衍射、扫描电子显微镜表征了膜的结构特征。结果表明,当阿魏酸添加量为0.1%(W/V)时,与对照组相比,膜的抗拉强度提高20.33%,水蒸气透过率降低20.58%,水溶性降低23.88%。傅利叶红外光谱(FTIR)显示,添加阿魏酸后酰胺带吸收峰发生红移,β-折叠转变为易变的随机螺旋分子结构,蛋白质的二级结构发生改变;差示扫描量热法(DSC)分析表明阿魏酸增强了分子间的相互作用,提高了膜的玻璃化转变温度,使膜具有更好的热稳定性;X-射线衍射(XRD)分析表明阿魏酸与蛋白质分子间发生共价交联,膜的结晶度提高;扫描电镜(SEM)结果显示复配阿魏酸后的米渣蛋白膜的表面结构更均匀平整。因此,阿魏酸的添加使米渣蛋白膜具有更优的性能和更稳定的空间结构。     

不同添加剂对酪蛋白可食膜理化性质的影响

酶可催化蛋白质分子内交联,多糖和多酚可与蛋白质发生分子间相互作用,3种添加剂均可改善蛋白质的功能性质。为提高酪蛋白(CA)可食膜的应用范围,研究了谷氨酰胺转氨酶(TG酶)、表没食子儿茶素没食子酸酯(EGCG)和壳聚糖(CS)对酪蛋白可食膜结构及性能的影响。测定膜溶液的粒径和电位,对复合膜机械性能、色泽和水蒸气透过率等指标进行表征。结果表明,TG酶、EGCG和CS对膜结构均影响显著,6组样品中添加EGCG和CS的样品(CAEC组)性能最佳。添加TG酶后,膜的机械性能有所提高,而水阻隔能力下降。添加EGCG后膜颜色变暗,抗拉伸强度提高,而韧性下降。添加CS后膜的抗拉伸强度提升,韧性也下降。同时添加EGCG和CS后,所得薄膜拥有良好的抗拉伸强度、水阻隔能力和较强的自由基清除能力。通过向酪蛋白膜中引入TG酶、EGCG和CS,赋予CA可食膜良好的机械性能和自由基清除能力,提高其水阻隔能力。研究结果对开发新型蛋白基可食膜具有一定意义。     

分子印迹固相萃取膜-高效液相色谱法检测粮谷中腈菌唑残留

以腈菌唑为模板分子,丙烯酰胺(AM)为功能单体,乙二醇二甲基丙烯酸酯(EDMA)为交联剂,模板分子与功能单体最佳浓度配比为1∶2,制备出对腈菌唑具有高选择性的分子印迹固相萃取膜。通过紫外光谱试验考察了制备印迹膜时致孔剂的选择和分子印迹膜印迹次数对液体通过性的影响。建立了基于分子印迹固相萃取膜-高效液相色谱法测定粮谷中腈菌唑残留的方法。样品经乙腈提取,固相萃取膜净化,经C18柱分离,紫外检测210 nm。结果表明,腈菌唑在0.3~20μg/m L浓度范围内有良好的线性关系(r=0.999 4),平均回收率在80.2%~86.0%之间,相对标准偏差(RSD)≤3.3%(n=5),检出限为1.2μg/g。该方法选择性强、灵敏、可靠,适用于粮谷等复杂基质中腈菌唑的残留检测。     

化学交联与酶法交联对鱼糜-明胶复合膜性质的影响

研究比较了戊二醛和谷氨酰胺转氨酶(TGase)对鱼糜-明胶复合膜的性质改良效果。当戊二醛的含量为蛋白质量的0.025%～0.1%时,膜的抗拉伸强度(TS)没有显著差异,但断裂延伸率(EAB)却随着戊二醛含量的增加而上升,当戊二醛含量增加到0.2%时,TS和EAB都出现了下降。然而,膜的TS和EAB都随着TGase的添加逐渐增加。虽然戊二醛和TGase都可以使蛋白发生交联,导致膜的固形物溶解率(FS)和蛋白溶解率(PS)下降,但是戊二醛的添加效果明显优于TGase。此外,戊二醛的添加会使膜的颜色变黄,而TGase不仅不会影响膜的色泽,还可以提高膜的透明性能。根据SDS-PAGE的结果,发现戊二醛和TGase都会使膜中蛋白分子发生交联形成高分子聚合物。FT-IR的分析结果表明,蛋白分子间的氢键作用随着TGase的添加逐渐减弱,随着戊二醛的添加出现先减弱后增强的趋势。     

围食膜因子的分子结构及其功能

围食膜是昆虫中肠细胞分泌的非细胞性半透膜,主要由几丁质和蛋白质组成,依据蛋白质与围食膜结合的紧密程度,将可溶于强变性剂的蛋白称为围食膜因子(peritrophin)。本文综述了peritrophin-44、peritrophin-48、peritrophin-95、peritrophin-55、peritrophin-30、peritrophin-15、Ag-Aper1和IIM的序列结构、生化功能及其在害虫防治中的作用,并提出了新的围食膜分子结构模型。     

基于纳米金信号放大的分子印迹膜-SPR传感器检测奶制品中的三聚氰胺

采用分子印迹和SPR信号放大技术,构建一种以嵌有纳米金的分子印迹膜为识别元件,用于检测奶制品三聚氰胺的分子印迹膜-表面等离子体共振传感器(MIM-SPR)。首先利用表面热引发聚合的方法在SPR金片表面合成嵌有纳米金的分子印迹膜,再对MIM-SPR传感器的吸附响应性、选择性、再生性及精准度进行评价。该传感器的响应值与三聚氰胺在1×10-2~2μg/m L浓度范围内线性关系良好,R2=0.998。加标浓度在0.5~1.5μg/m L范围内的回收率为86.43%~107.11%,RSD小于6.0%(n=5)。结果表明该方法操作简单、选择性和再生性好、精准度高,可用于奶制品三聚氰胺残留量的测定。     

类分子印迹纳米多孔膜修饰电极制备三聚氰胺电化学传感器

采用电化学法在充蜡石墨电极上,原位修饰了一种基于三聚氰胺/纳米银/聚槲皮素的类分子印迹-纳米多孔膜。场发射扫描电镜、X-射线光电子能谱、红外光谱和电化学验证了该类分子印迹-纳米多孔膜为三维网状结构。该纳米多孔膜修饰电极对三聚氰胺显示良好的选择性富集作用,氧化峰(0.17V)电流和三聚氰胺的浓度在1×10-7～1×10-5mol/L范围内,呈良好线性关系,检出限为1×10-8mol/L(3σ)。该修饰电极有较好的抗干扰能力,可用于牛奶样品中三聚氰胺的测定。     

Effect of Protein Fermentation Products on Gut Health Assessed in an In Vitro Model of Human Colon (TIM-2)

Scope

Western type of diets are characterized by high animal protein intake and are associated with various chronic inflammatory diseases. With a higher protein consumption, excess undigested protein will reach the colon and be subsequently metabolized by gut microbiota. Depending on the type of protein, fermentation in the colon generates different metabolites with varying biological effects. This study aims to compare the impact of protein fermentation products from different sources on gut health.

Methods and results

Three high protein diets (vital wheat gluten [VWG], lentil, or casein) are submitted to the in vitro model of colon. Fermentation of excess lentil protein for 72 h results in highest production of short-chain fatty acids and lowest production of branched-chain fatty acids. Exposure of Caco-2 monolayers or Caco-2 monolayers co-cultured with THP-1 macrophages to luminal extracts of fermented lentil protein results in less cytotoxicity of Caco-2 monolayers and less damage to barrier integrity, when compared to VWG and casein. Lowest induction of interleukin-6 is observed in THP-1 macrophages after treatment with lentil luminal extracts, which is identified to be regulated by aryl hydrocarbon receptor signaling.

Conclusion

The findings indicate that protein sources affect the health effects of high protein diet in the gut.     

脂肪酶GZEL及其C-端截断突变体对磷脂单分子膜的吸附动力学研究

基于单分子层技术研究了禾谷镰孢菌脂肪酶GZEL对不同磷脂单分子膜的吸附动力学。同时,采用截断突变方法分析C-端肽段(编号为269-319的氨基酸)缺失后对脂肪酶GZEL界面吸附动力学参数的影响。研究发现,脂肪酶GZEL对不同磷脂单分子层的吸附动力学参数(吸附常数k_a,解离常数k_d,吸附平衡常数K_(Ads))与磷脂单分子膜的种类及初始表面压力密切相关。尽管如此,相比于野生型GZEL,269-319肽段缺失后导致酶蛋白对不同磷脂单分子膜的吸附常数k_a均显著降低,而解离常数k_d则显著升高,两者共同导致酶蛋白对于磷脂单分子膜的亲和力K_(Ads)显著降低。野生型GZEL对不同磷脂单分子膜的选择性顺序为磷脂酰丝氨酸磷脂酰胆碱磷脂酰乙醇胺。而269-319肽段缺失后,酶蛋白对于这三种磷脂单分子膜的亲和力则表现为无显著差异。以上结果表明269-319肽段在脂肪酶GZEL的界面吸附及对磷脂选择性方面均发挥有重要作用。     

Assessment of the lupin seed glucose-lowering protein intestinal absorption by using in vitro and ex vivo models

A lupin seed glycoprotein, termed γ-conglutin, has previously been found to display insulin-mimetic activity in myocyte models and reduce plasma glucose concentration when orally administered to both rats and humans. To envisage the possible metabolic fate of this bioactive protein, we used in vitro cell and ex vivo tissue models to monitor its transit through the intestinal barrier. Caco-2 cell monolayers and rat intestinal everted sacs were treated with purified γ-conglutin and the protein was immuno-assayed by chemi-luminescence-enhanced Western blotting. The in vitro approach showed that the intact protein can transit from the apical to the basolateral side of the cell monolayers. The unmodified lupin protein was also detected inside the intestinal everted sacs. Proper controls of cell monolayer and sac integrity ruled out the possibility of protein passive leakage.     

Production and transepithelial transportation of angiotensin-I-converting enzyme (ACE)-inhibitory peptides from whey protein hydrolyzed by immobilized Lactobacillus helveticus proteinase

Lactobacillus helveticus LB 10 proteinases immobilized with sodium alginate were used to hydrolyze whey protein to produce angiotensin-I-converting enzyme (ACE)-inhibitory peptides. The generated hydrolysates were tested for ACE-inhibitory activity and for their ability to be transported across Caco-2 cell monolayers. Using a response surface method, we determined that a proteinase concentration of 7.55 mg/mL, sodium alginate concentration of 2.03 g/100 mL, and glutaraldehyde concentration of 0.39% were found to be the optimal immobilization conditions. Compared with free proteinase, the immobilized proteinase had significantly higher pH, thermal and storage stability, and reusability. Whey protein hydrolysates were fractionated by gel filtration chromatography and ACE-inhibitory peptide mixtures were transported across Caco-2 cell monolayers in a human intestinal-absorption model. The di- and tripeptides KA, EN, DIS, EVD, LF, AIV, and VFK (half-maximal inhibitory concentrations (mean ± standard deviation) of 1.24 ± 0.01, 1.43 ± 0.04, 1.59 ± 0.27, 1.32 ± 0.05, 1.60 ± 0.39, 2.66 ± 0.02, and 1.76 ± 0.09 mmol/L, respectively) were detected on the basolateral side of the Caco-2 cell monolayer using ultra-performance liquid chromatography-tandem mass spectrometry. These results highlight that ACE-inhibitory peptides are present on the basolateral side of the Caco-2 cell model after transportation of whey protein hydrolysate across the Caco-2 cell membrane.     

单分子膜研究的方法及现状

单分子膜一般是将两亲性物质溶解在铺展溶剂中制成铺展溶液，然后铺展在底液上而得的。在其性质的研究中，测定表面压－面积关系曲线是最基本、也是最重要的一种研究手段。但是，仅有表面压－面积曲线所能了解的信息是远远不够的，因此必须结合另一些手段进行研究，测定表面粘度就是其中很重要的一种。而随着现代光谱仪器的更新、发展，光学方法已日益成为单分子膜研究的一个重要途径。     

Partially Hydrolyzed Soy Protein Shows Enhanced Transport of Amino Acids Compared to Nonhydrolyzed Protein across an Intestinal Epithelial Cell Monolayer

Consumption of protein hydrolysates has been proposed to stimulate muscle anabolism more than intact (nonhydrolyzed) proteins via accelerated delivery of amino acids for muscle protein synthesis (MPS). We evaluated whether the rate of amino acid uptake and transport across intestinal cells was enhanced for soy protein hydrolysates versus nonhydrolyzed soy protein. Intact and partially hydrolyzed proteins were subject to simulated gut digestion and applied to the apical surface of Caco‐2 monolayers. Basolateral media was harvested after 3 h and quantitatively analyzed for free amino acids using ion‐exchange chromatography and comparison to an included reference standard. Basolateral concentrations of all amino acids were higher (mean 32%) for hydrolyzed versus nonhydrolyzed protein with the greatest differences in histidine, lysine, and valine. Scale‐up production of the soy protein hydrolysate did not diminish its enhanced absorption properties. These data support the hypothesis that hydrolyzed soy protein may provide dietary amino acids that are more rapidly transported across the intestinal epithelium versus intact soy protein. This would be important under conditions where rapid and increased levels of amino acids are needed such as in the stimulation of MPS.     

Structural characteristics of adsorbed protein and monoglyceride mixed monolayers at the air–water interface

Monolayer technique has been used successfully to study the properties of mixed emulsifiers spread at the air–water interface. In this contribution we are concerned with the analysis of structural characteristics of mixed monolayers formed by milk proteins (β-casein and β-lactoglobulin) and monoglycerides (monopalmitin and monoolein), and how these differ depending on the method used for the formation of the mixed film at the air–water interface (spreading, penetration or by spreading of a lipid into a previously adsorbed protein film). Measurements of the π-A isotherm were obtained in Langmuir- and Wilhelmy-type film balances coupled with Brewster angle microscopy (BAM). The π-A isotherm deduced for adsorbed β-lactoglobulin monolayer in this work is practically the same as that obtained directly by spreading. However, for β-casein the adsorbed monolayer is more condensed than the spread one. For protein–monoglyceride mixed films, the π-A isotherms for adsorbed and spread monolayers at surface pressure (π) higher than the equilibrium spreading pressure of protein (π_e^protein) are practically coincident, a phenomenon which may be attributed to protein displacement by the monoglyceride from the interface. At π<π_e protein and monoglyceride coexist at the interface and the adsorbed and spread π-A isotherms (monolayer structure) are different. Monopalmitin has a higher capacity than monoolein for the displacement of protein from the air–water interface. However, some degree of interaction exists between proteins and monoglycerides and these interactions are higher for adsorbed than for spread films. The topography of the monolayer corroborates these conclusions.     

Mixed biopolymers at interfaces: Competitive adsorption and multilayer structures

Mixed biopolymer layers are commonly involved in the stabilization of food emulsions and foams. The interfacial composition and structure of mixed layers are predominantly determined by two mechanistic phenomena—competitive adsorption from mixed solution and cooperative adsorption into multilayers. The surface-active protein components typically dominate primary layers around droplets and bubbles, and the interacting polysaccharides form outer secondary stabilizing layers. This article reviews progress in understanding the factors controlling the nanoscale structure and physico-chemical properties of adsorbed layers in colloidal systems containing mixtures of biopolymers. Contributions from different experimental techniques are described, with particular attention directed towards the role of surface shear rheology in providing information on competitive adsorption of proteins and macromolecular interactions at fluid interfaces. We also consider here the phenomenon of phase separation in mixed protein monolayers, the balance of thermodynamic and kinetic factors in determining biopolymer layer properties, and the involvement of electrostatic interactions in the stabilization of emulsions by protein–polysaccharide complexes.     

哈维氏弧菌磷脂酶D对不同磷脂单分子层的吸附动力学研究

基于单分子层技术研究了哈维氏弧菌来源磷脂酶D(Vh PLD)对不同磷脂单分子层的吸附动力学。探究初始表面压力条件对VhPLD吸附不同磷脂单分子层(磷脂酰胆碱(PC)、磷脂酰乙醇胺(PE)、磷脂酰丝氨酸(PS)、磷脂酰甘油(PG)和磷脂酰肌醇(PI))吸附动力学参数(k_a、k_d、K_(Ads))的影响规律。结果表明:VhPLD对磷脂单分子层的吸附动力学参数与磷脂单分子层初始表面压力密切相关;在15 m N/m条件下,VhPLD对不同磷脂单分子层吸附偏好性顺序为PC PG PS PE=PI;在20 m N/m条件下,VhPLD对不同磷脂单分子层吸附偏好性顺序为PG PI PC PS PE;在25 m N/m条件下,VhPLD对不同磷脂单分子层吸附偏好性顺序则转变为PC PS PI PE=PG。     

Some implications of nanoscience in food dispersion formulations containing phospholipids as emulsifiers

This contribution is concerned with phospholipid films in relation to food dispersions such as emulsions and foams. Structural, morphological and surface rheological characteristics of dipalmitoyl phosphatidylcholine (DPPC) and dioleoyl phosphatidylcholine (DOPC) monolayers were determined at the air–water interface at 20 °C and at pH 5, 7, and 9, by means of surface pressure (π)–area (A) isotherms coupled with Brewster angle microscopy (BAM), atomic force microscopy (AFM) and surface dilatational and shear rheometry. From the π–A isotherms it was deduced that DPPC monolayers show structural polymorphism at the air–water interface. DOPC monolayers formed a liquid-expanded (LE) structure under all experimental conditions, a consequence of the weak molecular interactions due to the double bond in the hydrocarbon chain. Electrostatic interactions between film-forming components influence the monolayer structure. BAM and AFM images corroborate, at a microscopic and at nanoscopic level respectively, the structural polymorphism deduced from the π–A isotherm for DPPC monolayers as a function of surface pressure and the pH of the aqueous phase. However, the homogeneous morphology of DOPC monolayers at a microscopic level, as observed by BAM, shows structural heterogeneity at a nanoscopic level when observed by AFM. The relative monolayer thickness increases with surface pressure and is a maximum at the collapse point, especially for DPPC monolayers. The results confirm that the interfacial rheological characteristic measured under dilatation and shear conditions are very dependent on the structural characteristics and morphology of the phospholipids (DPPC and DOPC) monolayers.