固定化酶化学发光型葡萄糖传感器法测定食品中的葡萄糖

制成了固定化酶化学发光型葡萄糖传感器,此传感器可用于对葡萄糖的在线快速检测。用MCM-41介孔分子筛固定葡萄糖氧化酶(GOD),制成GOD酶柱,并用于流动注射化学发光分析。酶柱的使用条件为:柱温40℃,葡萄糖溶液pH=6.0,流速3 r/min。测定的线性范围为1～200 mg/L,检出限为0.2 mg/L,10次测定的RSD为1.8%,回收率在97.81%～102.0%。测定结果与国标方法的测定结果无显著性差异。     

化学发光法在食品分析中的应用

论述近10年来化学发光法在食品分析中的应用情况，主要涉及含氮化合物、金属、油脂氧化、类激素、幅照食品和糖等食品成分，同时对化学发光的基本反应和试剂作了介绍。     

海藻酸钠固定化乳酸菌促熟干酪效果的研究

对海藻酸钠固定化乳酸菌促熟干酪的效果进行了研究。结果表明，在干酪粒中添加10^6CFU／g固定化乳酸菌，水溶性氮（WSN）、三氯乙酸氮（TCASN）和磷钨酸氮（PTASN）含量较对照组明显增大；ADV值在添加固定化乳酸菌干酪中成熟初期变化不大，45d后明显增大；感官评定结果表明，干酪粒中添加10^5CFU／g固定化乳酸菌干酪成熟60d时风味和质地较好，可比对照组干酪成熟期缩短30d左右。     

固定化酶及其在食品工业中的应用

固定化酶是酶工程的核心，它有利于实现酶的重复使用及产物与酶的分离。本文综述了固定化酶的发展简史、性质和固定方法。介绍了固定化酶中应用的载体。并对该技术在食品工业中的应用进行了重点介绍。     

葡萄糖氧化酶与过氧化氢酶的共固定化研究

研究了环氧树脂在共固定化葡萄糖氧化酶(GOD)和过氧化氢酶(CAT)中的应用,利用筛选出固定化效果最好的环氧树脂ES-4,考察加酶量、p H及磷酸钠缓冲液离子浓度对共固定化效果的影响。结果表明:共固定化的最佳条件是:GOD 0.5 m L、CAT 1.0 m L、磷酸钠缓冲液p H6.5、浓度1.0 mol/L,固定24 h后固定化葡萄糖氧化酶酶活回收率为35%,过氧化氢酶酶活回收率为43%。连续操作15批次后,酶活力仍能保持初始酶活的73%。该研究为工业化利用固定化酶生产葡萄糖酸钠提供了新的思路。     

磁性壳聚糖微球固定化碱性蛋白酶的酶学性质

以Fe3O4和壳聚糖为原料,以戊二醛为交联剂制备磁性壳聚糖微球,固定化碱性蛋白酶,并对其品貌及结构性质进行观察和分析。结果表明:壳聚糖微球具有良好的球形外貌,大小约为15nm,固定化后粒子大小约为20nm;红外光谱分析证实Fe3O4已被壳聚糖包裹;固定化酶前后粒子晶形完整,均具有良好的磁响应能力和超顺磁性。固定化酶与游离酶相比,最适温度从60℃下降到50℃;最适pH值从10升至11;固定化酶的Km为5.85×10-4mg/mL,游离酶的Km为6.06×10-4mg/mL。     

磁性微球固定化Alcalase酶与水解酪蛋白动力学模型的建立

采用化学沉积过程,以羟基Fe3O4为核、SiO2为壳进行核壳包被,再以氨基硅烷化试剂和戊二醛为交连试剂实现Alcalase酶的固定化,电镜及红外谱图分析作为表征手段,并将其用于酪蛋白的水解过程,通过双曲线回归模型建立水解全过程动力学模型。     

壳聚糖固定化纤维素酶的条件优化

采用Plackett-Burman试验设计和正交试验设计对影响固定化纤维素酶活的因素进行了筛选和优化.探讨了纤维素酶、壳聚糖、乙酸、乙醇、戊二醛、氢氧化钠等试剂对固定化效果的影响,筛选出制备固定化纤维素酶过程中的主要影响因素.结果表明:壳聚糖、乙酸、乙醇浓度、酶浓度分别为15g/L、2.0%、40%、100%时,以壳聚糖为载体制备固定化纤维素酶的活力最高,成型效果最好.     

固定化酵母在山西陈醋生产中的应用

就固定化酵母在山西陈醋生产中的应用、优势及规模化生产的效果作了总结，结果表明：该技术用在山西陈醋生产工艺中，具有显著提高食醋出品率、耐高温、抗杂菌、发酵稳定、能安全度夏的特点。     

新型酶固定化技术研究进展

酶的固定化技术是酶工程的核心技术之一,它的出现降低了酶制剂的成本,促进了酶的工业化应用.对新型的酶固定化技术进行了综述,包括共价固定法、氨基酸置换法、抗体偶联法、生物素-亲和素亲和法、酶与金属离子的连接和疏水定向固定法及其他技术处理方法,并对固定化技术的应用前景进行了展望.     

Flow injection chemiluminescent determination of clenbuterol using GoldMag particles as carrier

A novel flow injection chemiluminescent (CL) enzyme immunoassay for clenbuterol analysis based on GoldMag particles is described. GoldMag is a new type of super-paramagnetic Fe₃O₄/Au composite particle used as a carrier in a flow injection CL system. Clenbuterol conjugated with ovalbumin (OVA) was immobilized onto GoldMag particles and the particles fixed in a micro-channel by an external electromagnetic field. The clenbuterol test sample and clenbuterol polyclonal antibody (Ab) were injected into the channel and incubated with GoldMag particles. Clenbuterol, immobilized on the magnetic particle surfaces, competes for polyclonal antibodies with clenbuterol in the test sample. The free Ab or Ab combined with the clenbuterol sample was washed away and the magnetic particles conjugated with Ag-Ab left in the micro-channel. Horseradish peroxidase (HRP)-labelled goat anti-rabbit immunoglobulin G (IgG) was added and reacted with clenbuterol polyclonal antibodies; excess goat anti-rabbit-HRP was then washed off. When chemiluminescent reagents were injected into the channel, emitted light from the magnetic particle surface was measured and recorded using a photomultiplier-based apparatus. The linear range of this novel method was 0.01-0.1 ng g^-1 and recovery of clenbuterol was 85-105% with a RSD of 3.2% (n = 11).     

Enhancing activity and stability of Burkholderia cepacia lipase by immobilization on surface-functionalized mesoporous silicates

Burkholderia cepacia lipase was immobilized on various types of phenyl-functionalized mesoporous silicates (MPS). MPS, anchored with a phenyl group on the silica wall and with three dimensional (3D) mesoporosity, showed highest lipase adsorption capacity and best activities both in aqueous and organic reagents.     

Evaluation of alkaline phosphatase detection in dairy products using a modified rapid chemiluminescent method and official methods

Alkaline phosphatase is a ubiquitous milk enzyme that historically has been used to verify adequate pasteurization of milk for public health purposes. Current approved methods for detection of alkaline phosphatase in milk include the use of enzyme photoactivated substrates to give readings in milliunits per liter. The U.S. and European public health limit for alkaline phosphatase in pasteurized drinks is 350 mU/liter. A modified chemiluminescent method, fast alkaline phosphatase, was compared with the approved fluorometric and chemiluminescent alkaline phosphatase methods to determine whether the modified method was equivalent to the approved methods and suitable for detecting alkaline phosphatase in milk. Alkaline phosphatase concentrations in cow's, goat's, and sheep's milk and in flavored drinks and cream were determined by three methods. Evaluations in each matrix were conducted with pasteurized samples spiked with raw milk to produce alkaline phosphatase concentrations of 2 to 5,000 mU/liter. The tests were performed by the method developer and then reproduced at a laboratory at the National Center for Food Safety and Technology following the criteria for a single laboratory validation. The results indicated that the fast alkaline phosphatase method was not significantly different from the approved chemiluminescent method, with a limit of detection of 20 to 50 mU/liter in all the studied matrices. This modified chemiluminescent method detects alkaline phosphatase in the 350 mU/liter range with absolute differences from triplicate data that are lower and within the range of the allowed intralaboratory repeatability values published for the approved chemiluminescent method.     

Latex agglutination assays for detection of non-O157 Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145

Latex agglutination assays utilizing polyclonal antibodies were developed for the top six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups. Rabbit antisera were affinity purified through protein A/G columns, and the isolated immunoglobulins (IgGs) were covalently immobilized onto polystyrene latex particles. The resulting latex-IgG complex had a protein (IgG) load of 0.20 to 0.28 mg/ml in a 1% latex suspension. Optimum conditions for the agglutination assay consisted of utilizing 20 μm l of latex-IgG reagent containing 2.0 to 2.8 μm g IgG in a 0.5% latex suspension. Agglutination or flocculation was observed almost instantly after mixing the colonies with the latex-IgG, indicating STEC strains. More than 100 target and nontarget strains were tested in more than 3,000 test replicates. All target organisms produced positive results, but three antisera (anti-O26, anti-O103, and anti-O145) cross-reacted with some other STECs. The anti-O103 and anti-O145 latex reagents cross-reacted with O26 strains, and the anti-O26 cross-reacted with O103 strains. The latex-IgG reagents are stable for at least 1 year and are easy to prepare. These agglutination assays can be used for identification of presumptive non-O157 STEC colonies from agar media. The techniques used to prepare the latex reagents also can be utilized for testing other STEC serogroups, other E. coli serotypes, or other pathogens to ensure safe foods to consumers.     

Determination of Functional Groups in Glucose Isomerase

Following the introduction and widespread application of immobilized glucose isomerase for the industrial production of isosyrup (HFCS), the most significant challenge facing experts is to solve the problem of increasing the fructose content. In addition to well-known procedures another approach may be the changing of the equilibrium of the enzymatic reaction by modifying the active centre of the enzyme. Histidyl-, ϵ-amino-, arginyl-, tyrosyl- and tryptophyl- amino acid side chains in the active centre have been modified by means of selective protein reagents. The results were evaluated by kinetic methods and the role of functional groups of amino side chains has been determined. A new hypothesis is proposed for the mechanism of enzymatic reactions.     

化学发光免疫分析方法在食品安全检测中的 研究进展

化学发光免疫分析方法作为一项具有高特异性和高灵敏度的免疫分析方法,经过近几十年的发展,目前已在食品安全检测领域得到了良好的应用。本文根据不同化学发光免疫分析方法所选择标记物的不同,将化学发光免疫分析方法分为化学发光免疫分析、化学发光酶免疫分析和电化学发光免疫分析三个类别,并对各方法常用的化学发光标记物及分析方法原理进行介绍。同时,综述了化学发光免疫分析方法在农兽药残留、生物毒素、违禁添加物及微生物检测方面的应用进展情况。随着化学发光免疫分析方法在新型化学发光标记物及化学发光增敏体系方面取得进一步的研究进展,其在食品安全检测领域必将得到更为广泛的应用。     

Phage-borne peptidomimetics accelerate the development of polyclonal antibody-based heterologous immunoassays for the detection of pesticide metabolites

Competitive immunoassays for the detection of small analytes, such as pesticides and their metabolites, use haptens that compete with the target compounds for binding to the antibody. This competing hapten can be either the same as the immunizing hapten (homologous assay) or structurally modified mimics of the immunizing hapten (heterologous assay). Polyclonal antibody-based heterologous immunoassays have shown superior sensitivities to homologous ones, butthe synthesis of heterologous haptens may be time-consuming, requiring expertise in synthetic chemistry. In this work we demonstrate that phage display peptide libraries can be used as a source of phage-borne peptidomimetics to facilitate the development of sensitive heterologous assays. Different strategies for the isolation of these peptides were explored using two metabolites of pyrethroid insecticides. The sensitivities of the best competitive phage heterologous enzyme-linked immunosorbent assays were 13 fold and 100 fold better than the homologous assay, for the glycine conjugate of trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid and 3-phenoxybenzoic acid, respectively. The phage particles were highly versatile as tracer reagents, allowing the use of enzymatic, chemiluminescent, or immuno-polymerase chain reaction detection. The data presented here shows a new systematic procedure that enables the fast generation of several competing haptens for the rapid development of sensitive heterologous immunoassays.     

有机磷农药多残留检测化学发光标记物的 合成及鉴定

目的 以O,O-二乙基硫代磷酰氯和鲁米诺为原料, 合成一种新型的化学发光标记物, 用于后续的二乙氧基类有机磷农药多残留化学发光免疫法检测。方法 取鲁米诺和O,O-二乙基硫代磷酰氯溶于二氯甲烷中, 以三乙胺作为缚酸剂, 冰浴下反应8 h后分出有机层, 产物经柱层析纯化, 即得到化学发光标记物。结果 产物经紫外、红外鉴定, 实验结果表明反应产物即为所需的化学发光标记物, 产物在BPCL上进行发光特性测定, 表现出较强的发光能力, 最大发光强度达到9000 mV。结论 本方法合成的化学发光标记物具有较强的发光能力, 能用于后续的二乙氧基类有机磷农药多残留的化学发光免疫检测。     

Simultaneous determination of chloramphenicol,florfenicol and florfenicol amine in ham sausage with a hybrid chemiluminescent immunoassay

A novel chemiluminescent immunoassay utilising two types of primary antibodies (murine monoclonal antibody and rabbit polyclonal antibody) and two types of horseradish peroxidase–labelled secondary antibodies was established for simultaneously detecting multiple amphenicol residues in ham sausage. After combining the extract procedure of the target amphenicol into one simplified method, this hybrid chemiluminescent immunoassay could screen chloramphenicol (CAP), florfenicol (FF) and its metabolite florfenicol amine (FFA) at the same time by adding the corresponding secondary antibody. Ham sausage samples were analysed by using this hybrid immunoassay, with LODs of CAP being 0.01 μg kg^?1, of FF being 2.8 μg kg^?1 and of FFA being 3.0 μg kg^?1. The applicability of the proposed method has been validated by determining CAP, FF and FFA in ham sausage samples with satisfactory results. Good recoveries and high correlation with traditional enzyme-linked immunosorbent assay and LC-MS/MS results illustrated that the developed hybrid chemiluminescent immunoassay could screen high-throughput ultra-trace amphenicol residues effectively at one time.     

化学发光法测定卷烟主流烟气中的氮氧化物

探讨了一种用化学发光法测定卷烟主流烟气中NOx 的方法 ,即将每口新鲜主流烟气经剑桥滤片过滤后引入到一个减压的混合室 ,经充分混合后 ,用氮氧化物分析仪根据化学发光原理测定卷烟主流烟气中氮氧化物。该方法的CV <5 % ,回收率 >98% ,并用外标法定量测定了 6种牌号卷烟主流烟气中的氮氧化物。该方法快速、重复性好、回收率高 ,适用于卷烟烟气中的氮氧化物的分析