堇叶碎米荠低聚肽制备工艺优化及结构特征、氧化活性研究

目的：制备堇叶碎米荠低聚肽，并拓展其应用范围。方法：以堇叶碎米荠为原料，使用碱性蛋白酶酶解，采用超滤膜进行分离浓缩，冷冻干燥后获取堇叶碎米荠低聚肽粉末，并对该低聚肽的结构和抗氧化活性进行分析。结果：堇叶碎米荠蛋白水解的最佳工艺条件为酶解温度50 ℃、酶解pH 10、加酶量4%、底物质量分数2%，此条件下水解度为17.9%；堇叶碎米荠低聚肽的蛋白质和多肽含量分别为49.01%和42.87%；该低聚肽在200~220 nm有较强的吸收带，具有酰胺键的特征吸收峰，二级结构以β-转角为主，相对分子质量＜1 000的约占90%；当堇叶碎米荠低聚肽质量浓度为20 mg/mL时，其羟自由基、ABTS自由基、DPPH自由基清除率分别为84.54%，98.22%，60.33%。结论：试验优化的堇叶碎米荠低聚肽制备工艺合理可行，堇叶碎米荠低聚肽具有抗氧化活性。     

硒在堇叶碎米荠中的赋存形态及分布规律

使用水、氯化钠、乙醇和氢氧化钠等不同提取液分别提取堇叶碎米荠中的蛋白质、多糖、核酸等有机硒大分子及无机硒,并采用氢化物发生-原子荧光法(HG-AFS)测定其硒含量,研究硒在堇叶碎米荠中的赋存形态及分布规律。结果表明:堇叶碎米荠不同部位硒含量分布规律为茎叶根;叶片中有机硒的含量不仅高于无机硒,且有机硒转化率达到60%以上,硒多糖是堇叶碎米荠有机硒赋存形态的主要组成部分,占样品总硒的28.74%,其次为硒蛋白,硒蛋白组分中水溶性蛋白结合硒含量最高,占总蛋白含硒量的24.04%。     

碎米荠硒肽的制备及其体外抗氧化活性分析

本文以恩施碎米荠为原料,采用酶法制备具有抗氧化活性的碎米荠硒肽.以蛋白提取率为指标,通过单因素实验和正交试验优化了碎米荠蛋白的提取条件;以DPPH·、ABTS+·清除率为考察指标,优化了碎米荠蛋白的酶解参数,利用超滤设备对碎米荠蛋白酶解液进行分级分离,确定了碎米荠硒肽制备的最佳方案;考察了碎米荠硒肽的还原力、Fe2+螯...     

超聚硒植物堇叶碎米荠研究进展

在发现堇叶碎米荠具有强大聚硒能力后的二十多年中,对其富硒机制、营养品质、食用安全、生物活性的研究取得了较大进展。在人工强化栽培条件下,其硒含量最高可达9741.05mg/kg,有机硒占比达到85.11%,以硒代半胱氨酸为主。多组学研究揭示了一些与硒耐受有关的基因和代谢途径。堇叶碎米荠富含蛋白质、维生素、矿物质等营养成分,经急性和亚慢性毒性试验以及食用历史的大样本调查,证明实际属于无毒物质。体外试验和动物模型试验证实了富硒碎米荠提取物具有抗氧化、抑制肝癌细胞、改善仔猪肠道功能、提高蛋鸡产蛋、缓解疲劳等功能。通过提取工艺研究使硒蛋白提取实现得率75.4%、纯度77%、总硒含量(9097.33±35.66)mg/kg,采用固定酶提取硒肽,其有机硒可达2914 mg/kg。与其他硒源相比,堇叶碎米荠硒以有机硒为主,其清除自由基的能力强于无机硒。堇叶碎米荠硒蛋白能增强小鼠血液中的谷胱甘肽过氧化物酶和超氧化物歧化酶活力,抑制丙二醛的生成。另外,堇叶碎米荠硒肽能改善大鼠D-半乳糖诱导的代谢紊乱、记忆退化、海马体中的神经元损伤、减少神经炎症反应,对高脂食物引起肥胖以及代谢紊乱具有保护效应。     

壶瓶碎米荠多糖硫酸化结构修饰及抗氧化活性研究

为探索壶瓶碎米荠多糖结构修饰以及修饰后多糖的生物活性变化规律,利用三氧化硫-吡啶法对壶瓶碎米荠多糖进行了硫酸化结构修饰,得到了五种不同取代度的硫酸化壶瓶碎米荠多糖,取代度分别为0.46、0.55、0.69、0.72、0.80。通过傅立叶变红外光谱初步对其改性效果进行了分析,在此基础上研究了改性壶瓶碎米荠多糖的抗氧化活性。结果显示：硫酸化改性壶瓶碎米荠多糖可以改善壶瓶碎米荠多糖的抗氧化活性,其中取代度为0.80的硫酸化壶瓶碎米荠多糖在ABTS自由基、羟自由基、DPPH自由基上具有较好的清除能力。该研究结果为壶瓶碎米荠多糖的结构以及活性研究提供了一定的试验基础。     

碎米荠蛋白制备工艺及抗氧化活性

以恩施堇叶碎米荠为原料,研究碎米荠硒蛋白的提取工艺,探讨碎米荠蛋白的抗氧化活性,结果表明:提取温度为50℃,料液质量体积比为1 g:40 mL,先后用去离子水和0.1 mol/L的NaOH溶液分别提取8 h。在此工艺条件下蛋白质得率最高,为75.4%,其中水提蛋白质质量分数为26.5%,硒质量分数为1 748μg/g;碱提蛋白质质量分数为52.3%,硒质量分数为1 513μg/g。碎米荠碱提蛋白经Sephadex G-100凝胶层析柱分离得到两个组分,分别为峰1和峰2,硒质量分数分别为668μg/g和650μg/g,通过研究碎米荠蛋白对羟自由基(·OH)和1-1-二苯基-2-三硝基苯肼自由基(·DPPH)的清除能力,发现碎米荠蛋白均有一定的清除自由基的能力。     

富硒碎米荠不同提取物抗氧化性能研究

为进一步开发利用恩施堇叶碎米荠,通过比较测定3种碎米荠提取物:碎米荠碱提物(CE)、碎米荠富硒蛋白(SPR)和碎米荠富硒多肽(SPI)对1,1-二苯基-2-三硝基苯肼自由基(DPPH·)、羟自由基(OH·)、2'-联氨-双-3-乙基苯并噻唑啉-6-磺酸自由基(ABTS+·)和超氧阴离子自由基(O_2~-·)的清除能力,考察3种富硒碎米荠提取物的体外抗氧化性能,并与富硒酵母(SY)、无机硒(IS)和非富硒多肽与无机硒混合物(SPIS)进行比较。结果表明:SPI清除4种自由基能力最强,其对DPPH·、OH·、ABTS~+·和O_2~-·的清除率分别为87.5%,50.3%,99.7%,45.7%,IS清除4种自由基能力最弱,均不超过14%。通过比较SPR、SPI、CE、SY、IS和SPIS对4种自由基的清除率及半抑制浓度(IC_(50)),发现有机硒清除自由基的能力强于无机硒。     

壶瓶碎米荠粗黄酮产品的抗氧化研究

以DPPH自由基、超氧阴离子自由基、羟自由基和总还原力为指标，测定体外抗氧化能力；以小鼠血清中SOD、CAT、GSH-Px活性和MDA含量为测定指标，测定体内抗氧化能力。结果显示：壶瓶碎米荠含硒粗黄酮对自由基具有一定的清除作用，在实验考察的自由基中，除超氧阴离子外其余均显示出良好的抗氧化效果且随着剂量的增加呈现出良好的线性；动物实验中壶瓶碎米荠两种粗黄酮均能提高小鼠SOD、CAT、GSH-Px活性，降低MDA含量并呈现出良好的线性效果，含硒粗黄酮抗氧化效果显著高于普通粗黄酮，与阳性对照VC效果相似。当壶瓶碎米荠含硒粗黄酮剂量达到100 mg/kg时，芦丁实验组、粗黄酮实验组小鼠体重降低，出现轻微毒害作用，而含硒粗黄酮处理组体重差异不显著，从侧面说明硒具有一定增强免疫力的效果。     

壶瓶碎米荠多糖的提取、分离及抗氧化活性研究

研究壶瓶碎米荠多糖的提取、分离纯化方法,并分析其体外抗氧化活性。试验结果表明,通过超声波、酶辅助提取壶瓶碎米荠多糖的最佳工艺条件:液料比30 ∶ 1,超声时间25 min,纤维素酶添加量2%,提取温度90 ℃,提取时间120 min,测得实际值为48.53 mg/g。采用Svage法脱蛋白7次,多糖的损耗率较小。经DEAE-52纤维素层析得到4种组分多糖。体外抗氧化活性分析表明,壶瓶碎米荠粗多糖具有一定的还原能力,对ABTS自由基、羟基自由基的清除能力较强。     

响应面试验优化复合酶法提取碎米荠多糖工艺及其抗氧化活性

利用人工种植的碎米荠为原料,研究其粗多糖的提取工艺参数及抗氧化活性。首先对提取条件的单因素进行优化,在单因素试验基础上,进行提取条件的响应面优化。单因素优化条件为:质量分数2%复合酶(m(纤维素酶)∶m(果胶酶)=2∶1)、酶解时间90 min、酶解温度60℃、酶解pH 4.0。响应面优化结果为:酶解时间91.8 min、酶解温度57.1℃、酶解pH 4.17。在此条件下,碎米荠粗多糖提取率最高,粗多糖提取率预测值为4.14%,验证实验得到实际粗多糖的平均提取率为4.07%;与理论预测值相比,其相对误差约为1.62%。抗氧化活性研究结果显示,碎米荠多糖具有抗氧化活性,且效果优于VC。该实验结果为碎米荠多糖的提取以及多糖的性质研究提供理论依据。     

辣椒制品大肠杆菌O157及志贺氏菌污染调查与风险评估

掌握辣椒制品大肠杆菌O157及志贺氏菌的污染状况,为开展辣椒制品安全风险评估,企业及产品分类管理提供必要的依据。参照GB/T4789.36—2008全自动酶联荧光免疫分析仪筛选法对辣椒制品大肠杆菌O157进行快速筛选试验,大肠杆菌0157检验结果为阳性时,参照GB/T4789.36—2008常规培养法进行大肠杆菌O157分离鉴定确认。参照GB/T4789.5—2003对辣椒制品志贺氏菌进行检验。可疑菌株用API20E生化鉴定试剂盒及VITEK2compact30全自动细菌鉴定分析系统进行生化鉴定,综合生化试验和血清学的试验结果报告。共抽890份样品、包括4类辣椒制品、涉及18个省(区、市)的194家生产企业,进行了大肠杆菌O157和志贺氏菌检验,均未检出目标菌,表明大肠杆菌O157和志贺氏菌在辣椒制品中污染风险较小。     

The DNA Sequence of the Escherichia coli O22 O-Antigen Gene Cluster and Detection of Pathogenic Strains Belonging to E. coli Serogroups O22 and O91 by Multiplex PCR Assays Targeting Virulence Genes and Genes in the Respective O-Antigen Gene Clusters

Multiplex polymerase chain reaction (PCR) assays were developed for detection of pathogenic strains belonging to Escherichia coli serogroups O22 and O91. The O-antigen gene cluster of E. coli O22 was sequenced to identify genes that could be employed as targets for serogroup-specific PCR assays. The wzx and wzy genes in the O-antigen gene clusters of E. coli O22 and E. coli O91 were selected as target genes. The assays were serogroup-specific when tested against 72 E. coli O22 strains and 57 E. coli O91 strains isolated from food, humans, and animals, representative strains belonging to 168 E. coli O serogroups and non-E. coli bacteria. Furthermore, 72 E. coli O22 strains and 57 E. coli O91 strains isolated from food, water, animals, and humans were tested by the PCR for the presence of six and 19 virulence genes, respectively, associated with pathogenic E. coli strains. Based on the PCR screening results, multiplex PCR assays targeting the O22 wzy gene and the cnf-1 and sfa genes in E. coli O22 and the O91 wzy gene, conserved sequences of stx ₁ and stx ₂ genes, and the astA and cdt-III genes in E. coli O91 were developed to detect and identify pathogenic strains belonging to serogroups O22 and O91. Furthermore, E. coli O22 and O91 were detected by multiplex PCR assays targeting the wzx or wzy genes and conserved sequences of the stx ₁ and stx ₂ genes in ground beef samples inoculated with approximately two colony-forming units (CFU)/25 g after 18-h enrichment. The results demonstrate that the E. coli O22 and O91 wzx and wzy gene sequences were specific for the respective serogroups and can be used as diagnostic markers for rapid identification of these serogroups as an alternative to serotyping. The multiplex PCR assays targeting the O22 and O91 wzx and wzy genes and virulence genes can be used to identify and to detect pathogenic strains of these serogroups in food and fecal samples. Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

大肠杆菌O157微孔板法与经典方法的比较研究

大肠杆菌O157:H7是目前国内外极为关注的食源性致病菌,快速检测大肠杆菌O157:H7显得尤为重要.特针对9大类食品分别进行了传统培养法和3M TecraTM微孔板法的对比实验,并选择了其它血清型大肠杆菌和其他种属细菌进行了特异性的检验.在199份阳性样品中, 3M TecraTM大肠杆菌O157微孔板法的检出率为100%,传统培养法则为97.5%,3M TecraTM大肠杆菌O157微孔板法的灵敏度达到1 CFU/25g(mL),且该方法具有快速、简便、高效、准确的特点,适宜大规模化食品样品大肠杆菌O157检测.     

双抗夹心ELISA检测食品中大肠杆菌O157:H7方法研究

研究获得纯化抗大肠杆菌O157:H7IgY抗体,经检测10mg/ml纯化IgY抗体的效价为1:320;以大肠杆菌O157:H7免疫新西兰大耳白兔,获得兔抗大肠杆菌O157:H7IgG抗体,效价达1:25600。以兔抗大肠杆菌O157:H7IgG抗体稀释3200倍作为捕获抗体,抗大肠杆菌O157:H7IgY抗体为检测抗体建立双抗夹心ELISA方法检测大肠杆菌O157:H7,正交试验分析表明,捕获抗体于37℃包被2h、不封闭、抗原与捕获抗体于37℃结合2h、检测抗体浓度为0.25mg/ml、与抗原于37℃结合1h为最优反应条件。该方法对纯培养菌液检出限为105CFU/ml,具有良好的敏感性及特异性。染菌样品经在EC增菌液中选择性培养后进行双抗夹心ELISA检测,接种量为0.1～1CFU/g(ml)的样品在培养12h后可检出阳性反应,1～10CFU/g(ml)的样品在培养8h后可检出阳性反应。     

Efficacy of High Hydrostatic Pressure Treatment in Reducing Escherichia coli O157 and Listeria monocytogenes in Alfalfa Seeds

ABSTRACT: The application of high hydrostatic pressure (HHP) technology as a seed decontamination technique was evaluated. Alfalfa seeds inoculated with Escherichia coli O157 and Listeria monocytogenes were air-dried and subjected to independent HHP treatments of 275 to 575 MPa for 2 min or at 475 MPa for 2 to 8 min (40°C). There were 1.4-log and 2.0-log reductions in E. coli O157 populations at 575 MPa (2 min) and 475 MPa (8 min), respectively. However, these treatments caused only 0.8-log and 1.1-log reductions in L. monocytogenes counts. Treated seeds took longer to germinate, achieving germination rate of up to 34%, whereas 95% of the control germinated. Results suggest that L. monocytogenes is more resistant to the bactericidal effects of HHP than E. coli O157. Although HHP treatments achieved a greater reduction in E. coli O157, it was at the expense of seed germination. Overall, our results indicate that although HHP treatments reduced the populations of E. coli O157 and L. monocytogenes in alfalfa seeds, they did not completely eliminate these microorganisms.     

Occurrence of Escherichia coli O157, O111 and O26 in raw ewe's milk and performance of two enrichment broths and two plating media used for its assessment

The occurrence of Escherichia coli O157, O111 and O26 in 159 raw ewe's milk samples was examined. Sample-aliquots were incubated simultaneously in TSB added with yeast extract (YETSB) and mTSB with novobiocin (N-mTSB). Serogroup-specific immunomagnetic separation (IMS) was then used and IMS beads were plated in a cefixime tellurite (CT)-containing media (CT-SMAC, CT-SBMAC and CT-RMAC for E. coli O157, O111 and O26, respectively) and E. coli O157:H7 chromogenic ID agar. A sweep of confluent growth from each medium was examined for the presence of E. coli O157 and O111 using PCR, and for E. coli O26 using a latex agglutination test. Enumeration of E. coli O157 and O111 was performed in the samples tested positive for the correspondent serogroup using the most probable number (MPN) method combined with PCR. Percentage occurrences of E. coli O157, O111 and O26 were 18.2, 8.2 and 5.7, respectively. Mean E. coli O157 and O111 levels were 0.22 and < 0.04 MPN/mL, respectively. Enrichment in YETSB resulted in higher detection rates of E. coli O157 and O26 than in N-mTSB. When YETSB was used as enrichment broth and for these last two serogroups, the analysis of the confluent growth from the CT-media gave more positive results than that from E. coli O157:H7-ID medium.     

Comparison of a real-time PCR and an IMS/culture method to detect Escherichia coli O26 and O111 in minced beef in the Republic of Ireland

This study compared an immuno-magnetic separation (IMS)/culture method and a real-time PCR method to detect Verocytotoxigenic Escherichia coli (VTEC) serovar O26 and/or O111 in minced beef. A total of 65 samples were examined, 40 of which were frozen beef samples previously established as containing E. coli O157, and 25 were samples of fresh minced beef, purchased from butcher shops in the Dublin area. After selective enrichment, all samples were (a) subjected to IMS, plated on differential media and identified as E. coli O26 or O111 using biochemical and immuno-logical methods; and (b) subjected to DNA extraction and real-time PCR analysis using primers and probes against E. coli O111 and O26 serovar specific genes, and verotoxin genes. Overall, from the 65 minced beef samples collected, three were positive for E. coli O26 by real-time PCR, with only one of these samples positive for E. coli O26 by the culture method. One sample was positive for E. coli O111 by both real-time PCR and the culture method. The two samples found positive for E. coli O26 by real-time PCR method but not by culture method belonged to the group of frozen beef samples, indicating that the previously developed culture method for the detection of E. coli O26 may not be suitable for the detection of freeze injured cells. In conclusion, this study highlights the role of beef meat in the transmission of non-O157 VTEC. The results of the study emphasize that the analyses for emergent pathogens should be included in food safety surveillance systems and that the development of standard methods for the detection of E. coli O26 and O111 in routine food testing is needed in order to reduce the consumer exposure to contaminated food.     

不同粒径纳米金标记二抗增强表面等离子共振检测E. coli O157:H7

基于双通道表面等离子共振（surface plasmon resonance,SPR）传感器,结合不同粒径的金纳米粒子（Aunanoparticle,AuNPs）标记多克隆抗体（polyclonal antibody,PAb）作为第二抗体,采用氨基偶联的方法将PAb固定在传感器表面作为第一抗体,采用三明治夹心法进行了检测大肠杆菌O157∶H7（E. coli O157∶H7）的研究。考察3 种粒径的AuNPs作为标记物,增强SPR响应信号检测E. coli O157∶H7的能力。结果表明,增强效果最佳的AuNPs粒径为17.79 nm,其可检测到的E. coli O157∶H7的最低浓度为10 CFU/mL。     

Primers for Amplifying an Alanine Racemase Gene Fragment to Detect E. coli Strains in Foods

Specific oligonucleotide primers for detecting Escherichia coli in various foods were designed based upon the conserved sequences of the E. coli air gene from positions 322 to 345 and from 664 to 687. Bacteria and food samples were treated at 100°C for 10 min in 1% Tween 20 containing 5% NaCl and 1 mM EDTA, then used as templates for polymerase chain reaction (PCR). The oligonucleotide primers were specific to E. coli, except for Shigella species, when tested with 67 strains of E. coli, including such serotypes as O157:H7 and O111, and 32 strains of non-E. coli species. The oligonucleotide primers could prove useful for detecting E. coli in beef, chicken, pork, tomato, soybean, potato, cow's milk, and egg.