实时荧光定量PCR法鉴定发酵乳中嗜热链球菌

该研究通过系统发育树分析确定目标基因,根据目的基因设计特异性引物和探针,建立一种能够快速准确鉴定发酵乳中嗜热链球菌的实时荧光定量聚合酶链式反应（RT-fqPCR）法,通过特异性、灵敏性和抗干扰实验对所建立方法进行验证,并使用该方法对市售的60份标识含有嗜热链球菌的发酵乳样品进行检测。结果表明,recA基因具有种间特异性,种间差异率＞10%,以其为目的基因建立的RT-fqPCR方法能够特异性的检测嗜热链球菌;绝对灵敏度达1 pg/μL,相对灵敏度达103 CFU/mL;在培养物水平和基因组水平抗干扰能力良好。采用该方法从60份标识含有嗜热链球菌的发酵乳样品中均能检测出嗜热链球菌,说明实时荧光定量PCR方法能够快速、准确的对发酵乳中嗜热链球菌进行检测。     

基于微滴数字PCR技术的婴幼儿配方乳粉中双歧杆菌定量检测及应用

应用微滴式数字聚合酶链式反应（droplet digital polymerase chain reaction，ddPCR）技术，建立婴幼儿配方乳粉中双歧杆菌定量检测方法。以双歧杆菌的单拷贝特异性基因rpsL为目标基因设计引物探针，对ddPCR条件进行优化，考察方法的特异性、灵敏度和重复性，并与平板计数方法进行对照验证。结果表明，建立的方法具有良好的特异性、灵敏性和重复性。细菌纯培养液的检出限为296 CFU/mL，模拟样品检出限为7 300 CFU/g，不与10 种近缘乳酸菌发生交叉反应，且重复性较好，采用已建立的ddPCR方法和平板计数方法对市售婴幼儿配方乳粉样品进行检测，2 种方法测定值结果偏差小于10%，结果一致性较好。本研究建立的ddPCR方法对婴幼儿配方乳粉中的双歧杆菌定量检测能够更快速、灵敏、准确，具有一定的应用前景。     

实时荧光PCR法鉴定食品中双歧杆菌

根据双歧杆菌属16S rRNA基因的保守区序设计特异性引物和探针,建立一种鉴定食品中双歧杆菌属的实时荧光聚合酶链式反应（polymerase chain reaction,PCR）检测方法。对方法的特异性、灵敏度、重复性和体系抗干扰能力进行验证,最后采用该方法对市售25 份标示含双歧杆菌样品进行检测。结果表明：该检测方法可特异性检测双歧杆菌属细菌,对近缘的乳杆菌属、链球菌属及食品中常见菌群包括大肠杆菌、金黄色葡萄球菌、沙门氏菌等均无扩增。双歧杆菌DNA检测绝对灵敏度可达到2 pg,相对灵敏度可达到104 CFU/mL。重复性测试表明相对标准偏差小于1%。同时进行了杂菌干扰检测实验,在培养物水平和纯基因组DNA水平上将青春双歧杆菌ATCC15703与大肠杆菌ATCC25922混合进行检测,检出Ct值较纯菌检测时无显著影响,表明建立的荧光PCR方法抗干扰能力良好。对25 份市售实际样品进行测试,有5 份标识含有“双歧杆菌”的样品未检测出双歧杆菌成分。本研究所建立的实时荧光PCR法能准确、快速检测食品中双歧杆菌属细菌。     

实时荧光PCR法鉴定食品中双歧杆菌

根据双歧杆菌属16S rRNA基因的保守区序设计特异性引物和探针,建立一种鉴定食品中双歧杆菌属的实时荧光聚合酶链式反应(polymerase chain reaction,PCR)检测方法。对方法的特异性、灵敏度、重复性和体系抗干扰能力进行验证,最后采用该方法对市售25份标示含双歧杆菌样品进行检测。结果表明:该检测方法可特异性检测双歧杆菌属细菌,对近缘的乳杆菌属、链球菌属及食品中常见菌群包括大肠杆菌、金黄色葡萄球菌、沙门氏菌等均无扩增。双歧杆菌DNA检测绝对灵敏度可达到2 pg,相对灵敏度可达到10~4 CFU/m L。重复性测试表明相对标准偏差小于1%。同时进行了杂菌干扰检测实验,在培养物水平和纯基因组DNA水平上将青春双歧杆菌ATCC15703与大肠杆菌ATCC25922混合进行检测,检出Ct值较纯菌检测时无显著影响,表明建立的荧光PCR方法抗干扰能力良好。对25份市售实际样品进行测试,有5份标识含有“双歧杆菌”的样品未检测出双歧杆菌成分。本研究所建立的实时荧光PCR法能准确、快速检测食品中双歧杆菌属细菌。     

利用Real-time PCR技术分离鉴定酸乳中的乳双歧杆菌(Bifidobacterium Animalis Subsp.Lactis)

目的:利用Real-time PCR建立酸乳中乳双歧杆菌的快速精准鉴定方法。方法:选取乳双歧杆菌atpD基因,利用Primer Express 3.0.1设计引物探针,进而验证引物探针的特异性、灵敏度和抗干扰能力,最后应用到17种酸乳样品的检测。结果:所设计的引物探针可以有效地扩增出乳双歧杆菌,而对于20种乳酸菌和18种常见的致病菌都不能扩增,具有良好的特异性;其灵敏度在DNA水平可以达到1 pg/μL,在菌浓度水平可以达到10³ cfu/mL;并且不受酸乳中的嗜热链球菌和保加利亚乳杆菌的干扰,具有很好的抗干扰能力。利用RT-PCR技术能很好地鉴定17种酸乳中乳双歧杆菌。结论:该研究建立了酸乳中快速精准鉴定乳双歧杆菌的方法,对食品中双歧杆菌鉴定起到积极推动作用。     

婴幼儿乳粉内混合益生菌检测方法的研究

通过对比分析动物双歧杆菌和鼠李糖乳杆菌对万古霉素和莫匹罗星锂盐的抗性研究,确定了乳粉内含有动物双歧杆菌和鼠李糖乳杆菌的检测方法。结果表明:对万古霉素而言,鼠李糖乳杆菌具有较高的耐性,动物双歧杆菌具有极低的耐受性;对莫匹罗星锂盐而言,鼠李糖乳杆菌耐受性低,动物双歧杆菌耐受性高。在遇到样品中既含有动物双歧杆菌,又含有鼠李糖乳杆菌,需要分别对两种菌单独计数时,抗生素抑制法检测结果比不同需氧条件法和GB/T4789.35-2010检测法结果更准确,更能反应样品中动物双歧杆菌和鼠李糖乳杆菌的实际数量。     

基于环介导等温扩增技术快速检测阪崎肠杆菌

为实现婴幼儿配方乳粉中阪崎肠杆菌快速检测,建立等温荧光扩增方法特异性检测阪崎肠杆菌。针对阪崎肠杆菌特异性保守ompA基因设计3套引物,通过引物筛选和反应体系优化,建立阪崎肠杆菌快速检测的实时荧光环介导等温扩增(Loop-mediated isothermal amplification,LAMP)方法。以人工污染方式检验该方法在乳粉中阪崎肠杆菌检测灵敏度和稳定检测限,并对比实时荧光LAMP法和国标法在48份市售婴幼儿配方乳粉中的实际检测效果。结果显示,筛选ompA-2引物扩增效率最优,且与常见食源性致病菌无交叉反应;建立的实时荧光LAMP法对人工污染乳粉样品中的阪崎肠杆菌检测灵敏度、稳定检测限分别为10~2 cfu/g和10~3 cfu/g;48份市售乳粉样品进行增菌12 h,实时荧光LAMP法检测结果与传统国标培养结果一致。建立的实时荧光LAMP方法将为阪崎肠杆菌快速检测提供有力手段。     

婴儿配方乳粉中阪崎克罗诺杆菌解旋酶恒温基因扩增检测方法的建立

目的：建立一种检测婴儿配方乳粉中阪崎克罗诺杆菌的解旋酶恒温基因扩增方法。方法：根据阪崎克罗诺杆菌ITS基因设计特异性引物,优化解旋酶恒温基因扩增法反应条件UvrD helicase、T4 gp32的浓度,人工添加阪崎克罗诺杆菌确定检出限,多种致病菌在建立的解旋酶恒温基因扩增体系中扩增验证特异性,电泳检测扩增产物。结果：解旋酶恒温基因扩增法检测婴儿配方乳粉中阪崎克罗诺杆菌得到与设计序列长度一致的100 bp基因片段,检出限为10 CFU/g,优化反应条件UvrD helicase、T4 gp32的终浓度分别为0.1、5.0 μg。结论：解旋酶恒温基因扩增法用于检测婴儿配方乳粉中阪崎克罗诺杆菌的特异性强、灵敏度高、耗时短,为婴儿配方乳粉中阪崎克罗诺杆菌的快速检测提供了新的方法。     

添加扩增内标的PCR方法快速检测食品中的阪崎克罗诺杆菌

为了解决常规PCR方法检测阪崎克罗诺杆菌时因检测过程中环境和食品理化因素的影响而导致的假阴性结果的产生,本研究以细菌16S rRNA基因为扩增内标对照,以阪崎克罗诺杆菌特异性基因grx B为靶基因设计了一对特异性引物,并通过优化PCR反应条件,最终建立了一种添加扩增内标的阪崎克罗诺杆菌PCR检测方法,可以指示PCR过程中因存在DNA聚合酶抑制剂而导致的假阴性结果。通过对20种细菌进行PCR检测显示,该方法对阪崎克罗诺杆菌具有良好的特异性。灵敏度实验结果表明,该检测方法对阪崎克罗诺菌纯DNA模板的检测灵敏度为2.15×102fg/μL,对阪崎克罗诺杆菌纯培养物的检测灵敏度为9.4×103CFU/m L。对人工污染婴幼儿奶粉的检测结果显示,阪崎克罗诺杆菌接种量为0.94 CFU/g的婴幼儿奶粉样品经过8 h增菌培养后,即可检出。食品样品检测结果表明,不添加扩增内标的PCR检测方法中出现的假阴性结果可被本方法检出。该检测方法特异性强、灵敏度较高,能消除阪崎克罗诺杆菌常规PCR检测方法中可能出现的假阴性结果,适用于婴幼儿配方乳粉、乳制品等食品中阪崎克罗诺杆菌的快速检测。     

基于荧光定量PCR法鉴定婴幼儿配方羊奶粉中牛源性成分

婴幼儿配方乳粉的配料表中必须如实标明使用的乳制品原料的动物性来源。建立了以线粒体DNA12SrRNA为目标基因,用荧光定量PCR的方法快速鉴定婴幼儿配方羊奶粉中牛源性成分的方法,并采用此方法检测了20个婴幼儿配方羊奶粉。其中2个样品未检出牛源性成分,18个样品检出牛源性成分。此方法可用于婴幼儿配方乳粉标签标识的日常监管。     

Rapid discrimination of Bifidobacterium animalis subspecies by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Currently, the species Bifidobacterium animalis consists of two subspecies, B. animalis subsp. lactis and B. animalis subsp. animalis. Among these two subspecies, B. animalis subsp. lactis is especially important because it is widely used in the manufacture of probiotic dairy products. The application of these microbes in the food industry demands fast, accurate and low cost methods to differentiate between species and strains. Although various genotypic methods have been employed to discriminate between these two subspecies, they are not easily adapted for rapid identification in the industry. The purpose of this study was to evaluate the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to differentiate between the two subspecies of B. animalis, and for discrimination at strain level. We identified twenty-three strains of B. animalis at subspecies and strain level by genotypic methods and by proteomics using MALDI-TOF MS. The proteomics identification by MALDI-TOF was nearly identical to that obtained by genotypic identification using comparison of tuf and atpD gene sequences, and single-nucleotide polymorphisms (SNPs), insertions, and deletions (INDELs). We identified four protein markers, L1, L2, A1, and A2, which are useful for discriminating between both subspecies. Proteomics identification using MALDI-TOF MS was therefore an accurate method for discriminating and identifying these bacteria. Given the speed in which this method is achieved (~20 min including sample preparation), MALDI-TOF MS is promising as a tool for rapid discrimination of starter cultures and probiotics.     

Use of tuf gene-based primers for the PCR detection of probiotic Bifidobacterium species and enumeration of bifidobacteria in fermented milk by cultural and quantitative real-time PCR methods

Due to the increasing use of bifidobacteria in probiotic products, it is essential to establish a rapid method for the qualitative and quantitative assay of the bifidobacteria in commercial products. In this study, partial sequences of the tuf gene for 18 Bifidobacterium strains belonging to 14 species were determined. Alignment of these sequences showed that the similarities among these Bifidobacterium species were 82.24% to 99.72%. Based on these tuf gene sequences, 6 primer sets were designed for the polymerase chain reaction (PCR) assay of B. animalis subsp. animalis, B. animalis subsp. lactis, B. bifidum, B. breve, B. longum subsp. infantis, B. longum subsp. longum, and the genus of Bifidobacterium, respectively. These Bifidobacterium species are common probiotic species present in dairy and probiotic products. When each target Bifidobacterium spp. was assayed with the designed primers, PCR product with expected size was generated. In addition, for each target species, more than 70 bacterial strains other than the target species, including strains of other Bifidobacterium species, strains of Lactobacillus spp., Enterococcus spp., and other bacterial species, all generated negative results. PCR assay with primers specific to B. animalis subsp. lactis and B. longum subsp. longum confirmed the presence of these Bifidobacterium species in commercial yogurt products. In addition, for each product, enumeration of the bifidobacteria cells by culture method with BIM-25 agar and the quantitative real-time PCR showed similar cell counts. Such results indicated that within 15-d storage (4 °C) after manufacture, all the bifidobacteria cells originally present in yogurt products were viable and culturable during the storage.     

The viability of three probiotic organisms grown with yoghurt starter cultures during storage for 21 days at 4°C

This study investigated the viability of probiotic ( Lactobacillus acidophilus LA5, Lactobacillus rhamnosus LBA and Bifidobacterium animalis subsp . lactis BL-04) in milk fermented with Lactobacillus delbrueckii subsp . bulgaricus LB340 and Streptococcus thermophilus TAO (yoghurt – Y). Each probiotic strain was grown separately in co-culture with Y and in blends of different combinations. Blends affected fermentation time(s), pH and firmness during storage at 4°C. The product made with Y plus B. animalis subsp . lactis and L. rhamnosus had counts of viable cells at the end of shelf life that met the minimum required to achieve probiotic effect. However, L. acidophilus and L. delbrueckii subsp . bulgaricus were inhibited.     

Antibiotic resistance of lactic acid bacteria and Bifidobacterium spp. isolated from dairy and pharmaceutical products

The outlines of antibiotic resistance of some probiotic microorganisms were studied. This study was conducted with the double purpose of verifying their ability to survive if they are taken simultaneously with an antibiotic therapy and to increase the selective properties of suitable media for the isolation of samples containing mixed bacterial populations. We isolated from commercial dairy and pharmaceutical products, 34 strains declared as probiotics, belonging to the genera Bifidobacterium and Lactobacillus, and 21 strains of starter culture bacteria. All the microorganisms have been compared by electrophoresis of the soluble proteins for the purpose of identifying them. A Multiplex-PCR with genus- and species-specific primers was used to detect for Bifidobacterium animalis subsp. lactis presence. All bifidobacteria were B. animalis subsp. lactis except one Bifidobacterium longum. Sometimes the identification showed that the used strain was not the one indicated on the label. The lactobacilli were Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus delbrueckii subsp. bulgaricus. The streptococci were all Streptococcus thermophilus. The minimal inhibitory concentration (MIC) of 24 common antibiotic substances has been valued by the broth microdilution method. All tested strains were susceptible to ampicillin, bacitracin, clindamycin, dicloxacillin, erytromycin, novobiocin, penicillin G, rifampicin (MIC(90) ranging from 0.01 to 4 microg/ml); resistant to aztreonam, cycloserin, kanamycin, nalidixic acid, polymyxin B and spectinomycin (MIC(90) ranging from 64 to >1000 microg/ml). The susceptibility to cephalothin, chloramphenicol, gentamicin, lincomycin, metronidazole, neomycin, paromomycin, streptomycin, tetracycline and vancomycin was variable and depending on the species.     

Varying influence of the autolysin, N-acetyl muramidase, and the cell envelope proteinase on the rate of autolysis of six commercial Lactococcus lactis cheese starter bacteria grown in milk

The autolysin, N-acetyl muramidase (AcmA), of six commercial Lactococcus lactis subsp. cremoris starter strains and eight Lc. lactis subsp. cremoris derivatives or plasmid-free strains was shown by renaturing SDS-PAGE (zymogram analysis) to be degraded by the cell envelope proteinase (lactocepin; EC 3.4.21.96) after growth of strains in milk at 30 degrees C for 72 h. Degradation of AcmA was less in starter strains and derivatives producing lactocepin I/III (intermediate specificity) than in strains producing lactocepin I. This supports previous observations on AcmA degradation in derivatives of the laboratory strain Lc. lactis subsp. cremoris MG1363 (Buist et al. Journal of Bacteriology 180 5947-5953 1998). In contrast to the MG1363 derivatives, however, the extent of autolysis in milk of the commercial Lc. lactis subsp. cremoris starter strains in this study did not always correlate with lactocepin specificity and AcmA degradation. The distribution of autolysins within the cell envelope of Lc. lactis subsp. cremoris starter strains and derivatives harvested during growth in milk was compared by zymogram analysis. AcmA was found associated with cell membranes as well as cell walls and some cleavage of AcmA occurred independently of lactocepin activity. An AcmA product intermediate in size between precursor (46 kDa) and mature (41 kDa) forms of AcmA was clearly visible on zymograms, even in the absence of lactocepin I activity. These results show that autolysis of commercial Lc. lactis subsp. cremoris starter strains is not primarily determined by AcmA activity in relation to lactocepin specificity and that proteolytic cleavage of AcmA in vivo is not fully defined.     

双向电泳技术鉴别牛羊乳品真实性

采用双向电泳技术对牛羊乳品的蛋白质指纹进行分析。首先通过全乳图谱分析牛羊乳的差异蛋白,然后针对婴幼儿配方粉、乳清粉等开展方法特异性研究,通过婴幼儿配方粉添加实验开展方法灵敏度研究,最后对市售配方粉、液态乳、酸乳等进行检测。总体上,双向电泳技术信息丰度高,信息直观,重复性良好,能准确分辨牛羊乳及乳清,灵敏度达到5%。通过检测,8 份市售样品中有1 份酸羊乳实际为牛乳,图谱蛋白指纹信息清晰,直观反映产品真实性。     

双蛋白益生菌发酵乳的研制与品质分析

为丰富发酵乳的品类并满足消费者的多样化需求,本研究以一定比例的纯牛乳和花生蛋白粉为原料,添加益生菌制作了一种双蛋白益生菌发酵乳。将纯牛乳与不同比例的花生蛋白混合,巴氏灭菌后,将混合物与发酵物一起发酵（嗜热链球菌S10:动物双歧杆菌乳亚种菌株V9:干酪乳杆菌Zhang=10:10:1）,从感官特性、酸度、粘度、持水力及益生菌活菌数等角度,评价了贮藏期内28 d实验组及对照组发酵乳的品质及贮藏特性。结果表明,花生蛋白:牛乳蛋白=3:7时,发酵乳酸甜适口、色香兼具、组织细腻且凝聚性好,感官评分为（94±3）分,发酵时间5.4 h。经4℃下贮藏28 d后,3:7实验组的后酸化程度与对照组相比较弱,内聚性及黏度指数均高于两个对照组（分别为-20.24±1.77 g、-20.63±2.79 g·s）。与此同时,3:7实验组的粘度最高可达（1085±100）m Pa·s,持水力可平均稳定在75%左右,发酵乳中的干酪乳酪杆菌Zhang及动物双歧杆菌乳亚种V9的活菌数均可保持在1×107 CFU/mL以上。因此,通过花生蛋白替代部分牛乳蛋白来制作发酵乳的工艺是可行的,产品在28 d贮藏期内能够保持良好的...     

牛β-乳球蛋白胶体金免疫层析试纸条的研制及检测应用

研制了一种可快速检测羊乳及制品中牛源β-乳球蛋白(β-lactoglobulin,β-lg)的胶体金免疫层析试纸条。通过杂交瘤技术制备牛β-lg特异性单克隆抗体(monoclonal antibody,mAb),利用柠檬酸钠还原氯金酸,形成30 nm胶体金颗粒,并用于标记牛β-lgmAb。采用竞争法研制免疫层析试纸条,将胶体金标记的牛β-lgmAb包被于金标垫,牛β-lg和羊抗鼠IgG标记于硝酸纤维素膜分别作为检测线(T线)和质控线(C线),牛β-lg和二抗的最佳包被浓度均为1.0 mg/mL。制得的单克隆抗体纯度都在90%以上,效价均在10000以上且特异性较好。该试纸条对牛β-lg的检测限(limit of detection,LOD)值为3.13μg/mL,对牛源α-CN,牛源β-CN,牛源κ-CN,牛源α-LA,BSA均未产生交叉反应,对脱脂羊乳粉中掺杂脱脂牛奶粉的LOD值为5%,并用该方法对市售羊奶及配方羊奶粉进行分析,检测结果与商品化的ELISA试剂盒一致。该方法前处理快速简单,可在5 min内对牛β-lg进行检测,可用于羊乳制品的现场快速检测。