乳和乳制品中嗜冷菌及其耐热酶的危害与控制研究

嗜冷菌是乳低温贮藏中的主要腐败菌,嗜冷菌及其代谢酶的存在直接影响原料乳的质量,阐述了嗜冷菌及其耐热酶类对乳及乳制品的危害,并通过分析嗜冷菌的适冷机理和影响酶活的因素,探讨控制嗜冷菌及其代谢酶的方法,以保障原料乳质量。     

原料乳中嗜冷菌快速检测新技术研究进展

嗜冷菌在原料乳低温储藏过程中能大量繁殖,其生长过程中产生的脂肪酶和蛋白酶对乳制品后期储藏的风味品质有很大影响。因此,针对原料乳中嗜冷菌的检测,国内外开展了大量的研究,其中快速、自动化的现代技术蓬勃发展。本文在整理这些主要技术的基础上,重点介绍了近些年来发展较快且灵敏度高的嗜冷菌快速检测新技术及其优缺点,并对未来该领域快速检测技术的发展做出展望。     

乳及乳制品中嗜冷菌的多样性及其腐败危害研究进展

乳是一种营养丰富的物质,同时也是微生物生长繁殖的理想培养基。生乳的质量是影响乳制品产业链的关键因素,随着低温储存和冷链运输技术的发展,生乳中大部分细菌的生长受到抑制,但嗜冷菌的生长并未受到抑制,并逐步成为生乳中的优势菌。生乳在冷藏运输或储存期间,嗜冷菌依旧可以生长繁殖,其分泌的蛋白酶和脂肪酶可耐高温,经过巴氏杀菌或超高温灭菌处理后依旧保持活性,因此,了解嗜冷菌的多样性及其产生的酶对提高乳及乳制品质量、减少腐败和浪费具有重要作用。本文通过介绍乳及乳制品中嗜冷菌污染现状及腐败危害研究,旨在为乳及乳制品行业的风险评估提供背景信息,从源头控制嗜冷菌对生乳的浪费,保证乳及乳制品的品质。     

利用竞争酶联免疫吸附法测定牛血清白蛋白鉴别掺假牛乳

利用竞争酶联免疫吸附法(cELISA)定量分析牛乳中的牛血清白蛋白,以建立一种检测掺假牛乳的通用方法。通过对添加不同成分掺假牛乳样品的检测,分析了竞争酶联免疫吸附法测定牛血清白蛋白的特异性;通过对稀释倍数不同的掺假牛乳样品进行测定,验证了竞争酶联免疫吸附法测定牛血清白蛋白的可行性。结果表明:该法在测定掺假牛乳时不受添加物的影响,只与牛乳稀释程度有关,因而该法用于掺假牛乳的检测具有广泛适用性。     

免疫分析技术在乳及乳制品抗生素类兽药残留检测中的应用

为了预防与治疗乳牛疾病,抗生素类兽药被广泛应用于乳牛的饲养过程中。而滥用抗生素导致牛奶中抗生素残留超出最大允许残留量,不仅会对牛奶品质造成影响,而且可能会危害到牛奶饮用者的身体健康。因此,如何快速、准确地检测乳与乳制品中抗生素类兽药残留是乳品检测行业和企业亟需解决的问题。本文主要介绍了牛乳中兽药残留的来源、危害及其现状,阐述了酶联免疫分析技术、胶体金免疫层析技术的检测原理及其在应用过程中所具有的不同特点,最后对2种方法的优势与不足进行简要总结,并对免疫分析法在乳与乳制品抗生素类兽药残留检测中的应用前景做出展望。     

嗜冷菌对长货架期乳制品的影响

<正> 随着加工规模的不断扩大,生奶从收集到加工往往需要存放一至数天,为了防止生奶的变质而广泛采用了冷藏方法,利用冷藏方法保存生奶导致生奶中嗜冷菌占主导地位,成为影响产品质量的重要危害因素。 牛乳及乳制品中的主要嗜冷菌 国际乳业联合会提出,凡是在7℃以下能生长的细菌即称为低温菌,而在20℃以下能繁殖的细菌叫嗜冷菌,牛乳中的低温菌有:假单胞菌属,无色杆菌属,黄杆菌属,产碱杆菌属和一部分大肠菌群,此外,一部分乳酸菌,微球菌,酵母菌和霉     

牛奶中的耐热酶

牛奶大多采用低温贮藏,但嗜冷菌仍会生长繁殖。虽然利用巴氏消毒和超高温瞬时灭菌等方法可以有效抑杀嗜冷菌,但其衍生出来的耐热酶会仍然存在,还会影响牛奶活性。针对那些无法实现无菌操作的奶源国家和地区,嗜冷菌的影响就会更加明显,加速牛奶的凝固、变苦、变质。笔者主要梳理目前有关耐热酶的研究,并对未来发展进行展望。     

牛乳过敏原β-乳球蛋白检测方法的研究进展

目的食物过敏已成为全球性的食品安全问题,欧美等发达国家均要求对食品中的过敏原成分进行标识,其中就包括作为八大过敏性食物之一的牛乳及其乳制品。β-乳球蛋白是牛乳中的主要过敏原,约占乳清蛋白的50%,牛乳总蛋白的10%,并且约有82%的牛乳过敏患者对β-乳球蛋白过敏,因此其可以作为检测食品中是否含牛乳蛋白的一个有效的标志物。建立灵敏、可靠的β-乳球蛋白检测方法,对牛乳过敏原标识及保障牛乳过敏人群的安全消费具有重要意义。本文主要综述了牛乳β-乳球蛋白的高效液相色谱法、超高效液相色谱法、液相色谱-质谱联用法、酶联免疫吸附法、免疫层析法、电化学免疫法、蛋白微阵列法、等离子体共振法等色谱学和免疫学检测方法的研究进展,并对未来发展方向作出展望,以期促进牛乳β-乳球蛋白等过敏原检测方法的研究与开发。     

免疫学技术在食品安全快速检测中的应用研究进展

随着人们对于食品安全问题的关注程度不断增加,食品安全快速检测方法得到了广泛应用。目前常用的食品安全快速检测技术包括免疫学技术、酶抑制技术、传感器技术、生物芯片技术等。免疫学检测技术具有灵敏度高、特异性强、方便、快速和经济等优点,在食品安全快速检测中发挥了重要的作用。免疫学技术在食品安全领域广泛应用的主要有免疫吸附法和免疫层析法两大体系,其中免疫吸附法以酶联免疫吸附检测法(enzyme-linked immunosorbent assay,ELISA)最常用,而免疫层析法则以胶体金免疫层析技术(colloidal gold immunochromatographic assay,GICA)为代表。本文介绍了免疫学技术在食品安全快速检测中应用的原理及特点,并对酶联免疫吸附检测技术和胶体金免疫层析技术近几年来在食品安全快速检测中的应用进展进行了综述。     

乳制品中嗜热链球菌的快速检测

利用血清学方法检测乳制品中的嗜热链球菌。首先，通过动物免疫方法制备了小鼠及家兔抗嗜热链球菌(Streptococcus thermophilus)的免疫血清。然后，用此血清经酶联免疫吸附测定法(ELISA法)对乳制品中嗜热链球菌进行检测。用间接ELISA法及双夹心ELISA法对乳制品中的嗜热链球菌进行检测。结果表明，两种方法均可测出乳制品中嗜热链球菌，其最低检测量可达10^7mE^-1。这一结果为乳制品中嗜热链球菌的血清学快速检测提供了参考。     

微生物法在原料奶验收中的应用

对微生物法的应用进行了研究。采用对抗生素最敏感的嗜热链球菌为实验菌，从产酸能力和凝乳时间两个因素来检测牛乳中抗生素的残留，并定量得出该嗜热链球菌对青霉素类抗生素的最低检出量为0.04单位。结果表明，该方法操作简便。     

微生物法在原料奶验收中的应用

对微生物法的应用进行了研究。采用对抗生素最敏感的嗜热链球菌,从产酸能力和凝乳时间两个因素来检测牛乳中抗生素的残留,并定量得出该嗜热链球菌的最低检出量为0．04单位。结果表明,该方法操作简便。     

原料奶嗜冷菌快速检测方法研究进展

嗜冷菌作为冷藏原料奶中生长的优势菌群,在原料奶贮存过程中会大量繁殖,并产生及其耐热的脂肪酶和蛋白酶,这些酶类在高温处理后仍会有残留,并在奶制品储藏过程中继续分解其中的脂肪和蛋白质,导致产品的风味和质地产生变化。因此,研发出一种即快速又实用的嗜冷菌计数技术成为乳品行业关注的焦点。本文主要介绍了国内外对原料奶中嗜冷菌快速检测的一些方法,并将各个方法在工业应用上的优缺点进行了比较。     

An inter-laboratory ring trial for the detection and isolation of Mycobacterium avium subsp. paratuberculosis from raw milk artificially contaminated with naturally infected faeces

There is a need for standardised, robust, reproducible molecular and culture methods to achieve clarification of the inactivation of Mycobacterium avium subsp. paratuberculosis (Map), the causative microbial agent of Johne's disease, in (faecally) contaminated milk and other food products such as meat. This study assessed the performance of a commercially available Map DNA extraction kit for milk Adiapure and accompanying PCR detection kit Adiavet alongside 'in-house' molecular and culture methods in an inter-laboratory ring trial using raw milk spiked with Map-infected faeces. The combined Adiapure-Adiavet Map DNA extraction and detection kit consistently detected 30 copies of IS900 (equivalent to approximately 2 cells) ml(-1) raw milk, when used in four different laboratories. Improvements in sensitivity and ease of use for 'in-house' Map detection were observed when the Adiapure extraction kit was combined with 'in-house' detection assays. Detection by real-time PCR methods, using the commercial extraction and detection systems, resulted in an overall detection rate of 100%, 90%, 85% and 25% for respective Map concentrations of 300, 30, 3 and 0.3 copies of IS900ml(-1) raw milk. Map, at 300 copies of IS900 (equivalent to approximately 20 Map cells) ml(-1) raw milk, was recovered from all samples cultured in mycobacteria growth indicator tube (MGIT) medium, from 10 of 12 samples on Herrold's egg yolk medium (HEYM) and not recovered from any samples using BACTEC medium. In conclusion, the Adiapure DNA extraction kit allows for sensitive and easy detection of Map in raw milk. The extraction method can form a candidate part of essential methodology and real-time PCR can further increase the sensitivity of the detection method. Moreover, MGIT medium is promising for culture-dependent detection of Map from raw milk.     

Short communication: appropriate and alternative methods to determine viable bacterial counts in cow milk samples

Farm milk consumption is reported to be inversely related to the development of asthma and atopy in children and it has been hypothesized that microorganisms in milk might contribute to this protective effect. The GABRIEL study was designed to investigate this hypothesis in a large population of European children, calling for a rapid alternative to classical culture techniques to determine bacteriological properties of milk samples. One objective was to evaluate 2 different rapid methods to determine bacteriological properties in a large number of cow milk samples collected under field conditions. BactoScan (Foss Analytical, Hillerød, Denmark), an automated standard flow cytometric method utilized for routine testing of milk quality, and TEMPO (bioMérieux, Marcy l’Etoile, France), an automated most-probable-number method, were used to assess the total viable bacterial count in farm and commercial milk samples. Both methods were compared with standard plate count method and each other. Measurements based on the TEMPO method were in good agreement with the standard plate count method and showed reliable results, whereas BactoScan results did not correlate with standard plate count measurements and yielded higher bacteria counts in heat-treated milk samples compared with raw milk samples. Most likely, these discrepant results were due to inferences with staining reactions and detection of bacteria in heat-treated milk samples. We conclude that, in contrast to the routinely used BactoScan method, the TEMPO method is an inexpensive and rapid alternative to standard culture methods suitable to assess total bacterial counts in processed and raw milk samples.     

原料乳中微生物的多样性

牛乳营养丰富,适合各种微生物的生长繁殖。该文介绍了原料乳中微生物的种类,综述了国内外近年来关于原料乳中微生物多样性的研究进展。并对非培养微生物学在研究原料乳中微生物多样性的应用进行了总结,高度评价了非培养方式的快速、敏感以及可定义纯培养方式无法发现的微生物的特点。     

生乳中体细胞快速检测技术的对比研究

目的 建立一种检测生乳中体细胞含量的快速检测方法。方法 利用细胞裂解液及荧光染料,对奶样中的体细胞、稳定脂肪球和蛋白、渗透进入体细胞并沾染DNA,荧光标记液快速渗入体细胞,与DNA结合后荧光量子产率提高,通过荧光信号检测系统检测荧光强度来测定生乳中的体细胞数量。结果 该方法与快速仪器法对比,相关系数达94%以上,两方法在统计学上无显著性差异(P>0.05)。与标准方法对比,针对同一样品的检测结果的Log差值小于0.25,两方法统计学上也无显著性差异(P>0.05)。对不同样品进行重复性检测,相对标准偏差均小于15%。结论 该快速检测技术具有较高的准确度和精密度,可用于生乳中体细胞数的快速定量检测,由于无需大型的检测设备及配套试剂,适合牧场和企业实验室的快速检测。     

Rapid and simple estimation of microbiological quality of raw milk using chromogenic Limulus amoebocyte lysate endpoint assay

A rapid chromogenic Limulus amoebocyte lysate (LAL) endpoint assay for the enumeration of total mesophilic microbial loads and coliforms was investigated as a means to assess the microbiological quality of raw milk. For experiment 1, raw milk samples (n = 25) were stored in a refrigerator (2 +/- 2 degrees C) and then analyzed at regular intervals (1, 5, 10, and 15 days). For experiment 2, fresh raw milk samples (n = 50) were tested to determine the utility of the LAL assay for fresh raw milk. The sample was diluted threefold in a 96-well microtiter plate with pyrogen-free water and assayed with a chromogenic LAL kit to find a final reaction point. The LAL results were compared with standard plate counts (SPC) and coliform counts determined by conventional plating methods. The results of the LAL assay were strongly correlated to conventional SPC (r2 = 0.93; n = 100) and were highly correlated to coliforms (r2 = 0.74; n = 100). A highly significant linear relationship (r2 = 0.82; n = 50) was also observed between the predicted SPC based on the LAL value and the actual SPC. The results of LAL testing were classified into one of seven contamination groups. The data set for SPC was effectively differentiated using the LAL technique (P < 0.01). The chromogenic LAL assay was found to be a rapid (within 16 min) and simple (not requiring specific instruments) method for monitoring microbial levels in raw milk. This method may be successfully implemented to rapidly determine highly microbial contaminated raw milk (> 3.0 log10 CFU/ml of SPC).     

Visual detection of melamine in raw milk using gold nanoparticles as colorimetric probe

We report the development of a simple and rapid colorimetric detection method for melamine in raw milk using gold nanoparticles as probe. This assay relies upon the distance-dependent optical properties of gold nanoparticles. In neutral media, melamine could rapidly induce the aggregation of gold nanoparticles, thereby resulting in red-to-blue (or purple) colour change. The concentration of melamine in raw milk can be determined by monitoring with the naked eye or a UV–vis spectrometer. The present limit of detection for melamine is 0.4 mg/L. The method is rather simple, and the whole process including sample pretreatment takes only 12 min at room temperature. The merits (such as simplicity, rapidity, low cost and visual colorimetry) make the proposed method specially useful for on-site screening melamine levels well below the current safety limit in raw milk.     

生鲜牛乳总微生物基因组DNA的提取

目的 建立高效快速提取生鲜牛乳中总微生物基因组DNA的方法。方法 以生鲜牛乳为原料, 采用SDS-蛋白酶K法裂解细胞, 酚氯仿有机抽提去除蛋白和醋酸钾溶液沉淀蛋白, 制备样品中总微生物基因组DNA。结果 以NET(Tris?HCl, EDTA, NaCl)作为裂解缓冲液, 蛋白酶K消化得到的基因组DNA纯度和产量较高, 耗时较短; 缓冲液选择NCT(NaCl, CaCl2, Tris?HCl)时, PCR产物特异性低于前者且产量较低。RNA酶消化对产品纯度影响不大且会降低产量。用醋酸钾(KAc)沉淀去除蛋白, 操作快速简单, 耗时最短, 但DNA产量最低。结论 SDS-蛋白酶K法提取的生鲜乳微生物总DNA可以作为牛乳样品进一步检测的分子基础。