基于图像计数酿酒酵母的统计分析

图像计数可快速定量微生物的活细胞浓度，但对其统计参数及定量结果的可靠性研究较少。该研究以酿酒酵母为材料，研究了自行设计的微型计数板图像计数方法中异常数、中位数、平均数、图像间菌体数量的相对标准偏差等统计量与计数图像数量的关系，比较了图像计数和GB 4789.15—2016平板计数定量的相对标准不确定度，以及结果的差异性和相关性等。结果表明，酿酒酵母10⁵～10⁷ CFU/mL时，图像计数可使用中位数、平均数统计活菌数量；10⁵ CFU/mL时需计数50个图像，10⁶～10⁷ CFU/mL时计数13个图像可准确获得酿酒酵母浓度。图像计数的相对标准不确定度(0.022～0.100)小于平板计数法(0.099)。在10⁵～10⁷ CFU/mL时，图像计数与平板计数对酿酒酵母定量的结果一致。     

TTC在乳及乳制品菌落总数检测中的应用

检测乳及乳制品菌落总数,为达到菌落在培养基中清晰可辨,在平板计数琼脂培养基中添加2,3,5-三苯基氯化四氮唑(TTC)。以灭菌乳、发酵乳、冰淇淋为样品基质,分别加入适当菌含量的大肠埃希氏菌、金黄色葡萄球菌、粪肠球菌、枯草芽胞杆菌工作菌悬液,制备适当浓度的TTC平板计数琼脂,同时以不添加TTC的平板计数琼脂作为对照,对同一样品进行菌落计数并同国标方法结果进行对比。TTC平板计数琼脂终浓度在0.001%~0.002%时能够使菌落显色,且与不添加TTC的对照组对比结果一致。在检测成分复杂的乳及乳制品样品时添加适宜浓度的TTC,有助于菌落的辨认,能够提高检验人员菌落计数的准确性,可用于检测乳及乳制品中菌落总数。     

发酵乳制品中乳酸菌的流式检测方案探索与研究

目的 建立基于流式细胞术的发酵乳制品中乳酸菌快速计数方法, 实现发酵乳制品中乳酸菌的快速定量检测。方法 使用5(6)-羧基荧光素二乙酸酯（5-(and 6)-Carboxyfluorescein diacetate,cFDA） 和碘化丙啶（Propidium iodid,PI）2种荧光染料对乳酸菌进行染色,经流式细胞仪检测乳酸菌的荧光信号,从而实现细菌定量计数。在分析过程中对染色剂浓度、荧光通道阈值进行优化,并将流式计数结果与现行国标方法进行对比研究。结果 在样品浓度为104 CFU/mL(g)时,通过0.05 μg/mL的PI和10 μmol/L的cFDA染色,在绿色荧光通道增益为100,红色荧光通道增益达到1000时,能够达到最佳的流式检测条件,且样品检测结果与现行国标检测方法存在显著的正相关关系（P<0.01）,2种方法测试结果相关系数值为0.949。结论 流式细胞术具有灵敏度高、检测时间短、稳定性高、重现性好等优点,对于货架期普遍较短,放行压力大的乳制品生产企业来说,能够缩短检测时间,实现产品快速放行上架。     

荧光图像分析法进行酿酒酵母生长过程活力分析

应用荧光标记结合显微图像分析技术检测酿酒酵母摇床培养过程中活力的变化规律,分析酿酒酵母在生长过程中不同时期的活力特点及其与活性关系.结果表明,本技术不但可获得酿酒酵母生长的活力水平的变化趋势,而且还可获得酿酒酵母分裂周期中蛋白质的分布变化情况,并通过对酿酒酵母的连续动态观察而获得酵母分裂时相的信息.说明图像分析法能够替代流式细胞仪作为酿酒酵母活力评价的重要手段.     

酪蛋白胶束结构及其对牛乳稳定性的影响

论述了酪蛋白的组成及两类具有代表性的酪蛋白胶束模型—亚胶束模型和内部结构模型,这两种模型可以较好地解释酪蛋白胶束的稳定性。pH值、酶、添加成分、工艺过程等能够影响酪蛋白胶束的稳定性,并直接影响到牛乳的稳定。这一方面有利于乳制品的生产如制作酸奶、干酪,一方面影响乳制品的品质。     

应用流式细胞技术快速定量检测UHT奶产品中的微生物

采用基于单个活细胞的新型荧光标记技术,精确区分UHT奶样品中的活菌细胞与死细胞以及其他大颗粒物质,并应用流式细胞技术(flow cytometry,FCM)对UHT奶产品进行微生物快速定量检测。通过与传统的平板计数检测方法进行比对,结果表明,FCM方法的检测范围为101~107CFU/mL,远远高于平板计数法。2种方法的计数结果相关性分析表明,在一定菌液浓度范围内,FCM方法的定量结果与平板计数法线性相关良好,且对不同类型菌种都能精确标记定量,是更为快速、准确的检测方法。     

计数标准微球分散及浓度对酿酒酵母计数的比较

针对计数标准微球在微生物计数中的微球聚集、使用浓度及统计数量进行分析比较。以计数标准微球和酿酒酵母ATCC9080为实验对象,研究了Tween 20、Tween 80对计数标准微球的分散效果以及标准微球浓度、统计数量对酿酒酵母计数结果的影响,并将标准微球计数结果与平板计数结果进行比较。实验表明计数标准微球在0.08% Tween 20或0.06% Tween 80中的分散效果适宜,可使分散指数为3.30。对每毫升105、106、107个酿酒酵母悬液进行计数时,统计的标准微球总数依次应不小于2 000、1 500、1 000个,统计的细胞总数分别应不小于200、400、2 600个。在每毫升105~107个酿酒酵母浓度范围,以每毫升106个标准微球最适宜,且对酿酒酵母的计数结果与平板计数结果呈线性关系（R2=0.996 9）。结果表明Tween 20或Tween 80可有效分散计数标准微球,采用分散的、适宜浓度的计数标准微球可对微生物悬液进行准确计数,明确了计数标准微球用于微生物计数的适宜参数,补充 GB/T 39730-2020标准微球使用条件。     

荧光微球免疫层析法定量检测牛乳中酪蛋白

目的研究用荧光微球免疫层析法定量检测牛乳中的酪蛋白。方法本文以酪蛋白为抗原,免疫新西兰大白兔制备抗酪蛋白的多克隆抗体,将纯化后的多克隆抗体通过EDC介导法与荧光微球进行偶联,将滤纸、样品垫、结合垫、NC膜和吸水纸组装成试纸条,用此荧光微球免疫层析法定量检测牛乳中的酪蛋白。结果试纸条在25 min内就能判定结果,最低检测限为100 ng/mL,该方法与BSA、OVA均无交叉反应,具有很好的特异性。检测酪蛋白浓度为100.0、500.0、1000.0 ng/mL的样品,试纸条的批内回收率分别为(89.03±5.2)%、(93.47±6.9)%和(91.2±7.8)%,批间回收率分别为(87.69±6.2)%、(92.73±8.3)%、(89.82±8.5)%。结论初步建立了一种快速、方便、高灵敏度的荧光微球免疫层析方法用以检测牛乳制品中的过敏原——酪蛋白。     

裂殖壶菌胞内油脂尼罗红荧光染色快速检测方法的研究

为了建立快速检测裂殖壶菌胞内油脂的方法,探讨了尼罗红荧光染色法检测裂殖壶菌油脂含量的检测条件。通过考察裂殖壶菌重悬液中添加二甲基亚砜体积分数、尼罗红染液用量、染色温度、染色时间及细胞密度对荧光强度的影响,确立最优检测条件,进一步考察细胞油脂含量与荧光强度的关系,从而建立尼罗红荧光检测法的定量关系。结果表明,最优检测条件为:每毫升裂殖壶菌重悬液中二甲基亚砜体积分数和尼罗红染液用量分别为25%和15μL,染色温度50℃,染色时间15 min。在最优检测条件下,细胞密度不超过0.218×10~8个/m L范围内,菌液油脂含量(X)与荧光强度(Y)呈现良好的线性关系,其线性关系式为Y=798.55X-3.44,相关系数R2为0.996 4。因此,采用尼罗红荧光染色法可以快速检测裂殖壶菌的油脂含量。     

荧光定量PCR法计数农家干酪中动物双歧杆菌乳酸亚种

分别利用荧光定量PCR及平板菌落计数法计数农家干酪中双歧杆菌的菌体数,通过对结果的比较分析,建立一种适用于快速、敏感、特异的检测干酪中双歧杆菌活菌数的方法。利用传统工艺制备双歧杆菌农家干酪,在其贮存期间分别采用荧光定量PCR及平板菌落计数法计数干酪中双歧杆菌的数量。其中,在利用荧光定量PCR法检测时,对影响PCR定量准确的因素进行系统研究,包括设计双歧杆菌引物,并对引物特异性进行评价、考察,从干酪基质中提取DNA的数量和质量,建立标准曲线。引物特异性验证结果表明,引物专一性强。采用试剂盒法从干酪样品中提取DNA的纯度较好,OD_(260)/OD_(280)均在1.75~1.82之间。除贮存第1天外,荧光定量PCR法计数结果比平板计数法高0.39%~2.25%,未见显著差异。荧光定量PCR具有灵敏、特异、简便和快速的特点,可用于干酪中双歧杆菌的定量检测。     

Fluorescence assessment of Lactococcus lactis viability

The reproduction and activity of lactic acid bacteria (LAB) are essential in their applications in the dairy industry and other fermentations. Traditionally used methods like plate counting and acidification tests require long incubation times and provide limited information. Fluorescence techniques provide possibilities for rapid assessment of cell physiology. We used traditional and fluorescence assays to assess the physiological condition of L. lactis subsp. lactis ML3 cultures that were exposed to various stress conditions. After exposure to some of the stress conditions, carboxyfluorescein (cF) labelling did not agree with plate counts. Therefore, a two-step method was developed in which cF labelling was followed by a lactose-energized efflux assay. The combined assay proved to be a good and rapid indicator for reproduction and acidification capacity of stressed L. lactis. This novel assay has potential for physiological research and dairy applications related to LAB.     

Assessment of the response of indigenous microflora and inoculated Bacillus licheniformis endospores in reconstituted skim milk to microwave and conventional heating systems by flow cytometry

Heat treatment is one of the most widely used processing technologies in the dairy industry. Its primary purpose is to destroy microorganisms, both pathogenic and spoilage, to ensure the product is safe and has a reasonable shelf life. In this study microwave volumetric heating (MVH) was compared with a conventional tubular heat exchanger (THE), in terms of the effects of each at a range of temperatures (75°C, 85°C, 95°C, 105°C, 115°C, and 125°C) on indigenous microflora viability and the germination of inoculated Bacillus licheniformis endospores in reconstituted skim milk. To assess the heat treatment–related effects on microbial viability, classical agar-based tests were applied to obtain the counts of 4 various microbiological groups including total bacterial, thermophilic bacterial, mesophilic aerobic bacterial endospore, and thermophilic aerobic bacterial endospore counts, and additional novel insights into cell permeability and spore germination profiles post-heat treatment were obtained using real-time flow cytometry (FC) methods. No significant differences in the plate counts of the indigenous microorganisms tested, the plate counts of the inoculated B. licheniformis, or the relative percentage of germinating endospores were observed between MVH- and THE-treated samples, at equal temperatures in the range specified above, indicating that both methods inactivated inoculated endospores to a similar degree (up to 70% as measured by FC and 5 log reduction as measured by plate counting for some treatments of inoculated endospores). Furthermore, increased cell permeability of indigenous microflora was observed by FC after MVH compared with THE treatment of uninoculated skim milk, which was reflected in lower total bacterial count at a treatment temperature of 105°C. This work demonstrates the utility of FC as a rapid method for assessing cell viability and spore inactivation for postthermal processing in dairy products and overall provides evidence that MVH is at least as effective at eliminating native microflora and inoculated B. licheniformis endospores as THE.     

SPIRAL SYSTEM AND LASER COLONY SCANNER FOR ENUMERATION OF MICROORGANISMS

Aerobic Plate Count (APC) and spiral plate (Spiral) methods using both manual counting (MC) and laser counting (LC) procedures were compared for pure bacterial, yeast, and mold cultures and raw milk samples. All four combinations of methods (APC-MC, APC-LC, Spiral-MC, and Spiral-LC) gave similar log₁₀ counts of studied pure microbial cultures, producing results that were not different for the purposes of practical microbiology. With bacterial and yeast cultures, counts differed by less than half a logarithmic cycle (the range of difference = -0.26 to +0.42), the range of difference being -0.03 to +0.62 for Aspergillus flavus and Penicillium camemberti molds. An exception was noticed with Rhizopus oligosporus mold when plates were read by laser due to large (10–15 mm) colony size. The difference between the readings made manually and by laser colony scanner was about one logarithmic cycle with both APC and Spiral methods .
Statistical analyses of the manually read results of bacterial and Saccharomyces cerevisiae spiral plates showed no differences at the 0.05 level of significance between the readings made by four or five persons .
For raw milk samples, Spiral-MC and Spiral-LC methods gave higher microbial numbers than APC-MC (63% of samples) and APC-LC (54% of samples) at 0.05 level of significance (p<0.001). Of the LC results, 75% were within a ± 0.5 logarithmic cycle range when compared to MC results, of which 98% were within the same range. All results were still within a ± 1.0 logarithmic range. When APC and Spiral for raw milk samples were analyzed, LC gave higher microbial numbers compared to MC: 71% of the APC-LC results were higher than APC-MC results, and 85% of Spiral-LC results were higher than Spiral-MC results at 0.05 level of significance (p<0.001). 77% of the results by either plating technique were within a ± 0.5 logarithmic range .     

Multispectral Fluorescence Imaging for the Identification of Food Products

A multispectral fluorescence imaging system was tested to identify four food products (maize, pea, soya bean and wheat). The system made it possible to record 12 images for each sample by a combination of various excitation and emission filters. Direct observation of images showed that the fluorescence of the four food products made identification possible, although more than one image was necessary to obtain satisfactory discrimination. The images were linearly combined and the most relevant images for identification were determined using a stepwise discriminant analysis and a mapping of the four food products was obtained. In the segmented images, the percentage of well-classified pixels was up to 98·8% for the four products.     

Influence of fluorescence of bacteria stained with acridine orange on the enumeration of microorganisms in raw milk

The staining of gram-positive and gram-negative cultures with acridine orange in metabolically active and inactive states was investigated using a Bactoscan, direct epifluorescent filter technique (DEFT), and standard plate count as the reference method. The evaluation of the bacterial cultures in the Bactoscan revealed a linear relationship between Bactoscan counts (pulses) and the quantity of pure culture suspension used. But the proper detection of bacteria with the fluorescence optic methods was dependent on the type of microorganism and the physiological state of the cells. The Bactoscan and DEFT underestimated the bacterial counts of gram-negative cultures as compared with standard plate counting. When stained with acridine orange, metabolically active bacteria showed more orange fluorescence and a lower percentage of green fluorescent cells as compared with inactive bacteria. Bactoscan pulse height analysis (PHA) diagrams, graphs of the detected pulses and their intensity, showed low pulses of inactive bacteria. Many of these weak pulses were eliminated from counting because of their faint fluorescent staining. In contrast, PHA diagrams of metabolically active microorganisms showed bright staining and, therefore, high pulses. A complete count of these bacteria was possible. These investigations point out that discrepancies between the fluorescence optical counting methods and the standard plate count depend strongly on the staining of the cultures with acridine orange and, therefore, on the type of microorganism and the metabolic state of the cells measured.     

乳酸菌菌粉定量计数方法研究

目的:以9株乳杆菌、6株双歧杆菌、3株球菌、1株凝结芽孢杆菌菌粉为研究对象,在国标GB 4789.35-2016的基础上,对稀释倍数、稀释液成分、培养基成分进行比较研究,考察对乳酸菌菌粉计数活菌数的影响。方法:采用稀释平板计数的方法,对不同的乳酸菌菌粉进行活菌计数。结果:初始乳酸菌菌粉样品稀释倍数对最终计数活菌数无明显影响,ISO稀释液对部分乳杆菌、双歧杆菌菌粉的活菌计数结果有显著提高(P<0.05);双歧杆菌菌粉采用TOS琼脂培养基的计数结果显著优于国标培养基(P<0.05);凝结芽孢杆菌菌粉采用改良芽孢计数培养基计数结果优于PCA和NA计数培养基。结论:平板计数方法中,稀释液中含有酪蛋白胨能提升双歧杆菌菌粉的活菌计数数量;TOS琼脂培养基更有利于双歧杆菌的增殖培养;改良芽孢计数培养基更有利于凝结芽孢杆菌的芽孢萌发增殖。     

水牛奶酪蛋白胶束结构的荧光光谱研究

利用内源荧光团色氨酸（Trp）和外源荧光探针8-苯胺基-1-萘磺酸（8-anilino-1-naphthalenesulfonic acid,ANS）的荧光特性对水牛奶酪蛋白胶束结构进行研究。结果表明：当蛋白质质量浓度较低时,酪蛋白胶束结构变化不明显,而较高的蛋白质质量浓度会破坏其胶束结构,使其疏水基团暴露;此外,在离子强度较大、pH值较低条件下,对酪蛋白胶束结构影响较大,使酪蛋白疏水基团暴露,酪蛋白微球先膨胀后聚集并形成酪蛋白胶束。     

Relationship between occurrence of mastitis pathogens in dairy cattle herds and raw-milk indicators of hygienic-sanitary quality

Mastitis is an inflammation of the mammary glands and in most cases it is caused by the presence of microorganisms. High mastitis rates in dairy cattle herds can cause an increase in total microorganism counts of bulk tank milk. The present paper was aimed at verifying whether the occurrence of mastitis in dairy cattle herds is reflected in raw-milk indicators of hygienic-sanitary quality. To observe the correlation among the analysed variables, we performed a logarithmical transformation (log10) of different indicator counts of raw milk and compared them with the occurrence of mastitis in dairy cattle herds. Few correlations were observed among mastitis cases in dairy cattle herds and the raw-milk indicators of hygienic-sanitary quality. We observed a negative correlation between the log10 of mesophilic aerobic plate counts and psychotropic aerobic plate counts when compared with the occurrence of no bacterial growth. The log10 of thermophilic aerobic plate counts and yeasts and mould aerobic plate counts presented a positive correlation with the cases of infectious mastitis and mastitis caused by Staphylococcus spp.     

An assessment of factors characterising the microbiology of Grana Trentino cheese,a Grana‐type cheese

In this study, microbiological aspects of Grana Trentino, a variant of Grana Padano cheese, were defined by plate counts, random amplified polymorphic DNA (RAPD) PCR genotying, 16S rRNA gene sequencing of bacterial isolates and PCR–denaturing gradient gel electrophoresis (PCR–DGGE). Results showed variability in monthly fluctuations of whey culture counts, differences in the diffusion of bacterial genotypes among producers and dairy plant‐specific microbial associations. Moreover, the presence of bacteria not previously reported in this cheese type was highlighted, including coagulase‐negative staphylococci and Lactobacillus sanfranciscensis‐like micro‐organisms.