原料乳中微生物检测技术的研究进展

牛乳是营养丰富的天然食品,被营养学家誉为"最完美的食品"、"白色血液",是自然界赐予我们最理想的天然食品之一。而原料乳质量好坏直接影响到后续加工乳制品,乳中微生物指标成为制约原料乳卫生指标的关键因素,所以该指标一直倍受乳品厂家及消费者的关注,如何快速检测出原料乳的微生物数量及来源问题则更加突出。文中主要讨论了与原料乳质量密切相关的几项微生物指标的鉴定方法,包括全自动微生物分析系统、多重PCR检测技术、脉冲场凝胶电泳技术、微生物荧光光电检测技术、DNA探针技术及生化反应快速检测试剂盒等,并提出了建立原料乳质量追溯体系,控制原料乳从被挤出到加工前各个环节上可能对原料乳造成微生物污染的因素,从根本上控制原料乳质量,保障原料乳质量安全。     

原料乳微生物特性对ESL牛乳货架期影响的研究

研究了ESL乳生产过程中对原料乳质量的要求。结果表明,ESL乳的生产中,在保证原料乳细菌总数≤5×105mL-1时,原料乳中的芽孢数应控制在≤5×103mL-1,同时,应选取低温菌数量和体细胞数量都较低的原料乳。     

原料乳中嗜冷菌菌群多样性及分析方法研究进展

嗜冷菌是原料乳中主要危害性腐败菌群之一,其种类繁多,菌群结构和多样性受到多种因素影响,这为原料乳中优势嗜冷菌的鉴定带来了困难.结合国内外文献,综述了储藏温度、储藏时间、地域和季节等因素对原料乳中嗜冷菌菌群结构和多样性的影响,原料乳中嗜冷菌可能的外界来源.评述了高通量测序、随机扩增多态性DNA、变性凝胶电泳等现代分子生物...     

低温储存期间原料乳微生物预测模型建立

本研究对秋季低温储存原料乳进行预测微生物学研究。使用Gompertz模型建立4~14℃储存的秋季原料乳中总菌落和嗜冷菌的生长动力模型。模型可以有效的模拟秋季原料乳中微生物的生长情况,模型的相关系数均大于0.972,可以用来预测微生物生长情况和达到控制下限的时间。研究表明嗜冷菌是低温储存的原料乳中优势微生物,在4~8℃储存温度下总菌落数与嗜冷菌的数量呈很好的相关性(R~2=0.931),可以通过总菌数推算出嗜冷菌的数量,保证乳制品的品质。      

不同季节原料乳中主要微生物和理化指标分析

研究了冬季、春季和夏季原料乳的主要理化指标和微生物指标。3个季节内,蛋白质、乳脂、乳糖和干物质的变化范围分别是3.38%～3.52%,3.98%～4.26%,4.80%～4.85%和12.78%～13.19%。理化指标检测结果表明冬季牛乳的营养成分高于春夏两季,并且3个季节的乳成分均高于生鲜牛乳的收购标准;此外,对原料乳中主要的微生物:总菌数、乳酸菌、大肠菌群、沙门氏菌、蜡样芽孢杆菌、单增李斯特菌和嗜冷菌的菌数进行了检测。结果中未检测到沙门氏菌、蜡样芽孢杆菌和单增李斯特菌,其他微生物质量分数均在可接受范围内,但是大肠菌群的出现说明需要建立相关的卫生质量标准。     

原料乳生产中空气源微生物的快速检测及 控制方法

本文概述了原料乳中微生物的来源、种类;空气中微生物的采集方法、检测方法以及如何控制原料乳中微生物。原料乳的安全性直接影响乳制品的安全,乳中微生物指标成为制约原料乳卫生指标的关键因素,因此该指标一直倍受乳品厂家及消费者的关注。保证原料乳的安全是保证乳品安全的前提。     

原料乳生产中空气源微生物的快速检测及控制方法

微生物的污染是影响乳制品安全的重要因素,主要以空气为媒介污染原料乳进而影响乳制品安全。本文概述了原料乳中微生物的来源、种类;空气中微生物的采集方法、检测方法以及如何控制原料乳中微生物。原料乳中可能污染的微生物有葡萄球菌、沙门氏菌、志贺氏菌、链球菌属、大肠杆菌等病原微生物和腐败菌、真菌。空气微生物的采样方法有自然沉降法和微生物采样器采样法,比较而知采样器采样法具有稳定、不受气候影响的优点;同时比较了固体和液体微生物采样器的优缺点。原料乳质量好坏直接影响到后续加工乳制品,乳中微生物指标成为制约原料乳卫生指标的关键因素,该指标一直倍受乳品厂家及消费者的关注。因此,保证原料乳的安全是保证乳品安全的前提。     

电导微生物技术快速测定原料乳菌落总数的研究

从乳品电导率微生物学的角度研究了快速测定原料乳菌落总数的原理。向计算机(MALTHUS计算软件)中一一对应输入平皿菌落计数值的log值与检测时间，当输入50组以上数据时曲线自动生成。将电导测定方法与常规方法测定结果相比较，结果较理想。电导率微生物检测提供了传统微生物测试所不能比拟的优点。结果表明，该方法具有快速测定、方便、资料自动搜查、允许样品随时插入测试的优点，原料奶得以更快速的监测，适合用于原料乳的微生物指标的质量控制。     

原料乳中腐败微生物对液态乳品质的影响及防控

乳因其营养丰富备受关注,被称为人体的“白色血液”,同时乳也是微生物良好的培养基,因此原料乳品质和液态乳加工过程都会受到严格把控,但目前液态乳在储藏期、流水线或货架期内发生腐败变质现象仍屡屡发生,引起行业对乳及乳制品安全的高度重视。原料乳作为液态乳生产的原料,是其品质保障的基础,原料乳中腐败微生物随着储藏时间的延长而大量繁殖,经过热处理后未被完全杀死的腐败菌及其产生的耐热酶仍会继续影响液态乳品质,此外生物膜的形成也为腐败菌在管道内残留提供了机会,加大了腐败微生物防控的难度。因此本文将从原料乳中腐败微生物多样性进行阐述并探究腐败微生物对于液态乳的不良影响,挖掘腐败微生物破坏液态乳可能存在的作用机制,总结现阶段国内外对腐败微生物的防控手段并探讨其商业应用价值,旨在为今后液态乳质量安全保障及延长货架期提供理论指导。     

壳寡糖对原料乳中微生物抑制作用的研究

通过研究壳寡糖对原料乳中细菌总数、嗜冷菌数、芽孢数及耐热芽孢数的抑制作用和不同浓度的壳寡糖对原料乳中美兰变色时间的影响,得出壳寡糖对原料乳中细菌总数和嗜冷菌数均有显著的抑制作用(P<0.05),对芽孢总数和耐热芽孢数没有显著的影响(P>0.05),并明显的延长原料乳中美兰的变色时间。     

原料乳生产过程中微生物污染的来源追溯研究

为研究原料乳微生物污染的来源,2011-2012年间,研究者对北京市原料乳产地的牛乳、牛体涂抹、挤乳设备涂抹等及生活用水、卧床土壤、饲料等进行了大量采样分析和调查,并针对采集样品中分离出的目的菌株进行了生化鉴定及分析.研究结果表明,该地区原料乳中的特征病原菌为金黄色葡萄球菌,对后期加工乳品质影响较大的微生物种类为嗜冷菌和芽孢杆菌属.在此基础上,研究者对原料乳和其他来源样品中的这三种菌利用16S rRNA分子生物学技术建立了目的菌株来源系统发育树,以此来进行基因序列比对分析,确定了原料乳中这三种菌的主要来源,为有效减少和控制该地区原料乳污染提供了参考.     

Effect of leaving milk trucks empty and idle for 6 h between raw milk loads

The US Pasteurized Milk Ordinance (PMO) allows milk tanker trucks to be used repeatedly for 24 h before mandatory clean-in-place cleaning, but no specifications are given for the length of time a tanker can be empty between loads. We defined a worst-case hauling scenario as a hauling vessel left empty and dirty (idle) for extended periods between loads, especially in warm weather. Initial studies were conducted using 5-gallon milk cans (pilot-scale) as a proof-of-concept and to demonstrate that extended idle time intervals could contribute to compromised raw milk quality. Based on pilot-scale results, a commercial hauling study was conducted through partnership with a Pacific Northwest dairy co-op to verify that extended idle times of 6 h between loads have minimal influence on the microbiological populations and enzyme activity in subsequent loads of milk. Milk cans were used to haul raw milk (load 1), emptied, incubated at 30°C for 3, 6, 10, and 20 h, and refilled with commercially pasteurized whole milk (load 2) to measure cross-contamination. For the commercial-scale study, a single tanker was filled with milk from a farm known to have poorer quality milk (farm A, load 1), emptied, and refilled immediately (0 h) or after a delay (6 h) with milk from a farm known to have superior quality milk (farm B, load 2). In both experiments, milk samples were obtained from each farm's bulk tank and from the milk can or tanker before unloading. Each sample was microbiologically assessed for standard plate count (SPC), lactic acid bacteria (LAB), and coliform counts. Selected isolates were assessed for lipolytic and proteolytic activity using spirit blue agar and skim milk agar, respectively. The pilot-scale experiment effectively demonstrated that extended periods of idle (>3 h) of soiled hauling vessels can significantly affect the microbiological quality of raw milk in subsequent loads; however, extended idle times of 6 h or less would not measurably compromise milk quality in subsequent loads in commercial tankers. Current tanker sanitation practices appear to be sufficient for maintaining raw milk SPC, LAB, and coliform levels, which are important measures of milk quality.     

Short communication: Inactivation of microbial contaminants in raw milk La Serena cheese by high-pressure treatments

La Serena cheese, a Spanish variety made from Merino ewes’ raw milk, has a high pH value, low salt content, and high moisture, conditions that are all favorable for growth and survival of contaminating microorganisms, including pathogens. To improve its microbiological quality and safety, high-pressure treatments at 300 or 400 MPa for 10 min at 10°C were applied to 2 batches of La Serena cheese on d 2 or 50 of ripening. Cheese treated on d 2 at 300 MPa showed viable aerobic counts that were 0.99 log units lower than those for control cheese on d 3 and showed counts of enterococci, coagulase-positive staphylococci, gram-negative bacteria, and coliforms that were 2.05, 0.49, 3.14, and 4.13 log units lower, respectively, than control cheese. For cheese treated on d 2 at 400 MPa, the respective reductions in counts were 2.02, 2.68, 1.45, 3.96, and 5.50 log units. On d 60, viable aerobic counts in cheese treated on d 50 at 300 MPa were 0.50 log units lower than those in control cheese, and counts of enterococci, gram-negative bacteria, and coliforms were 1.37, 2.30, and 4.85 log units lower, respectively. For cheese treated on d 50 at 400 MPa, the respective reductions in counts were 1.29, 1.98, 4.47, and > 5 log units. High-pressure treatments at 300 or 400 MPa on d 2 or 50 reduced significantly the counts of undesirable microorganisms, improving the microbiological quality and safety of La Serena cheese immediately after treatment and at the end of the ripening period.     

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。     

Low pressure CO2 storage of raw milk: microbiological effects

The effects of holding raw milk under carbon dioxide pressures of 68 to 689 kPa at temperatures of 5, 6.1, 10, and 20°C on the indigenous microbiota were investigated. These pressure-temperature combinations did not cause precipitation of proteins from the milk. Standard plate counts from treated milks demonstrated significantly lower growth rate compared with untreated controls at all temperatures, and in some cases, the treatment was microcidal. Raw milk treated with CO₂ and held at 6.1°C for 4 d exhibited reduced bacterial growth rates at pressures of 68, 172, 344, and 516 kPa; and at 689 kPa, demonstrated a significant loss of viability in standard plate count assays. The 689-kPa treatment also reduced gram-negative bacteria and total Lactobacillus spp. The time required for raw milk treated at 689 kPa and held at 4°C to reach 4.30 log₁₀ cfu/mL increased by 4 d compared with untreated controls. Total coliform counts in the treated milk were maintained at 1.95 log₁₀ cfu/mL by d 9 of treatment, whereas counts in the control significantly increased to 2.61 log₁₀ cfu/mL by d 4 and 2.89 log₁₀ cfu/mL by d 9. At d 8, Escherichia coli counts had not significantly changed in treated milk, but significantly increased in the control milk. Thermoduric bacteria counts after 8 d were 1.32 log₁₀ cfu/mL in treated milk and 1.98 log₁₀ cfu/mL in control milk. These data indicated that holding raw milk at low CO₂ pressure reduces bacterial growth rates without causing milk protein precipitation. Combining low CO₂ pressure and refrigeration would improve the microbiological quality and safety of raw milk and may be an effective strategy for shipping raw single strength or concentrated milk over long distances.     

原料奶分级收购标准的研究

以乳品企业所辖区域收购奶户的牛奶为研究对象,对原料奶理化指标、菌落总数进行检测,并对2009年原料奶的理化平均指标、菌落总数平均指标进行汇总分析.结果表明,原料奶的各理化指标在一年12个月平均指标不等,各地区之间也存在差异,河北和徐州地区原料奶的脂肪、蛋白质、全脂乳固体、非脂乳固体呈现规律性变化.该研究为企业原料奶分级提供了理论依据,建立了企业不同月份的分级收购标准.     

Microbial community dynamics of a blue-veined raw milk cheese from the United Kingdom

A commercial blue-veined cheese made from unpasteurized milk was examined by conventional culturing and PCR denaturing gradient gel electrophoresis analysis of the bacterial community 16S rRNA genes using 3 primer sets, V3, V4V5, and V6V8. Genomic DNA for amplification was extracted directly from raw milk, starter culture, cheese at different stages of production, fully ripened cheese, and from the cultured cells grown on various media. The outer rind was sampled separately from the inner white core and blue veins. A diverse microbiota containing Lactococcus lactis ssp. lactis, Lactobacillus plantarum, Lactobacillus curvatus, Staphylococcus gallinarum, Staphylococcus devriesei, Microbacterium sp., Sphingobacterium sp., Mycetocola sp., Brevundimonas sp., Enterococcus faecalis, Proteus sp., and Kocuria sp. was detected in the raw milk using culturing methods, but only Lactococcus lactis ssp. lactis, Lactobacillus plantarum, and Enterococcus faecalis survived to the final cheese and were detected both in the core and the rind. Using PCR denaturing gradient gel electrophoresis analysis of the cheese process samples, Staphylococcus equorum and Enterococcus durans were found in the rind of prepiercing samples but not in the core and veins; after piercing, these species were found in all parts of the cheese but survived only in the rind when the cheese was fully ripened. Brevibacterium sp., Halomonas sp., Acinetobacter sp., Alkalibacterium sp., and Corynebacterium casei were identified only by PCR denaturing gradient gel electrophoresis and not cultured from the samples. Brevibacterium sp. was initially identified in the cheese postpiercing (core and veins), Halomonas sp. was found in the matured cheese (rind), and Acinetobacter sp., Alkalibacterium sp., and Corynebacterium casei were also found in the prepiercing samples (rind) and then found through the subsequent process stages. The work suggests that in this raw milk cheese, a limited community from the milk survive to the final cheese, with salt addition and handling contributing to the final cheese consortium.     

The effect of raw milk microbial flora on the sensory characteristics of Salers-type cheeses

The sensory characteristics of Salers Protected Denomination of Origin raw-milk cheeses are linked to the biochemical composition of the raw material (milk) and to the resultant microbial community. To evaluate the influence of the microbial community on sensory characteristics, Salers-type cheeses were manufactured with the same pasteurized milk, reinoculated with 3 different microbial communities from 3 different filtrates from microfiltered milks. Each cheese was subjected to microbial counts (on selective media), biochemical tests, and volatile and sensory component analyses at different times of ripening. Adding different microbial communities to specimens of the same (biochemically identical) pasteurized milk lead to different sensory characteristics of the cheeses. Cheeses with fresh cream, hazelnut, and caramel attributes were opposed to those with fermented cream, chemical, and garlic flavors. The aromatic compounds identified (esters, acids, alcohols, and aldehydes) in these cheeses were quite similar. Nevertheless, one milk was distinguished by a higher content of acetoin, and lower 2-butanone and 3-methylpentanone concentrations. Over the production period of 1 mo, the different cheeses were characterized by the same balance of the microbial population assessed by microbial counts on different media. This was associated with the stability of some sensory attributes describing these cheeses. Nevertheless, there was no linear correlation between microbial flora data and sensory characteristics as measured in this study.     

Single-molecule real-time sequencing reveals differences in bacterial diversity in raw milk in different regions and seasons in China

The quality of raw milk is a key factor influencing the whole dairy processing chain. The richness and diversity of bacteria in raw milk affect its quality and safety. However, traditional microbial detection methods mainly depend on the known microbe culture and are often time consuming. Thus, the development of efficient ways for supervising any possible microbiological contamination is desiderated. In the current work, single-molecule real-time (SMRT) sequencing, developed by Pacific Biosciences (PacBio), was applied to acquire long reads and applied for discrimination of bacteria at species level. Forty samples of raw milk obtained from Beijing, Hebei, Inner Mongolia, Shanghai, and Guangdong in China during summer, autumn, and winter were investigated. Among 35 bacteria species identified in these samples, Acinetobacter albensis, Pseudomonas gessardii, Pseudomonas weihenstephanensis, and Rahnella inusitata were the bacteria with the highest relative abundance in the overall sample, whereas the bacteria with the highest relative abundance in raw milk samples of different origins and seasons are different. Significant differences in bacterial richness and bacterial community diversity in raw milk grouped according to different production areas and different sampling seasons were confirmed by Welch's t-test. Interestingly, the transport distance and transport time positively correlated with the relative abundance of Pseudomonas weihenstephanensis, suggesting that the content of this bacteria was expected to be a standard for evaluating the freshness of raw milk. Pathogens Bacillus cereus and Klebsiella pneumoniae were detected in most samples, indicating that the raw milk was at risk of contamination by pathogenic bacteria. Moreover, the findings of this study provide important evidence for quality and safety monitoring and biological control of raw milk.