Institut des Nutraceutiques et des Aliments Fonctionnels (INAF), Université Laval, Quebec, Canada.
Int J Food Microbiol. 2010 Mar 31;138(1-2):108-18. doi: 10.1016/j.ijfoodmicro.2010.01.008. Epub 2010 Jan 20.
Spoilage bacteria in milk are controlled by treatments such as thermization, microfiltration and addition of carbon dioxide. However, little information is known about the changes in microbial communities during subsequent cold storage of treated milk. Culture-dependent methods and a direct molecular approach combining 16S rRNA gene clone libraries and quantitative PCR (Q-PCR) were applied to obtain a better overview of the structure and the dynamics of milk microbiota. Raw milk samples were treated by the addition of carbon dioxide (CO(2)), thermization (TH) or microfiltration (MF) and stored at 4 degrees C or 8 degrees C up to 7d. Untreated milk (UT) was used as a control. Psychrotrophic and staphylococci bacteria were enumerated in the milk samples by culture methods. For the molecular approach, DNA was extracted from milk samples and 16S rRNA gene was amplified by PCR with universal primers prior to cloning. The Q-PCR method was used to evaluate the dynamics of dominant bacterial species revealed by clone library analysis of 16S rRNA gene. Comparison of the 16S rRNA gene sequence indicated that the two most abundant operational taxonomic units (OTU), determined at 97% identity, belonged to the class Gammaproteobacteria (40.3% of the 1415 sequences) and Bacilli (40%). Dominant bacterial species in UT, CO(2) and TH milk samples at day 3 were affiliated with Staphylococcus, Streptococcus, Clostridia, Aerococcus, Facklamia, Corynebacterium, Acinetobacter and Trichococcus. Dominant bacterial species detected in MF milk were Stenotrophomonas, Pseudomonas and Delftia, while Pseudomonas species dominated the bacterial population of UT, CO(2) and MF milk samples at day 7. Staphylococcus and Delftia were the dominant bacterial species in thermized milk. Q-PCR results showed that populations of S. aureus, A. viridans, A. calcoaceticus, C. variabile and S. uberis were stable during 7d of storage at 4 degrees C. Populations of P. fluorescens, S. uberis and total bacteria increased in UT and CO(2) milk samples during 7d of storage at 8 degrees C and were noticeable from day 3. This study shows new microbial species which can develop during cold storage after milk treatment and contributes to identifying causes of reduced shelf life and deterioration of technological properties of milk during storage.
牛奶中的腐败细菌可以通过热灭菌、微滤和添加二氧化碳等方法进行控制。然而，对于处理后的牛奶在随后的冷藏过程中微生物群落的变化，人们知之甚少。本研究应用基于培养的方法和直接的分子方法（结合 16S rRNA 基因克隆文库和定量 PCR（Q-PCR）），以更好地了解牛奶微生物群落的结构和动态。将原始牛奶样品用添加二氧化碳（CO2）、热灭菌（TH）或微滤（MF）的方法进行处理，并在 4°C 或 8°C 下储存，最长可达 7 天。未处理的牛奶（UT）用作对照。通过培养方法对牛奶样品中的嗜冷菌和葡萄球菌进行计数。对于分子方法，从牛奶样品中提取 DNA，并用通用引物通过 PCR 扩增 16S rRNA 基因，然后进行克隆。使用 Q-PCR 方法评估通过克隆文库分析 16S rRNA 基因揭示的优势细菌物种的动态。16S rRNA 基因序列比较表明，在 97%的同一性水平上，两个最丰富的操作分类单元（OTU）属于γ变形菌纲（40.3%的 1415 个序列）和芽孢杆菌纲（40%）。在第 3 天，UT、CO2 和 TH 牛奶样品中的优势细菌种属与葡萄球菌、链球菌、梭菌、微球菌、法克氏菌、棒状杆菌、不动杆菌和毛螺菌有关。在 MF 牛奶中检测到的优势细菌种属为 Stenotrophomonas、Pseudomonas 和 Delftia，而在第 7 天，假单胞菌种属主导了 UT、CO2 和 MF 牛奶样品的细菌种群。金黄色葡萄球菌和 Delftia 是热灭菌牛奶中的优势细菌种属。Q-PCR 结果表明，在 4°C 下储存 7 天期间，金黄色葡萄球菌、A. viridans、A. calcoaceticus、C. variabile 和 S. uberis 的种群保持稳定。在 8°C 下储存 7 天期间，荧光假单胞菌、S. uberis 和总细菌在 UT 和 CO2 牛奶样品中的种群增加，并且从第 3 天开始变得明显。本研究表明，在牛奶处理后冷藏期间可能会出现新的微生物种属，并有助于确定导致货架期缩短和储存过程中牛奶技术特性恶化的原因。
