Callon Cécile, Duthoit Frédérique, Delbès Céline, Ferrand Marion, Le Frileux Yves, De Crémoux Renée, Montel Marie-Christine
INRA-Unité de Recherches Fromagères URF545, INRA, 36 Rue de Salers, 15000 Aurillac, France.
Syst Appl Microbiol. 2007 Nov;30(7):547-60. doi: 10.1016/j.syapm.2007.05.004. Epub 2007 Jul 2.
The microbial communities in milks from one herd were evaluated during 1-year of lactation, using molecular methods to evaluate their stability and the effect of breeding conditions on their composition. The diversity of microbial communities was measured using two approaches: molecular identification by 16S and 18S rDNA sequencing of isolates from counting media (two milks), and direct identification using 16S rDNA from clone libraries (six milks). The stability of these communities was evaluated by counting on selective media and by Single Strand Conformation Polymorphism (SSCP) analysis of variable region V3 of the 16S rRNA gene and variable region V4 of the 18S rRNA gene. One hundred and eighteen milk samples taken throughout the year were analyzed. Wide diversity among bacteria and yeasts in the milk was revealed. In addition to species commonly encountered in milk, such as Lactococcus lactis, Lactococcus garvieae, Enterococcus faecalis, Lactobacillus casei, Leuconostoc mesenteroides, Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus caprae, Staphylococcus equorum, Micrococcus sp., Kocuria sp., Pantoea agglomerans and Pseudomonas putida, sequences were affiliated to other species only described in cheeses, such as Corynebacterium variabile, Arthrobacter sp., Brachybacterium paraconglomeratum, Clostridium sp. and Rothia sp. Several halophilic species atypical in milk were found, belonging to Jeotgalicoccus psychrophilus, Salinicoccus sp., Dietza maris, Exiguobacterium, Ornithinicoccus sp. and Hahella chejuensis. The yeast community was composed of Debaryomyces hansenii, Kluyveromyces lactis, Trichosporon beigelii, Rhodotorula glutinis, Rhodotorula minuta, Candida pararugosa, Candida intermedia, Candida inconspicua, Cryptococcus curvatus and Cryptococcus magnus. The analyses of microbial counts and microbial SSCP profiles both distinguished four groups of milks corresponding to four periods defined by season and feeding regime. The microbial community was stable within each period. Milks from winter were characterized by Lactococcus and Pseudomonas, those from summer by P. agglomerans and Klebsiella and those from autumn by Chryseobacterium indologenes, Acinetobacter baumanii, Staphylococcus, Corynebacteria and yeasts. However, the composition of the community can vary according to factors other than feeding. This study opens new investigation fields in the field of raw milk microbial ecology.
在一年的泌乳期内,对一群奶牛所产牛奶中的微生物群落进行了评估,采用分子方法评估其稳定性以及饲养条件对其组成的影响。使用两种方法测量微生物群落的多样性:通过对计数培养基(两份牛奶)中的分离株进行16S和18S rDNA测序进行分子鉴定,以及使用来自克隆文库的16S rDNA进行直接鉴定(六份牛奶)。通过在选择性培养基上计数以及对16S rRNA基因的可变区V3和18S rRNA基因的可变区V4进行单链构象多态性(SSCP)分析来评估这些群落的稳定性。对全年采集的118份牛奶样本进行了分析。结果显示牛奶中的细菌和酵母具有广泛的多样性。除了牛奶中常见的物种,如乳酸乳球菌、格氏乳球菌、粪肠球菌、干酪乳杆菌、肠系膜明串珠菌、表皮葡萄球菌、模仿葡萄球菌、山羊葡萄球菌、马肠葡萄球菌、微球菌属、考克氏菌属、成团泛菌和恶臭假单胞菌外,还发现了一些仅在奶酪中描述过的其他物种的序列,如可变棒状杆菌、节杆菌属、副凝聚短杆菌、梭菌属和罗氏菌属。发现了几种牛奶中不常见的嗜盐物种,属于嗜冷乔氏球菌、盐球菌属、海氏迪茨氏菌、嗜盐栖热袍菌、鸟氨酸球菌属和济州海杆菌。酵母群落由汉逊德巴利酵母、乳酸克鲁维酵母、白吉利丝孢酵母、粘红酵母、微小粘红酵母、近平滑念珠菌、中间念珠菌、隐匿念珠菌、弯曲隐球菌和大隐球菌组成。微生物计数分析和微生物SSCP图谱分析均区分出了四组牛奶,分别对应于由季节和饲养方式定义的四个时期。每个时期内微生物群落是稳定的。冬季牛奶的特征是含有乳酸乳球菌和假单胞菌,夏季牛奶的特征是含有成团泛菌和克雷伯菌,秋季牛奶的特征是含有产吲哚金黄杆菌、鲍曼不动杆菌、葡萄球菌、棒状杆菌和酵母。然而,群落组成可能会因饲养以外的因素而有所不同。本研究为生鲜乳微生物生态学领域开辟了新的研究方向。