A novel approach was developed to extract the extracellular DNA (eDNA), i.e. the free DNA outside the microbial cell, compared to the intracellular DNA (iDNA). The two DNA fractions were investigated in seven long-ripened cheeses. Among different buffer solutions tested, EDTA 0.5 M at pH 8 enabled a mild homogenization of cheese samples and the highest eDNA recovery. The extraction protocol was tested on single strains of lactic acid bacteria characterizing many Italian long-ripened cheeses, such as Streptococcus thermophilus, Lactobacillus helveticus, and Lactobacillus rhamnosus. The method resulted suitable for eDNA extraction because it minimized cell-lysis, avoiding the leakage of iDNA from the cells. The yields of eDNA, ranging from 0.01 to 0.36 µg g⁻¹ cheese, were generally higher than the iDNA, indicating that autolytic phenomena prevailed over intact cells after 8–12 months of ripening. In four of the seven cheeses, the same LAB species were detected in the eDNA and iDNA fractions by length-heterogeneity PCR, while in the remaining three samples, a higher number of species was highlighted in the eDNA compared to the corresponding iDNA. The sequential extraction of eDNA and iDNA can be applied to obtain additional information on the composition of the bacterial community in long-aged cheeses.

中文翻译：

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细胞外和细胞内DNA用于长成熟干酪的细菌分析。

与细胞内DNA（iDNA）相比，开发了一种新颖的方法来提取细胞外DNA（eDNA），即微生物细胞外部的游离DNA。在七个长期成熟的奶酪中研究了两个DNA组分。在测试的不同缓冲溶液中，pH 8的EDTA 0.5 M可使干酪样品温和匀浆并实现最高的eDNA回收率。提取方案在具有许多意大利长寿奶酪的特征的单一乳酸菌菌株上进行了测试，例如嗜热链球菌，瑞士乳杆菌和鼠李糖乳杆菌。该方法适用于eDNA提取，因为它可最大程度地减少细胞裂解，避免了iDNA从细胞中泄漏。eDNA的产量为0.01至0.36 µg g^-1奶酪通常高于iDNA，这表明成熟8至12个月后自溶现象普遍存在于完整细胞上。在七个奶酪中的四个中，通过长度异质性PCR在eDNA和iDNA馏分中检测到相同的LAB物种，而在其余三个样品中，与相应的iDNA相比，eDNA中突出显示了更多种类的物种。eDNA和iDNA的顺序提取可用于获得有关长寿奶酪中细菌群落组成的其他信息。