Graham Ken, Rea Rosemary, Simpson Paul, Stack Helena
Department of Biological Sciences, Cork Institute of Technology, Co. Cork, Ireland; Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland.
Department of Biological Sciences, Cork Institute of Technology, Co. Cork, Ireland.
J Microbiol Methods. 2017 Jan;132:99-105. doi: 10.1016/j.mimet.2016.11.016. Epub 2016 Nov 25.
Enterococci show higher proteolytic activities than other lactic acid bacteria and thus have received considerable attention in scientific literature in recent years. Proteolytic enzymes of enterococci have warranted the use of some species as starter, adjuncts or protective cultures and as probiotics, while in some strains they have also been linked with virulence. Consequently, the isolation and identification of proteolytic enterococci is becoming of increasing interest and importance. However, current screening methods for proteolytic enterococci can be time consuming, requiring a two-step procedure which may take up to 96h. This study describes a method, utilising Kanamycin Skim Milk Aesculin Azide (KSMEA) agar, for the isolation of proteolytic enterococci in one-step, thereby significantly reducing screening time. KSMEA combines the selective properties of Kanamycin Aesculin Azide Agar (KAA) with skim milk powder for the detection of proteolytic enterococci. Enterococci produced colonies with a black halo on KSMEA which were accompanied by a zone of clearing in the media when enterococci were proteolytic. KSMEA medium retained the selectivity of KAA, while proteolytic enterococci were easily distinguished from non-proteolytic enterococci when two known strains were propagated on KSMEA. KSMEA also proved effective at isolating and detecting enterococci in raw milk, faeces and soil. Isolates recovered from the screen were confirmed as enterococci using genus-specific primers. Proteolytic enterococci were present in the raw milk sample only and were easily distinguishable from non-proteolytic enterococci and other microorganisms. Therefore, KSMEA provides a rapid, one-step screening method for the isolation of presumptive proteolytic enterococci.
肠球菌比其他乳酸菌表现出更高的蛋白水解活性，因此近年来在科学文献中受到了相当多的关注。肠球菌的蛋白水解酶使一些菌株被用作发酵剂、辅助培养物或保护性培养物以及益生菌，而在一些菌株中，它们也与毒力有关。因此，蛋白水解性肠球菌的分离和鉴定变得越来越有意义和重要性。然而，目前用于筛选蛋白水解性肠球菌的方法可能很耗时，需要两步程序，可能需要长达96小时。本研究描述了一种利用卡那霉素脱脂乳七叶苷叠氮化物（KSMEA）琼脂一步分离蛋白水解性肠球菌的方法，从而显著缩短筛选时间。KSMEA将卡那霉素七叶苷叠氮化物琼脂（KAA）的选择性特性与脱脂奶粉结合起来用于检测蛋白水解性肠球菌。肠球菌在KSMEA上产生带有黑色晕圈的菌落，当肠球菌具有蛋白水解活性时，培养基中会出现透明圈。KSMEA培养基保留了KAA的选择性，当两种已知菌株在KSMEA上繁殖时，蛋白水解性肠球菌很容易与非蛋白水解性肠球菌区分开来。KSMEA还被证明在从生牛奶、粪便和土壤中分离和检测肠球菌方面是有效的。使用属特异性引物将从筛选中回收的分离株确认为肠球菌。仅在生牛奶样品中存在蛋白水解性肠球菌，并且很容易与非蛋白水解性肠球菌和其他微生物区分开来。因此，KSMEA为推定的蛋白水解性肠球菌的分离提供了一种快速的一步筛选方法。
