Cheddar cheese production uses mixed starters composed of Lactococcus lactis subsp. cremoris strains with complementary or competing enzymatic activity. However, strain interactions within the same subspecies are difficult to investigate by conventional microbiological methods. This study uses fluorescent RNA arbitrarily primed PCR (FRAP-PCR) to analyze the association of three L. lactis subsp. cremoris strains (LL074, LL225, and LL390 with proteinase types: PI, PIII, and PI/PIII, respectively) by monitoring whole transcription profiles. The effect of strain association was demonstrated by distinguishing profiles obtained with pure cultures from those obtained with defined mixed cultures. Both strains LL225 and LL390 dominated culture activity when in dual culture with strain LL074. The three-strain starter was also dominated by LL225 and LL390, in an approximately equal ratio which was stable over 35 generations. Strain LL225 had a stronger inhibitory effect on the growth of strain LL074 than LL390 did, showing incompatibility between strains LL225 and LL074. A new economic and semi-quantitative single-nucleotide polymorphism detection technique was developed and validated the results obtained by FRAP-PCR. Strain disequilibrium detected by FRAP-PCR could be related to inhibition of strain LL074 with a PI-type proteinase by the LL225 strain with a PIII-type proteinase. Strain compatibility could be characterized using these methods, leading to an improved understanding of mixed culture association of lactococcal strains.

摘要 切达干酪生产使用由具有竞争酶活性或互补酶活性的Lactococcus lactis subsp. cremoris菌株组成的混合发酵剂。然而,使用传统的微生物方法很难调查相同亚种中不同菌株间的相互作用。本研究采用荧光RNA任意引物PCR (FRAP-PCR),分析了三个菌株L. lactis subsp. cremoris (LL074, LL225,LL390,分别带有蛋白酶型 PI,PIII,PI/PIII)的联系。菌株间的作用通过区分纯培养和已知的混合培养物来阐明。当LL225和LL390分别与LL074进行两菌株发酵时,LL225和LL390决定培养活力。三菌株的混合发酵剂也是由LL225和LL390控制,稳定性可超过35代。菌株LL225与LL390相比,对菌株LL074的生长具有更强的抑制能力。LL225和LL074显示了不相容性。本文建立了一个新型、经济、半定量SNP(单核苷酸多态性)检测,并通过FRAP-PCR方法对结果进行了验证。由 FRAP-PCR检测的菌株不稳定与LL074菌株(PI型蛋白酶)被LL225 菌株(PIII型蛋白酶)抑制有关。通过这些方法可以确定菌株的兼容性,也增加了对混合培养物中乳球菌株联系的理解。