Resistance to bacteriocin Lcn972 improves oxygen tolerance of Lactococcus lactis IPLA947 without compromising its performance as a dairy starter

Abstract

Background: Lactococcus lactis is the main component of the mesophilic starters used in cheese manufacture. The success of milk fermentation relies on the viability and metabolic activity of the starter bacteria. Therefore, robust strains able to withstand the harsh conditions encountered during cheese manufacture and starter production are demanded. In this work, we have applied adaptive evolution under cell envelope stress imposed by the cell wall active bacteriocin Lcn972 to evolve strains with more robust phenotypes.

Results: Consecutive exposure of the starter strain L. lactis IPLA947 to Lcn972 yielded a stable mutant, L. lactis R5, with enhanced survival when challenged with hydrogen peroxide. L. lactis R5 exhibited faster growth rates in aerobic fermentations in broth and was able to acidify milk to a lower pH in aerated milk cultures. The improved behavior of L. lactis R5 in the presence of oxygen did not translate into a better performance in the presence of heme (i.e. respiration metabolism) or into higher survival during storage at cold temperatures or after freeze-drying compared to the wild type L. lactis IPLA947. L. lactis R5 retained the same milk acidification rate and no changes in the consumption of lactose and production of organic acids were noticed. However, the profile of volatile compounds revealed a significant increase in 3-hydroxy-2-butanone (acetoin) in curds manufactured with L. lactis R5.

Conclusions: Based on our results, L. lactis R5 can be proposed as a suitable dairy starter with improved survival under oxidative stress and enhanced metabolic traits. The results support the notion that adaptive evolution under cell envelope stress might be useful to generate strain diversity within industrial L. lactis strains.

Keywords: Adaptive evolution; Bacteriocin; Dairy starter; Lactococcus lactis; Lcn972; Oxidative stress.

Fig. 1

Survival of L. lactis IPLA947 (WT) and its Lcn972R derivatives L. lactis R2, R3 and R5 after exposure to 5 mM H₂O₂ for 2 h at 30 °C. Nt: CFU/ml after treatment; No: CFU/ml control. Results are the average of three independent cultures. Error bars are standard deviations. (*) P < 0.05; (**) P < 0.01, significantly different from L. lactis IPLA947

Fig. 2

Optical density (OD₆₀₀) (a) and CFU/ml (b) of batch and aerated cultures of L. lactis IPLA947 (grey bars) and the Lcn972R derivative L. lactis R5 (white bars) at the end of the exponential phase. Results are the average of two independent cultures. Error bars are standard deviations. (*) P < 0.05; (***) P < 0.001, significantly different from L. lactis IPLA947

Fig. 3

Cell morphology of L. lactis IPLA947 and the Lcn972R derivative L. lactis R5. Phase contrast microscopy of L. lactis IPLA 947 (a) and L. lactis R5 (b) sampled during growth in the presence of oxygen. Length (c) and width (d) of cells of L. lactis IPLA947 (grey) and L. lactis R5 (white) from exponentially and stationary (24 h) aerated cultures and exponentially growing batch cultures. (*) P < 0.05; (***) P < 0.001, significantly different from L. lactis IPLA947

Fig. 4

Decrease of pH in L. lactis IPLA947 (black squares) and the Lcn972R mutant L. lactis R5 (white squares) in aerated cultures in milk. Results are the average of two independent cultures. Error bars are standard deviations. (*) P < 0.05, significantly different from L. lactis IPLA947

Fig. 5

Survival during cold storage of L. lactis IPLA947 (grey) and the Lcn972R derivative L. lactis R5 (white). (a) Viability of batch and aerated cultures in M17 broth stored for 5 weeks at 4 °C. (b) Viability after freeze-drying and stored at 4 °C under air. Results are the average of two independent experiments. Error bars are standard deviations

Fig. 6

Volatile compounds detected by GC-MS in milk cultures of L. lactis IPLA947 (grey bars) and the Lcn972R mutant L. lactis R5 (white bars). Results are the mean ± standard deviation of two independent cultures. IS, internal standard (*) P < 0.05, significantly different from L. lactis IPLA947