Purpose: This study evaluated the in situ expression of the nisin A gene (NisA) of the novel NisA+ Lactococcus lactis subsp. cremoris M78 strain during Graviera cheese processing.
Methods: First, survival and growth kinetics of a mixed LAB commercial starter culture (CSC) against strain M78 were evaluated in synthetic media and milk. The inhibitory activity of strain M78 was evaluated individually against each CSC strain assigned to Streptococcus thermophilus, Lactobacillus helveticus and Lc. lactis subspecies lactis and cremoris. Then Graviera cheeses were manufactured without (CSC) or with strain M78 (CSC+M78) added to thermized ewes’ milk under commercial conditions. The cheeses were sampled on the day of manufacture (day-0) and after 24 h (day-1). Total RNA was directly extracted from cheeses and the expression of NisA was evaluated by real-time reverse transcription-PCR.
Results: Growth kinetics of each CSC strain in co-culture with strain M78 showed that while lactococcal and thermophilic lactobacillic strains were inhibited strongly, streptococcal strains were inhibited weakly by nisin A in vitro. Importantly, NisA expression was detected in CSC+M78 cheese only, with its expression levels being 0.11- or 1.62-times higher in the curd (day-0) or 24-h cheese (day 1) compared to those of the reference gene, respectively.
Significance: This is the first report on quantitative expression of the NisA gene in Graviera cheese during real cheese manufacturing conditions using a wild, rarely isolated NisA+ Lc. lactis subsp. cremoris genotype as a bioprotective co-starter culture.