Growth, nisA Gene Expression, and In Situ Activity of Novel Lactococcus lactis subsp. cremoris Costarter Culture in Commercial Hard Cheese Production

Abstract

This study evaluated in situ expression of the nisA gene by an indigenous, nisin A-producing (NisA+) Lactococcus lactis subsp. cremoris raw milk genotype, represented by strain M78, in traditional Greek Graviera cheeses under real factory-scale manufacturing and ripening conditions. Cheeses were produced with added a mixed thermophilic and mesophilic commercial starter culture (CSC) or with the CSC plus strain M78 (CSC+M78). Cheeses were sampled after curd cooking (day 0), fermentation of the unsalted molds for 24 h (day 1), brining (day 7), and ripening of the brined molds (14 to 15 kg each) for 30 days in a fully controlled industrial room (16.5°C; 91% relative humidity; day 37). Total RNA was directly extracted from the cheese samples, and the expression of nisA gene was evaluated by real-time reverse transcription PCR (qRT-PCR). Agar overlay and well diffusion bioassays were correspondingly used for in situ detection of the M78 NisA+ colonies in the cheese agar plates and antilisterial activity in whole-cheese slurry samples, respectively. Agar overlay assays showed good growth (>8 log CFU/g of cheese) of the NisA+ strain M78 in coculture with the CSC and vice versa. The nisA expression was detected in CSC+M78 cheese samples only, with its expression levels being the highest (16-fold increase compared with those of the control gene) on day 1, followed by significant reduction on day 7 and almost negligible expression on day 37. Based on the results, certain intrinsic and mainly implicit hurdle factors appeared to reduce growth prevalence rates and decrease nisA gene expression, as well as the nisin A-mediated antilisterial activities of the NisA+ strain M78 postfermentation. To our knowledge, this is the first report on quantitative expression of the nisA gene in a Greek cooked hard cheese during commercial manufacturing and ripening conditions by using a novel, rarely isolated, indigenous NisA+ L. lactis subsp. cremoris genotype as costarter culture.

Effects of curd heating with or without previous milk pasteurisation on the microbiological quality and safety of craft‐made ‘Pasta Filata’ Kashkaval cheese curds

“…However in the absence of the Lb. helveticus starter from all raw milk curd samples, thermophilic LAB counts on MRS/45°C agar consisted of enterococci, as previously shown in other traditional Greek cheeses produced without thermophilic Lactobacillus starter species (Samelis et al 2010;Noutsopoulos et al 2017;Vandera et al 2019).…”

“…lactis ) starter LAB selected for growth on M17 agar at 42 °C and M17 and MRS agars at 22 and 30 °C, respectively (Noutsopoulos et al . ; Samelis and Kakouri ), were noted (Table ). However, the populations of thermophilic Lb.…”

“…; Noutsopoulos et al . ; Samelis and Kakouri ). In the absence of active commercial starters and depending on the maturation temperature, indigenous lactococci and enterococci dominate in Greek raw or thermised milks and fresh curds (Lianou et al .…”

“…The strong competitive growth and rapid acidification capacity of commercial S. thermophilus starters in fresh cheese curds made of pasteurised or thermised milk during fermentation followed by a shift towards the prevalence of mesophilic Lc. lactis starters during maturation at reduced temperatures are well documented (Beresford et al 2001;Noutsopoulos et al 2017;Samelis and Kakouri 2018). In the absence of active commercial starters and depending on the maturation temperature, indigenous lactococci and enterococci dominate in Greek raw or thermised milks and fresh curds (Lianou et al 2017;Samelis and Kakouri 2018).…”

“…paracasei and Lb. plantarum, occur in fresh raw or heat-treated milk cheese curds, but usually increase their prevalence by midto late maturation and in the mature (semihard or hard) cheese (Beresford et al 2001;Montel et al 2014;Noutsopoulos et al 2017), including the present Kashkaval cheeses at maturation (Pappa et al 2019).…”