Longitudinal Study of Lactococcus Phages in a Canadian Cheese Factory.

The presence of virulent phages is closely monitored during cheese manufacturing, as these bacterial viruses can significantly slow down the milk fermentation process and lead to low-quality cheeses. From 2001 to 2020, whey samples from cheddar cheese production in a Canadian factory were monitored for the presence of virulent phages capable of infecting proprietary strains of Lactococcus cremoris and Lactococcus lactis used in starter cultures. Phages were successfully isolated from 932 whey samples using standard plaque assays and several industrial Lactococcus strains as hosts. A multiplex PCR assay assigned 97% of these phage isolates to the Skunavirus genus, 2% to the P335 group, and 1% to the Ceduovirus genus. DNA restriction profiles and a multilocus sequence typing (MLST) scheme distinguished at least 241 unique lactococcal phages from these isolates. While most phages were isolated only once, 93 of them (out of 241, 39%) were isolated multiple times. Phage GL7 was isolated 132 times from 2006 to 2020, demonstrating that phages can persist in a cheese factory for long periods of time. Phylogenetic analysis of MLST sequences showed that phages could be clustered based on their bacterial hosts rather than their year of isolation. Host range analysis showed that Skunavirus phages exhibited a very narrow host range, whereas some Ceduovirus and P335 phages had a broader host range. Overall, the host range information was useful in improving the starter culture rotation by identifying phage-unrelated strains and helped mitigating the risk of fermentation failure due to virulent phages. IMPORTANCE Although lactococcal phages have been observed in cheese production settings for almost a century, few longitudinal studies have been performed. This 20-year study describes the close monitoring of dairy lactococcal phages in a cheddar cheese factory. Routine monitoring was conducted by factory staff, and when whey samples were found to inhibit industrial starter cultures under laboratory conditions, they were sent to an academic research laboratory for phage isolation and characterization. This led to a collection of at least 241 unique lactococcal phages, which were characterized through PCR typing and MLST profiling. Phages of the Skunavirus genus were by far the most dominant. Most phages lysed a small subset of the Lactococcus strains. These findings guided the industrial partner in adapting the starter culture schedule by using phage-unrelated strains in starter cultures and removing some strains from the starter rotation. This phage control strategy could be adapted for other large-scale bacterial fermentation processes.

Comparative transcriptome analysis of Lactococcus lactis subsp. cremoris strains under conditions simulating Cheddar cheese manufacture.

Gene expression in response to technological variations can influence fermentation and flavor generation in Cheddar cheese, and can vary from one lactococcal strain to another, perceived as differences in starter performance. The aim of this study was to determine the influence of cheese cooking temperature at 38 °C and salting on the transcriptional profiles of four closely related strains of L. lactis subsp. cremoris under simulated conditions of Cheddar cheese manufacture. Two responses could be distinguished, a core gene expression, corresponding to the common response of all strains and strain-specific response during the Cheddar simulating process. For the core gene expression after heating of inoculated milk at 38 °C, two groups of differentially expressed genes were identified: i) stress response and ii) carbohydrate and amino acid metabolism. The response to combined stresses of heat, acid and salt resulted in: i) general decrease of functions linked to cell division and metabolism, ii) specific responses related to stress such as the induction of genes coding for chaperones and proteases and iii) expression of prophage lytic systems for certain strains. Strain-specific responses were mainly observed in three of the four tested strains. These responses were the induction of genes related to osmotic stress or the release of CodY repression leading to the activation of oligopeptide transporters as well as the bcaT gene, related to amino acid degradation for the production of flavor. Comparing transcriptomes provides a core expression profile that contributes to understanding gene expression responses to environmental variations. The strain-specific responses identify predictive markers for the transcriptional state of starter strains before they enter the cheese ripening phase.

Evaluation of the genetic polymorphism among Lactococcus lactis subsp. cremoris strains using comparative genomic hybridization and multilocus sequence analysis.

Genetic diversity of Lactococcus lactis subsp. cremoris provides an important reservoir of industrial functions. Knowledge of strain diversity is an important step for the selection of starter cultures, because technological and sensorial attributes are strain-dependent and it may help to distinguish strains with particular technological properties and performances. In the present study, microarray-based comparative genomic hybridization (CGH) and multilocus sequence analysis (MLSA) were used to investigate the genetic variation among eight strains of Lactococcus lactis subsp. cremoris. The CGH analysis allows strain grouping and identification of absent or divergent genes involved in metabolism, amino acid biosynthesis, osmoregulation and proteolysis. The MLSA clustering of strains based on the partial sequence of eight genes shows good correlation with the CGH grouping. Strains HP, ATCC 19257 and Wg2 were clustered together, followed by E8, and finally SK11 was in a separate cluster. The combined information provides genetic markers for distinguishing between strains and their variants. By selecting strains according to their genetic profiles, compatible and complementary mixed starters can be formulated, opening new avenues for industrial applications.

Characterization of a galactokinase-positive recombinant strain of Streptococcus thermophilus.

The lactic acid bacterium Streptococcus thermophilus is widely used by the dairy industry for its ability to transform lactose, the primary sugar found in milk, into lactic acid. Unlike the phylogenetically related species Streptococcus salivarius, S. thermophilus is unable to metabolize and grow on galactose and thus releases substantial amounts of this hexose into the external medium during growth on lactose. This metabolic property may result from the inability of S. thermophilus to synthesize galactokinase, an enzyme of the Leloir pathway that phosphorylates intracellular galactose to generate galactose-1-phosphate. In this work, we report the complementation of Gal(-) strain S. thermophilus SMQ-301 with S. salivarius galK, the gene that codes for galactokinase, and the characterization of recombinant strain SMQ-301K01. The recombinant strain, which was obtained by transformation of strain SMQ-301 with pTRKL2TK, a plasmid bearing S. salivarius galK, grew on galactose with a generation time of 55 min, which was almost double the generation time on lactose. Data confirmed that (i) the ability of SMQ-301K01 to grow on galactose resulted from the expression of S. salivarius galK and (ii) transcription of the plasmid-borne galK gene did not require GalR, a transcriptional regulator of the gal and lac operons, and did not interfere with the transcription of these operons. Unexpectedly, recombinant strain SMQ-301K01 still expelled galactose during growth on lactose, but only when the amount of the disaccharide in the medium exceeded 0.05%. Thus, unlike S. salivarius, the ability to metabolize galactose was not sufficient for S. thermophilus to simultaneously metabolize the glucose and galactose moieties of lactose. Nevertheless, during growth in milk and under time-temperature conditions that simulated those used to produce mozzarella cheese, the recombinant Gal(+) strain grew and produced acid more rapidly than the Gal(-) wild-type strain.

Growth and morphology of thermophilic dairy starters in alginate beads.

The aim of this research was to produce concentrated biomasses of thermophilic lactic starters using immobilized cell technology (ICT). Fermentations were carried out in milk using pH control with cells microentrapped in alginate beads. In the ICT fermentations, beads represented 17% of the weight. Some assays were carried out with free cells without pH control, in order to compare the ICT populations with those of classical starters. With Streptococcus thermophilus, overall populations in the fermentor were similar, but maximum bead population for (8.2 x 10(9) cfu/g beads) was 13 times higher than that obtained in a traditional starter (4.9 x 10(8) cfu/ml). For both Lactobacillus helveticus strains studied, immobilized-cell populations were about 3 x 10(9) cfu/g beads. Production of immobilized Lb. bulgaricus 210R strain was not possible, since no increases in viable counts occurred in beads. Therefore, production of concentrated cell suspension in alginate beads was more effective for S. thermophilus. Photomicrographs of cells in alginate beads demonstrated that, while the morphology of S. thermophilus remained unchanged during the ICT fermentation, immobilized cells of Lb. helveticus appeared wider. In addition, cells of Lb. bulgaricus were curved and elongated. These morphological changes would also impair the growth of immobilized lactobacilli.