Monitoring Growth Compatibility and Bacteriocin Gene Transcription of Adjunct and Starter Lactic Acid Bacterial Strains in Milk

Abstract

When developing protective starter cultures for application in cheese technologies, monitoring growth interactions between starter and adjunct LAB species and in situ expression of bacteriocin genes in the mixtures, are crucial. This study firstly aimed to monitor growth of mixed LAB strain populations during milk model fermentations by microbial counts and real-time quantitative PCR (qPCR). The primary starter Streptococcus thermophilus ST1 and costarter Lactococcus lactis subsp. cremoris M78 strains served as the basic starter composite co-inoculated in all milk treatments. Adjunct bacteriocinogenic Enterococcus faecium strains KE82 and GL31 and a ripening Lactiplantibacillus plantarum H25 strain were added separately to the starter composite resulting in four LAB combination treatments. The second aim was to quantify gene transcripts of nisin and enterocins B and A synthesized by M78, KE82 and GL31, respectively, by real-time reverse transcription PCR (RT-qPCR) and to detect the in situ antilisterial effects of the cocultures. Adjunct LAB strains showed growth compatibility with the starter, since all of them exhibited a 2 to 3 log units increase in their population levels, compared to their initial inoculation levels, with ST1 prevailing in all treatments. KE82 grew more competitively than GL31 whereas cocultures with KE82 displayed the strongest in situ antilisterial activity. Nisin gene expression levels were higher at the exponential phase of microbial growth in all treatments. Finally, expression of nisin and enterocins A and B genes was interrelated indicating an antagonistic activity.

Safety Evaluation, Biogenic Amine Formation, and Enzymatic Activity Profiles of Autochthonous Enterocin-Producing Greek Cheese Isolates of the Enterococcus faecium/durans Group

“…All LAB strains were incubated at 30 • C for 24 h in MRS broth (Neogen Culture Media, LAB094, Heywood, UK) for two sequent transfers before use, except for S. thermophilus ST1, which was cultivated twice in M17 broth (Merck, Darmstadt, Germany) at 37 • C [24].…”

Growth and Biocontrol of Listeria monocytogenes in Greek Anthotyros Whey Cheese without or with a Crude Enterocin A-B-P Extract: Interactive Effects of the Native Spoilage Microbiota during Vacuum-Packed Storage at 4 °C

“…In general agreement with the above studies, in this study, the antilisterial effects of the CEntE in most Ent-treated whey cheese samples increased with delay, which means that afterward, the native LAB biota, dominated by atypical L. mesenteroides strains, reduced the cheese pH below 5.5 to 4.5 to enhance the combined enterocin A-B-P activity. It is well known that the in situ effectiveness of the enterocins A and B increases when they are copresent to act synergistically [45,46].…”

Biogenic Amine Formation in Artisan Galotyri PDO Acid-Curd Cheeses Fermented with Greek Indigenous Starter and Adjunct Lactic Acid Bacteria Strain Combinations: Effects of Cold (4 °C) Ripening and Biotic Factors Compromising Cheese Safety

“…Four combinations (A1, A2, A3, A4) of Greek indigenous adjunct and natural starter LAB strains previously monitored for their growth compatibility, bacteriocin gene tran-scription, and production in model sterile raw milk (SRM) fermentations [55], were tested for their ability to produce BAs in SRM in vitro and then in artisan Galotyri cheese in situ after fermentation and cold ripening. Briefly, A1 was the basic starter LAB combination, consisting of S. thermophilus CSL-ST1, a primary acidifying strain of natural origin derived from a freeze-dried CSC product (GRU IDC 01, Centro Sperimentale del Latte [CSL], Lodi, Italy) [56], and the novel, wild nisin A-producing (NisA+) Lc.…”

“…cremoris M78 (GenBank accession number JX402634) isolated from bulk raw ewes'/goats' milk in Epirus, Greece [57]. The A2, A3, and A4 combinations consisted of the A1 supplemented with one 'regional' indigenous adjunct (nonstarter) LAB strain each; A2 with the m-Ent+ (entA-entB-entP) E. faecium KE82 (MW644969) from a naturally fermented and ripened (3-month-old) Epirus Graviera cheese; A3 with the enterocin A-producing (entA) E. faecium GL31 (MW709884) from an artisan-type Galotyri PDO retail cheese; and A4 with L. plantarum H25, a multi-functional strain from a ripening (5-week-old) Graviera cheese fermented with a mixed thermophilic and mesophilic CSCs [54,55].…”

“…Portions (100mL each) of heat-sterilized (121 • C for 5 min) raw ewe's milk (SRM) in 250 mL Duran flasks, cooled and pre-tempered at 37 • C, were inoculated with each of the aforementioned LAB strain combinations, as described by Asimakoula et al [55]. Briefly, raw ewes' milk samples (2 liters each) were obtained from our collaborating traditional cheese plant (Pappas Bros., Skarfi E.P.E., Filippiada, Epirus, Greece); heat sterilization was conducted in the microbiology laboratory of the Dairy Research Department (DRD; Ioannina, Greece) to exclude interfering effects of the natural raw milk microbiota on the growth of the A1 to A4 strain combinations in model milk.…”