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

2017 ◽  
Vol 80 (12) ◽  
pp. 2137-2146 ◽  
Author(s):  
Dimitrios Noutsopoulos ◽  
Athanasia Kakouri ◽  
Eleftheria Kartezini ◽  
Dimitrios Pappas ◽  
Efstathios Hatziloukas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lactococcus Lactis ◽  
Starter Culture ◽  
Fold Increase ◽  
Raw Milk ◽  
Good Growth ◽  
Lactococcus Lactis Subsp ◽  
Quantitative Expression ◽  
Hard Cheese

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.

Behavior of Staphylococcus aureus in Culture Broth, in Raw and Thermized Milk, and during Processing and Storage of Traditional Greek Graviera Cheese in the Presence or Absence of Lactococcus lactis subsp. cremoris M104, a Wild, Novel Nisin A–Producing Raw Milk Isolate

2014 ◽  
Vol 77 (10) ◽  
pp. 1703-1714 ◽  
Author(s):  
JOHN SAMELIS ◽  
ALEXANDRA LIANOU ◽  
ELENI C. PAPPA ◽  
BOJANA BOGOVIČ-MATIJAŠIĆ ◽  
MARIA PARAPOULI ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Lactococcus Lactis ◽  
Starter Culture ◽  
Raw Milk ◽  
Culture Broth ◽  
Microbial Consortia ◽  
Aureus Strain ◽  
Lactococcus Lactis Subsp ◽  
Log Reduction ◽  
And Storage

This study was conducted to evaluate the behavior of Staphylococcus aureus during processing, ripening, and storage of traditional Greek Graviera cheese in accordance with European Union Regulation 1441/2007 for coagulase-positive staphylococci in thermized milk cheeses. Lactococcus lactis subsp. cremoris M104, a wild, novel nisin A–producing (NisA+) strain, also was evaluated as an antistaphylococcal adjunct. A three-strain cocktail of enterotoxigenic (Ent+) S. aureus increased by approximately 2 log CFU/ml when coinoculated (at approximately 3 log CFU/ml) in thermized Graviera cheese milk (TGCM; 63°C for 30 s) with commercial starter culture (CSC) and/or strain M104 at approximately 6 log CFU/ml and then incubated at 37°C for 3 h. However, after 6 h at 37°C, significant retarding effects on S. aureus growth were noted in the order TGCM+M104 > TGCM+CSC = TGCM+CSC+M104 > TGCM. Additional incubation of TGCM cultures at 18°C for 66 h resulted in a 1.2-log reduction (P < 0.05) of S. aureus populations in TGCM+M104. The Ent+ S. aureus cocktail did not grow but survived during ripening and storage when inoculated (at approximately 3 log CFU/g) postcooking into Graviera mini cheeses prepared from TGCM+CSC or TGCM+CSC+M104, ripened at 18°C and 90% relative humidity for 20 days, and stored at 4°C in vacuum packages for 2 months. A rapid 10-fold decrease (P < 0.05) in S. aureus populations occurred within the first 24 h of cheese fermentation. Reductions of S. aureus were greater by approximately 0.4 log CFU/g in CSC+M104 than in CSC only cheeses, concomitantly with the presence of NisA+ M104 colonies and nisin-encoding genes in the CSC plus M104 cheeses and their corresponding microbial consortia only. A high level of selective survival of a naturally nisin-resistant EntC+ S. aureus strain from the cocktail was noted in CSC+M104 cheeses and in coculture with the NisA+ M104 strain in M-17 broth. In conclusion, although S. aureus growth inhibition is assured during Graviera cheese ripening, early growth of the pathogen during milk curdling and curd cooking operations may occur. Nisin-resistant S. aureus strains that may contaminate Graviera cheese milks postthermally may be difficult to control even by the application of the NisA+ L. lactis subsp. cremoris strain M104 as a bioprotective adjunct culture.

Cell Growth Density and Nisin A Activity of the Indigenous Lactococcus lactis subsp. cremoris M78 Costarter Depend Strongly on Inoculation Levels of a Commercial Streptococcus thermophilus Starter in Milk: Practical Aspects for Traditional Greek Cheese Processors

2020 ◽  
Vol 83 (3) ◽  
pp. 542-551
Author(s):  
JOHN SAMELIS ◽  
ATHANASIA KAKOURI
Keyword(s):  
Lactococcus Lactis ◽  
Streptococcus Thermophilus ◽  
Raw Milk ◽  
Starter Cultures ◽  
Competitive Growth ◽  
Population Densities ◽  
Relative Population ◽  
Lactococcus Lactis Subsp ◽  
Pure Cultures

ABSTRACT Mixed thermophilic and mesophilic commercial starter cultures (CSCs), particularly those including Streptococcus thermophilus as a primary milk acidifier, have been found to reduce growth and counteract in situ nisin A (NisA+) antilisterial effects by the novel, indigenous Lactococcus lactis subsp. cremoris M78 costarter in traditional Graviera thermized milk cheese curds. Therefore, this model challenge study evaluated growth and in situ NisA+ activity of strain M78 in coculture with S. thermophilus ST1 singly in sterilized raw milk (SRM). Strain ST1, derived from a CSC for cheese, was challenged at two inoculation levels (5 and 7 log CFU/mL) in SRM against 6 and 3 log CFU/mL of strain M78 and Listeria monocytogenes, respectively. Pure cultures of each strain and cocultures of strain ST1 with the CSC L. lactis LL2, in replacement of strain M78, served as controls. At the high (7-log) inoculation level, the rapid, competitive growth (>9.3 log CFU/mL) of S. thermophilus ST1 reduced growth of both L. lactis by at least 10-fold; the industrial strain LL2 retained slightly higher relative population densities (7.4 to 9.1%) than the wild NisA+ strain M78 (3.8 to 5.6%) after 6 h at 37°C, followed by an additional 66 h of incubation at 22°C. In full contrast, at the low (5-log) inoculation level, S. thermophilus ST1 failed to predominate in SRM at 6 h; thus, the starter lactic acid bacteria populations were reversed in favor of L. lactis. Notably, strain M78 retained higher relative population densities (83.0 to 90.1%) than the CSC strain LL2 (80.3 to 85.2%) at 22°C. Moreover, at the 5-log ST1 level, the direct and deferred in situ NisA+ activities of strain M78 were at similar levels with its pure culture with L. monocytogenes in SRM, whereas at the 7-log ST1 level, the respective NisA+ effects were counteracted. Hence, 10- to 100-fold lowered inoculation levels of CSC S. thermophilus are required to enhance the performance of the M78 costarter in traditional Greek cheese technologies. HIGHLIGHTS

Characterization of diacetin B, a bacteriocin fromLactococcus lactissubsp.lactisbv.diacetylactisUL720

1995 ◽  
Vol 41 (9) ◽  
pp. 832-841 ◽  
Author(s):  
D. Ali ◽  
C. Lacroix ◽  
R. E. Simard ◽  
D. Thuault ◽  
C. M. Bourgeois
Keyword(s):  
Lactic Acid ◽  
Amino Acid ◽  
Lactococcus Lactis ◽  
High Performance ◽  
High Capacity ◽  
Raw Milk ◽  
Mass Spectrometry Analysis ◽  
Original Activity ◽  
Spectrometry Analysis ◽  
Lactococcus Lactis Subsp

Fourteen Lactococcus lactis strains showing inhibitory activity against Listeria innocua SICC 4202 were isolated from different French raw milks and raw milk cheeses and screened for bacteriocin production by the triple layer method under conditions that eliminate the effects of lactic acid and hydrogen peroxide. Three bacteriocinogenic strains (two Lactococcus lactis subsp. lactis bv. diacetylactis UL719 and UL720 and one Lactococcus lactis subsp. lactis UL730) were selected for their high capacity to inhibit the growth of various food pathogens, including Listeria monocytogenes, Staphylococcus aureus, and clostridial strains. The inhibitory compounds from these three strains are inactivated by selected proteases, indicating their protein nature. They retained their antibacterial activity after heat treatments of 100 °C for 60 min and 121 °C for 20 min, and in the pH range from 2 to 11. The bacteriocin diacetin B produced by strain UL720 has been purified by a pH-dependent adsorption–desorption procedure, followed by reverse-phase high performance liquid chromatography, with a yield of 1.25% of the original activity. Mass spectrometry analysis indicates that the pure peptide has a molecular mass of 4292.32 or 4490.28 Da, while amino acid sequencing allowed the identification of the primary structure of the bacteriocin composed of 37 amino acid residues. The structure of the peptide did not show similarity with other known bacteriocins from lactic acid bacteria.Key words: isolation, Lactococcus lactis subsp. lactis bv. diacetylactis, bacteriocin, diacetin B, purification.

Addition to Thermized Milk of Lactococcus lactis subsp. cremoris M104, a Wild, Novel Nisin A–Producing Strain, Replaces the Natural Antilisterial Activity of the Autochthonous Raw Milk Microbiota Reduced by Thermization

2014 ◽  
Vol 77 (8) ◽  
pp. 1289-1297 ◽  
Author(s):  
ALEXANDRA LIANOU ◽  
JOHN SAMELIS
Keyword(s):  
Lactococcus Lactis ◽  
Great Part ◽  
Raw Milk ◽  
Control Treatment ◽  
Lactococcus Lactis Subsp ◽  
Milk Samples ◽  
Bulk Milk ◽  
C 30 ◽  
Sterilized Milk ◽  
Cheese Production

Recent research has shown that mild milk thermization treatments routinely used in traditional Greek cheese production are efficient to inactivate Listeria monocytogenes and other pathogenic or undesirable bacteria, but they also inactivate a great part of the autochthonous antagonistic microbiota of raw milk. Therefore, in this study, the antilisterial activity of raw or thermized (63°C, 30 s) milk in the presence or absence of Lactococcus lactis subsp. cremoris M104, a wild, novel, nisin A–producing (Nis-A+) raw milk isolate, was assessed. Bulk milk samples were taken from a local cheese plant before or after thermization and were inoculated with a five-strain cocktail of L. monocytogenes (approximately 4 log CFU/ml) or with the cocktail, as above, plus the Nis-A+ strain (approximately 6 log CFU/ml) as a bioprotective culture. Heat-sterilized (121°C, 5 min) raw milk inoculated with L. monocytogenes was used as a control treatment. All milk samples were incubated at 37°C for 6 h and then at 18°C for an additional 66 h. L. monocytogenes grew abundantly (>8 log CFU/ml) in heat-sterilized milk, whereas its growth was completely inhibited in all raw milk samples. Conversely, in thermized milk, L. monocytogenes increased by 2 log CFU/ml in the absence of strain M104, whereas its growth was completely inhibited in the presence of strain M104. Furthermore, nisin activity was detected only in milk samples inoculated with strain M104. Thus, postthermal supplementation of thermized bulk milk with bioprotective L. lactis subsp. cremoris cultures replaces the natural antilisterial activity of raw milk reduced by thermization.

Use of a Nisin-Producing Starter Culture of Lactococcus lactis subsp. lactis To Improve Traditional Fish Fermentation in Senegal

2009 ◽  
Vol 72 (9) ◽  
pp. 1930-1934 ◽  
Author(s):  
MICHEL BAKAR DIOP ◽  
ROBIN DUBOIS-DAUPHIN ◽  
JACQUELINE DESTAIN ◽  
EMMANUEL TINE ◽  
PHILIPPE THONART
Keyword(s):  
Lactococcus Lactis ◽  
Pathogenic Bacteria ◽  
Starter Culture ◽  
Enteric Bacteria ◽  
Local Market ◽  
Spontaneous Fermentation ◽  
Ambient Temperatures ◽  
Lactococcus Lactis Subsp ◽  
Lean Fish ◽  
Fermentation Strategy

Lactococcus lactis subsp. lactis strain CWBI B1410, which produces various antibacterial compounds including organic acids and nisin, was used as a starter culture to improve the traditional Senegalese fish fermentation in which fish are mostly transformed to guedj by spontaneous fermentation for 24 to 48 h at ambient temperatures near 30°C followed by salting (with NaCl) and sun drying. Assays were performed on lean fish (Podamasys jubelini) and fat fish (Arius heudelotii) purchased at a local market. The total viable microbial counts in raw fillets of P. jubelini and A. heudelotii were 5.78 and 5.39 log CFU/g, respectively. Populations of enteric bacteria (which can include pathogenic bacteria) in P. jubelini and A. heudelotii were 4.08 and 4.12 log CFU/g, respectively. Spontaneous fermentation of raw fillets at 30°C led to the proliferation of enteric bacteria to 9 log CFU/g after 24 h in fermented P. jubelini and A. heudelotii fillets with pH values of 6.83 and 7.50, respectively. When raw fish fillets were supplemented with glucose (1%, wt/wt) and inoculated with Lactococcus lactis (107 CFU/g), the pH decreased to about 4.60 after 10 h at 30°C, and nisin activity was detected in juice from the fillets. Traditionally fermented fillets of P. jubelini and A. heudelotii contained enteric bacteria at higher levels of 4 and 2 log CFU/g, respectively, than did fillets of the same fish supplemented with glucose and fermented with the starter culture. These data suggest that this new fish fermentation strategy combined with salting and drying can be used to enhance the safety of guedj.

Sign in / Sign up

Export Citation Format

Share Document