Antilisterial activity of raw sheep milk from two native Epirus breeds: Culture-dependent identification, bacteriocin gene detection and primary safety evaluation of the antagonistic LAB biota.

Raw milk from native small ruminant breeds in Epirus, Greece, is a valuable natural source of autochthonous lactic acid bacteria (LAB) strains with superior biotechnological properties. In this study, two bulk milks (RM1, RM2) from two local sheep yards, intended for traditional Kefalotyri cheese production, were preselected for bacteriocin-like antilisterial activity by in vitro tests. Their antagonistic LAB biota was quantified followed by polyphasic (16S rRNA gene sequencing; IGS for Enterococcus; a multiplex-PCR for Leuconostoc) identification of 42 LAB (RM1/18; RM2/24) isolates further evaluated for bacteriocin encoding genes and primary safety traits. Representative isolates of the numerically dominant mesophilic LAB were Leuconostoc mesenteroides (10) in both RMs, Streptococcus parauberis (7) in RM2, and Lactococcus lactis (1) in RM1; the subdominant thermophilic LAB isolates were Enterococcus durans (8), E. faecium (6), E. faecalis (3), E. hirae (1), E. hermanniensis (1), Streptococcus lutetiensis (2), S. equinus (1) and S. gallolyticus (1). Based on their rpoB, araA, dsr and sorA profiles, six Ln. mesenteroides strains (8 isolates) were atypical lying between the subspecies mesenteroides and dextranicum, whereas two strains profiled with Ln. mesenteroides subsp. jonggajibkimchi that is first-time reported in Greek dairy food. Two RM1 E. faecium strain biotypes (3 isolates) showed strong, enterocin-mediated antilisterial activity due to entA/entB/entP possession. One E. durans from RM1 possessed entA and entP, while additional nine RM2 isolates of the E. faecium/durans group processed entA or entP singly. All showed direct (cell-associated) antilisterial activity only, as also both S. lutetiensis strains from RM2 did strongly. Desirably, no LAB isolate was ß-hemolyrtic, or cytolysin-positive, or possessed vanA, vanB for vancomycin resistance, or agg, espA, hyl, and IS16 virulence genes. However, all three E. faecalis from RM2 possessed gelE and/or ace virulence genes. In conclusion, all Ln. mesenteroides strains, the two safe, enterocin A-B-P-producing E. faecium strains, and the two antilisterial S. lutetiensis strains should be validated further as potential costarter or adjunct cultures in Kefalotyri cheese. The prevalence of α-hemolytic pyogenic streptococci in raw milk, mainly S. parauberis in RM2, requires consideration in respect to subclinical mastitis in sheep and the farm hygiene overall.

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.

Effective biopreservation measures are needed to control the growth of postprocess Listeria monocytogenes contamination in fresh whey cheeses stored under refrigeration. This study assessed growth and biocontrol of inoculated (3 log10 CFU/g) L. monocytogenes in vacuum-packed, fresh (1-day-old) or 'aged' (15-day-old) Anthotyros whey cheeses, without or with 5% of a crude enterocin A-B-P extract (CEntE), during storage at 4 °C. Regardless of CEntE addition, the pathogen increased by an average of 2.0 log10 CFU/g in fresh cheeses on day 15. Gram-negative spoilage bacteria also increased by an average of 2.5 log10 CFU/g. However, from day 15 to the sell-by date (days 35-40), L. monocytogenes growth ceased, and progressively, the populations of the pathogen declined in most cheeses. This was due to an unmonitored, batch-dependent natural acidification by spoilage lactic acid bacteria, predominantly Leuconostoc mesenteroides, which reduced the cheese pH to 5.5, and finally to ≤5.0. The pH reductions and associated declines in pathogen viability were greater in the CEntE-treated samples within each batch. L. monocytogenes failed to grow in cheeses previously 'aged' in retail for 15 days. Overall, high population levels (>7.5 log10 CFU/g) of psychrotrophic Enterobacteriaceae, particularly Hafnia alvei, were associated with an extended growth and increased survival of L. monocytogenes during storage.

Assessment of the Spoilage Microbiota during Refrigerated (4 °C) Vacuum-Packed Storage of Fresh Greek Anthotyros Whey Cheese without or with a Crude Enterocin A-B-P-Containing Extract.

Although fresh whey cheeses are prone to rapid deterioration, mainly by psychrotrophic Gram-negative bacteria and lactic acid bacteria (LAB), data on the specific spoilage species in traditional Greek whey cheeses are scarce. Therefore, this study quantified growth and characterized the primary spoilage bacteria in fresh Anthotyros whey cheeses stored at 4 °C in a vacuum for 40 days, without or with an added 5% (v/w) of an enterocin A-B-P crude extract (CEntE). Psychrotrophic Pseudomonas spp., Aeromonas spp., Hafnia spp. and Serratia spp. grew faster than LAB during early storage. However, LAB outgrew the Gram-negative bacteria and prevailed by mid to late storage in all cheese batches, causing a strong or milder batch-dependent natural acidification. Two major non-slime-producing and two minor biotypes of Leuconostoc-like bacteria, all identified as Leuconostoc mesenteroides by 16S rRNA sequencing, dominated the LAB association (76.7%), which also included four subdominant Carnobacterium maltaromaticum biotypes (10.9%), one Leuconostoc lactis biotype (3.3%) and few Lactococcus (1.6%), mesophilic Lactobacillus (0.8%) and Enterococcus (0.8%). Growth and distribution of LAB and Gram-negative species were strongly batch-dependent and plant-dependent. The CEntE neither retarded growth nor altered the whey cheese spoilage association but enhanced LAB growth and the declines of Gram-negative bacteria by late storage.

Assessment of the spoilage microbiota in minced free-range chicken meat during storage at 4 C in retail modified atmosphere packages.

This study assessed the evolution of spoilage microbiota in association with the changes in pH and concentrations of lactic and acetic acids in retail oxygen-free modified atmosphere (30:70 CO2/N2) packages (MAP) of minced free-range chicken meat during storage at 4 °C for 10 days. MAP retarded growth of spoilage lactic acid bacteria (LAB) below 6.5 log cfu/g and fully suppressed growth of pseudomonads, enterobacteria, enterococci, staphylococci and yeasts. Two distinct Latilactobacillus sakei strain biotypes were predominant and Leuconostoc carnosum, Carnobacterium divergens, Latilactobacillus fuchuensis and Weissella koreensis were subdominant at spoilage. The chicken meat pH ranged from 5.8 to 6.1. l-lactate (832 mg/100 g on day-0) decreased slightly on day-7. d-lactate remained constantly below 20 mg/100 g, whereas acetate (0-59 mg/100 g) increased 5-fold on day-7. All MAP samples developed off-odors on day-7 and a strong 'blown-pack' sulfur-type of spoilage on day-10. However, neither the predominant Lb. sakei nor other LAB or gram-negative isolates formed H2S in vitro, except for C. divergens.

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

Autochthonous single (Ent+) or multiple (m-Ent+) enterocin-producing strains of dairy enterococci show promise for use as bioprotective adjunct cultures in traditional cheese technologies, provided they possess no pathogenic traits. This study evaluated safety, decarboxylase activity, and enzymatic (API ZYM) activity profiles of nine Ent+ or m-Ent+ Greek cheese isolates previously assigned to four distinct E. faecium (represented by the isolates KE64 (entA), GL31 (entA), KE82 (entA-entB-entP) and KE77 (entA-entB-entP-bac31)) and two E. durans (represented by the isolates KE100 (entP) and KE108 (entP-bac31-cyl)) strain genotypes. No strain was ß-hemolytic or harbored vanA and vanB or the virulence genes agg, ace, espA, IS16, hyl, or gelE. All strains were of moderate to high sensitivity to ampicillin, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, penicillin, tetracycline, and vancomycin, except for the E. faecium KE64 and KE82 strains, which were resistant to erythromycin and penicillin. All cheese strains showed moderate to strong esterase-lipase and aminopeptidase activities and formed tyramine, but none formed histamine in vitro. In conclusion, all Ent+ or m-Ent+ strain genotypes of the E. faecium/durans group, except for the cyl-positive E. durans KE108, were safe for use as adjunct cultures in traditional Greek cheeses. Further in situ biotechnological evaluations of the strains in real cheese-making trials are required.

Growth Inhibitory and Selective Pressure Effects of Sodium Diacetate on the Spoilage Microbiota of Frankfurters Stored at 4 °C and 12 °C in Vacuum.

This study evaluated microbial growth in commercial frankfurters formulated with 1.8% sodium lactate (SL) singly or combined with 0.25% sodium diacetate (SDA), vacuum-packaged (VP) and stored at 4 °C and 12 °C. Standard frankfurters without SDA, containing 0.15% SL, served as controls (CN). Lactic acid bacteria (LAB) were the exclusive spoilers in all treatments at both storage temperatures. However, compared to the CN and SL treatments, SL + SDA delayed growth of LAB by an average of 5.1 and 3.1 log units, and 3.0 and 2.0 log units, respectively, after 30 and 60 days at 4 °C. On day 90, the SL + SDA frankfurters were unspoiled whereas the SL and CN frankfurters had spoiled on day 60 and day 30 to 60, respectively. At 12 °C, LAB growth was similar in all treatments after day 15, but strong defects developed in the CN and SL frankfurters only. Differential spoilage patterns were associated with a major reversal of the LAB biota from gas- and slime-producing Leuconostoc mesenteroides and Leuconostoc carnosum in the CN and SL frankfurters to Lactobacillus sakei/curvatus in the SL + SDA frankfurters. Thus, SL + SDA extends the retail shelf life of VP frankfurters by delaying total LAB growth and selecting for lactobacilli with a milder cured meat spoilage potential than leuconostocs, particularly under refrigeration.

Application of Enterococcus faecium KE82, an Enterocin A-B-P-Producing Strain, as an Adjunct Culture Enhances Inactivation of Listeria monocytogenes during Traditional Protected Designation of Origin Galotyri Processing.

ABSTRACT: The ability of the enterocin A-B-P-producing Enterococcus faecium KE82 adjunct strain to inactivate Listeria monocytogenes during protected designation of origin Galotyri processing was evaluated. Three trials were conducted with artisan cheeses made from traditionally "boiled" (85°C) ewe's milk. The milk was cooled at 42°C and divided in two treatments. A1 milk was inoculated with Streptococcus thermophilus ST1 and Lactococcus lactis subsp. cremoris M78, and A2 was inoculated with the basic starter ST1+M78 plus KE82 (step 1). All milks were fermented at 20 to 22°C for 24 h (step 2), and the curds were drained at 12°C for 72 h (step 3) and then salted with 1.5 to 1.8% salt to obtain the fresh Galotyri cheeses (step 4). These fresh cheeses were then ripened at 4°C for 30 days (step 5). Because artificial listerial contamination in the dairy plant was prohibited, samples of A1 and A2 cheese milk (200 mL) or curd (200 g) were collected after steps 1 through 5, inoculated with L. monocytogenes 10 (3 to 4 log CFU/mL or g), incubated at 37, 22, 12, and 4°C for predefined periods, and analyzed for microbial levels and pH. L. monocytogenes levels declined in all cheese curd portions contaminated after steps 2 through 5 (pH 4.36 to 4.84) when stored at 4 or 12°C for 15 days. The final net reductions in Listeria populations were 2.00-, 1.07-, 0.54-, and 0.61-log greater in the A2 than in the A1 curd portions after steps 2, 3, 4, and 5, respectively. In step 1, conducted to simulate the whole cheese milk fermentation process, L. monocytogenes levels declined by 1.47 log CFU/mL more in the A2 than in the A1 milk portions after 72 h at 22°C; however, slight growth (0.6 log CFU/mL) occurred during the first 6 h at 37°C. E. faecium KE82 was compatible with the starter culture and enhanced inactivation of L. monocytogenes during all steps of Galotyri cheese processing. The antilisterial effects of the combined acid and enterocin were the weakest in the fermenting milks, the strongest in the unsalted fermented curds, and declined again in the salted fresh cheeses.

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 lactic acid bacterial (LAB) species and in situ expression of bacteriocin genes in the mixtures is crucial. This study first aimed to monitor the growth of mixed LAB strain populations during milk model fermentations by microbial counts and real-time quantitative PCR. The primary starter strains, Streptococcus thermophilus ST1 and costarter Lactococcus lactis subsp. cremoris M78, served as the basic starter composite coinoculated in all milk treatments. Adjunct bacteriocinogenic Enterococcus faecium strains KE82 and GL31 and the 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 strains M78, KE82, and GL31, respectively, by reverse transcription-real-time quantitative PCR 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 2- to 3-log-unit increases 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, the expression levels of nisin and enterocin A and B genes were interrelated, indicating an antagonistic activity.

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.

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.

Structural enterocin gene profiles and mode of antilisterial activity in synthetic liquid media and skim milk of autochthonous Enterococcus spp. isolates from artisan Greek Graviera and Galotyri cheeses.

The presence of eight common structural enterocin genes, singly or in varying combinations, in the genome of 15 antagonistic Enterococcus spp. previously isolated from artisan Greek Graviera and Galotyri retail cheeses was tested and associated with the mode of enterocin (Ent+) antilisterial activity of each isolate in three liquid culture media. The isolates were assigned to nine distinct strain genotypes of E. faecium (4 strains), E. durans (2) and E. faecalis (3). All strains were non-hemolytic, except for a cyl-positive E. faecalis genotype isolated from Galotyri cheese, which was strongly listericidal. All other strains varied from being listeriostatic to weakly listericidal in MRS and M17 broth, whereas all failed to inhibit listerial growth in skim milk. Two E. faecium strains retained strong Ent+ activity following neutralization and filter-sterilization of their MRS or M17 co-culture supernatants, whereas, all others required contact or proximity of their viable cells with L. monocytogenes cells in order to display activity. Additional studies to evaluate safety and potential synergistic effects of each strain genotype with starter LAB species in real milk environments will reveal the most active and truly harmless Enterococcus genotypes to be applied as co-starter or bioprotective adjunct cultures in traditional Greek cheese technologies.

Major ecological shifts within the dominant nonstarter lactic acid bacteria in mature Greek Graviera cheese as affected by the starter culture type.

Traditional Greek Graviera cheese is often produced from thermized milk to control undesirable bacterial contaminants. Since thermization also reduces the desirable lactic acid bacteria (LAB) microbiota of raw milk, natural undefined or commercially defined starters are used. This study evaluated effects of the type of starter added to bulk thermized milk on the microbiology of mature (day-90) Graviera cheese. Cheeses produced with a natural starter culture (NSC) in non-concentrated yogurt-like form or a commercial starter culture (CSC) containing Streptococcus thermophilus and various Lactococcus lactis strains in concentrated freeze-dried form, were analyzed microbiologically, and 200 LAB isolates (100 from each type of cheese) were identified. The LAB microbiota of the mature CSC-cheeses was dominated by nonstarter strains of Lactobacillus paracasei and Lb. plantarum whereas indigenous Enterococcus faecium and E. durans strains of high phenotypic and genotypic diversity predominated in the respective NSC-cheeses. Populations of enterococci in CSC-cheeses were subdominant by 10 to 100-fold compared with those in NSC-cheeses; E. faecium was the most frequently isolated Enterococcus species from the mature CSC-cheeses. Sporadic or no isolates of other LAB species, including the commercial S. thermophilus and Lc. lactis starter strains in the CSC-cheeses and the natural S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus starter strains plus indigenous Lactococcus, Leuconostoc and E. faecalis in the NSC-cheeses, were detected. In conclusion, the replacement of the NSC with the CSC controlled growth of dairy enterococci in favor of mesophilic nonstarter lactobacilli during ripening. While safety concerns associated with the inefficiency of NSCs to prevent outgrowth of indigenous enterococci suggest that CSCs should be preferred by traditional Greek Graviera cheese processors, panel sensory evaluations showed that the NSC-ripened cheeses were of slightly lower appearance but of occasionally higher flavor scores than the CSC-ripened cheeses.

Approaches for enhancing in situ detection of enterocin genes in thermized milk, and selective isolation of enterocin-producing Enterococcus faecium from Baird-Parker agar.

Enterococci are naturally selected for growth in thermized ewes'/goats' milk mixtures used for traditional cooked hard cheese processing in Greece. A culture-independent PCR-based approach was applied to detect the presence of enterocin-encoding genes in naturally culture-enriched thermized milk (TM). Portions of TM (63 °C, 30 s) collected from a commercial cheese plant before addition of starters were fermented at 37 °C for 48 h to facilitate growth of indigenous enterococci. The multiple enterocin-producing (m-Ent+) Enterococcus faecium KE82 and the nisin A-producing Lactococcus lactis subsp. cremoris M104 served as bacteriocin-positive inocula in separate TM treatments. The PCR results revealed a constant presence of the enterocin A, B and P genes in TM fermented naturally at 37 °C. Eleven out of 42 (26.2%) lactic isolates from the enriched TM cultures without inoculation were Ent+ E. faecium assigned to three biotypes. Biotype I (4 isolates) included single entA possessors, whereas biotype II (5 isolates) and biotype III (2 isolates) were m-Ent+ variants profiling entA-entB-entP and entA-entB genes, respectively. Biotype II displayed the strongest antilisterial activity in vitro. Surprisingly, 85.7% (6/7) of the m-Ent+ E. faecium were selectively isolated from Baird-Parker agar, reflecting their natural resistance to 0.01% tellurite contained in the egg yolk supplement. No cytolysin-positive E. faecalis or other Ent+ Enterococcus spp. were isolated. In conclusion, commercially thermized Greek milk is a natural pool or 'reservoir' of antagonistic Ent+ or m-Ent+ E. faecium strains that can be easily detected and recovered by applying this PCR-based approach to naturally fermented milks or cheese products.

Hurdle factors minimizing growth of Listeria monocytogenes while counteracting in situ antilisterial effects of a novel nisin A-producing Lactococcus lactis subsp. cremoris costarter in thermized cheese milks.

The capacity of growth, survival, and adaptive responses of an artificial contamination of a three-strain L. monocytogenes cocktail in factory-scale thermized (65 °C, 30 s) Graviera cheese milk (TGCM) was evaluated. Bulk TGCM samples for inoculation were sequentially taken from the cheese making vat before process initiation (CN-LM) and after addition of a commercial starter culture (CSC), the CSC plus the nisin A-producing (NisA+) costarter strain Lactococcus lactis subsp. cremoris M78 (CSC + M78), and all ingredients with the rennet last (CSC + M78-RT). Additional treatments included Listeria-inoculated TGCM samples coinoculated with the NisA+ costarter strain M78 in the absence of the CSC or with the CSC in previously sterilized TGCM to inactivate the background microbiota (CSC-SM). All cultures were incubated at 37 to 42 °C for 6 h, followed by additional 66 h at 22 °C, and 48 h at 12 °C after addition of 2% edible salt. L. monocytogenes failed to grow and declined in all CSC-inoculated treatments after 24 h. In contrast, the pathogen increased by 3.34 and 1.46 log units in the CN-LM and the CSC-SM treatments, respectively, indicating that the background microbiota or the CSC alone failed to suppress it, but they did so synergistically. Supplementation of the CSC with the NisA+ strain M78 did not deliver additional antilisterial effects, because the CSC Streptococcus thermophilus reduced the growth prevalence rates and counteracted the in situ NisA+ activity of the costarter. In the absence of the CSC, however, strain M78 predominated and caused the strongest in situ nisin-A mediated effects, which resulted in the highest listerial inactivation rates after 24 to 72 h at 22 °C. In all TGCM treatments, however, L. monocytogenes displayed a "tailing" survival (1.63 to 1.96 log CFU/mL), confirming that this pathogen is exceptionally tolerant to cheese-related stresses, and thus, can't be easily eliminated.

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

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.

Enhanced Control of Listeria monocytogenes by Enterococcus faecium KE82, a Multiple Enterocin-Producing Strain, in Different Milk Environments.

Enterococcus faecium KE82, isolated from traditional Greek Graviera cheese, was identified in pure broth cultures in vitro as a multiple enterocin-producing bacterial strain possessing the structural entA, entB, and entP enterocin genes. E. faecium KE82 was further assessed for in situ antilisterial activity in raw milk (RM) and commercially thermized milk (TM; 63°C for 30 s) in the presence of the indigenous microbiota and in sterile raw milk (SRM; 121°C for 5 min) with or without the addition of two commercial starter culture (CSC) strains Streptococcus thermophilus and Lactococcus lactis . Growth of Listeria monocytogenes was completely inhibited in RM incubated at 37°C for 6 h, whereas the pathogen was significantly inactivated in RM+KE82 samples during further incubation at 18°C for 66 h. In contrast, L. monocytogenes levels increased by approximately 2 log CFU/ml in TM, but in TM+KE82 samples, pathogen growth was retarded during the first 6 h at 37°C followed by growth cessation and partial inactivation at 18°C. After 48 to 72 h, growth of L. monocytogenes in SRM+CSC samples decreased by 4 to 5 log CFU/ml compared with the SRM control, whereas additional 10-fold decreases in the pathogen were observed in SRM+CSC+KE82 samples. Reverse transcription PCR analysis of SRM+KE82 and SRM+CSC+KE82 samples confirmed that the entA and entB genes were transcribed, but entP gene transcription was not detected. All RM and SRM samples inoculated with E. faecium KE82 displayed strong in situ inhibitory activity against L. monocytogenes in well diffusion bioassays, whereas activity was weaker to undetectable in comparable or additional TM+KE82 samples; no milk sample without E. faecium KE82 had activity against L. monocytogenes . The findings of this study indicate that E. faecium KE82 is an antilisterial agent that could be used in traditional dairy foods because it concomitantly produces enterocins A and B in situ in milk.

Growth interactions and antilisterial effects of the bacteriocinogenic Lactococcus lactis subsp. cremoris M104 and Enterococcus faecium KE82 strains in thermized milk in the presence or absence of a commercial starter culture.

Traditional Greek cheeses are often produced from thermized milk (TM) with the use of commercial starter cultures (CSCs), which may not inhibit growth of Listeria monocytogenes completely. Therefore, this study evaluated the behavior of an artificial L. monocytogenes contamination in commercially TM (63 °C; 30 s) inoculated with a CSC plus Lactococcus lactis subsp. lactis M104 and/or Enterococcus faecium KE82, two indigenous strains producing nisin A and enterocin A and B, respectively. Inoculation treatments included TM with the CSC only, and TM without the CSC but with strain M104 alone, or combined with strain KE82. All treatments were incubated at 37 °C for 6 h followed by 66 h at 18 °C. L. monocytogenes grew by 0.66-1.24 log cfu/ml at 37 °C, whereas its further growth at 18 °C was retarded, suppressed, or accompanied by different inactivation rates, depending on each TM treatment. Strain M104 caused the greatest inactivation, whereas the CSC per se was the least effective treatment. Strain KE82 assisted the CSC in controlling pathogen growth at 37 °C, whereas both reduced the nisin A-mediated antilisterial activity of strain M104. Overall, the most 'balanced' treatment against L. monocytogenes was CSC+M104+KE82. Hence, this starter/co-starter combination may be utilized in traditional Greek cheese technologies.

Microbial Quality of and Biochemical Changes in Fresh Soft, Acid-Curd Xinotyri Cheese Made from Raw or Pasteurized Goat's Milk.

The microbiological quality of and changes in the main physicochemical parameters, together with the evolution of proteolysis, lipolysis and volatile profiles of soft Xinotyri, a traditional Greek acid-curd cheese (pH≈4.4, moisture 65%, salt 1%) made from raw (RMC) or pasteurized (PMC) goat's milk without starters, were evaluated during aerobic storage at 4 oC for 60 days. No statistically significant differences between the total nitrogen (TN) and nitrogen fraction (% of TN) contents, the degradation of intact αs- or ß-caseins, total free amino acid (FAA) contents, and the ratio of hydrophilic and hydrophobic peptides in the water-soluble fraction of RMC and PMC were found. Threonine, alanine and lysine were the principal FAAs. Oleic, palmitic, capric and caprylic acids, and ethyl hexonate, ethyl octanoate, ethyl decanoate, ethanol, 3-methyl butanol, phenyl ethyl alcohol and acetone were the most abundant free fatty acids and volatile compounds, respectively. Cheese lipolysis evolved slowly at 4 oC, and milk pasteurization had no significant effect on it. Mesophilic lactic acid bacteria (LAB) were predominant in fresh cheese samples. PMC samples had significantly lower levels of enterococci and enterobacteria than RMC samples, while yeasts grew at similar levels during storage at 4 oC. All cheese samples (25 g) were free of Salmonella and Listeria monocytogenes. Coagulase--positive staphylococci exceeded the 5-log safety threshold in fresh RMC samples, whereas they were suppressed (<100 CFU/g) in all PMC samples. Consequently, pasteurization of raw goat milk's and utilization of commercially defined or natural mesophilic LAB starters are recommended for standardizing the biochemical, microbial and safety qualities of fresh soft Xinotyri cheese.

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.

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 co-inoculated (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 z 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.

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.

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.

Characterization of a wild, novel nisin a-producing Lactococcus strain with an L. lactis subsp. cremoris genotype and an L. lactis subsp. lactis phenotype, isolated from Greek raw milk.

Several molecular taxonomic studies have revealed that many natural (wild) Lactococcus lactis strains of dairy origin which are phenotypically representative of the L. lactis subspecies lactis cluster genotypically within subspecies cremoris and vice versa. Recently, we isolated two wild nisin-producing (Nis(+)) L. lactis strains, M78 and M104, of the lactis phenotype from Greek raw milk (J. Samelis, A. Lianou, A. Kakouri, C. Delbès, I. Rogelj, B. B. Matijasic, and M. C. Montel, J. Food Prot. 72:783-790, 2009); strain M78 possess a novel nisin A sequence (GenBank accession number HM219853). In this study, the actual subspecies identity of M78 and M104 isolates was elucidated, using 16S rRNA and acmA (encoding lactococcal N-acetylmuramidase) gene and histidine biosynthesis operon polymorphisms and 16S rRNA and ldh (encoding lactate dehydrogenase) gene phylogenies. Except the acmA gene analysis, molecular tools revealed that isolates M78 and M104 clustered with strains of the cremoris genotype, including the LMG 6897(T) strain, while they were distant from strains of the lactis genotype, including the LMG 6890(T) strain. The two wild isolates had identical repetitive sequence-based PCR (rep-PCR), randomly amplified polymorphic DNA (RAPD), plasmid, and whole-cell protein profiles and shared high 16S rRNA (99.9%) and ldh (100%) gene sequence homologies. In contrast, they exhibited identical sugar fermentation and enzymatic patterns which were similar to those of the subspecies lactis LMG 6890(T) strain. To our knowledge, this is the first complete identification report on a wild L. lactis subsp. cremoris genotype of the lactis phenotype which is capable of nisin A production and, thus, has strong potential for use as a novel dairy starter and/or protective culture.