Assessing the proteolytic and lipolytic activities of single strains of mesophilic lactobacilli as adjunct cultures using a Caciotta cheese model system

Seventeen strains of mesophilic lactic acid bacteria, isolated from cheese (non-starter lactic acid bacteria, NSLAB) or sourdough, were used individually as adjunct cultures in a Caciotta cheese model system. Adjunct cultures were monitored by randomly amplified polymorphic DNA analysis and their cell counts mainly varied from ca. 9.0 to 8.0 log cfu g⁻¹ throughout 36 days of ripening. Adjunct cultures influenced differently cheese proteolysis. Both NSLAB and sourdough strains caused an extensive secondary proteolysis; however, some NSLAB strains produced the highest concentration of free amino acids. Principal component analysis (PCA) differentiated cheeses manufactured with NSLAB strains Lactobacillus parabuckneri B₉F_ST, Lb. paracasei B₆₁F₅, Lb. curvatus 2768 and Lb. rhamnosus ATCC 7469 based on the accumulation of Lys, Glu, Phe, Hist, Asp and Met. Assessment of cheese lipolysis showed that: (i) highest concentrations of free fatty acids (FFA) were found with NSLAB strains Lb. rhamnosus ATCC 7469 and Lb. casei subsp. pseudoplantarum 2742 (ca. 10 500 mg kg⁻¹); (ii) PCA differentiated cheeses manufactured with NSLAB strains Lb. rhamnosus ATCC 7469 and Lb. casei subsp. pseudoplantarum 2742 based on the accumulation of palmitic (C16:0) and linoleic (C18:2) acids, and those with Lb. curvatus 2768 and Lb. parabuckneri B₉F_ST based on the high concentration of short chain FFA; (iii) the cheese made with sourdough strain Lb. sanfranciscensis CB1 had the highest levels of unsaturated FFA.     

Proteolytic pattern and organic acid profiles of probiotic Cheddar cheese as influenced by probiotic strains of Lactobacillus acidophilus,Lb. paracasei,Lb. casei or Bifidobacterium sp.

Cheddar cheeses were produced with starter lactococci and Bifidobacterium longum 1941, B. lactis LAFTI^® B94, Lactobacillus casei 279, Lb. paracasei LAFTI^® L26, Lb. acidophilus 4962 or Lb. acidophilus LAFTI^® L10 to study the survival of the probiotic bacteria and the influence of these organisms on proteolytic patterns and production of organic acid during ripening period of 6 months at 4 °C. All probiotic adjuncts survived the manufacturing process of Cheddar cheese at high levels without alteration to the cheese-making process. After 6 months of ripening, cheeses maintained the level of probiotic organisms at >8.0 log₁₀ cfu g⁻¹ with minimal effect on moisture, fat, protein and salt content. Acetic acid concentration was higher in cheeses with B. longum 1941, B. lactis LAFTI^® B94, Lb. casei 279 and Lb. paracasei LAFTI^® L26. Each probiotic organism influenced the proteolytic pattern of Cheddar cheese in different ways. Lb. casei 279 and Lb. paracasei LAFTI^® L26 showed higher hydrolysis of casein. Higher concentrations of free amino acids (FAAs) were found in all probiotic cheeses. Although Bifidobacterium sp. was found to be weakly proteolytic, cheeses with the addition of those strains had highest concentration of FAAs. These data thus suggested that Lb. acidophilus 4962, Lb. casei 279, B. longum 1941, Lb. acidophilus LAFTI^® L10, Lb. paracasei LAFTI^® L26 and B. lactis LAFTI^® B94 can be applied successfully in Cheddar cheese.     

Contribution of autochthonous non-starter lactobacilli to proteolysis in Caciocavallo Pugliese cheese

Caciocavallo Pugliese cheese was manufactured according to a traditional protocol and with added autochthonous Lactobacillus paracasei subsp. paracasei and L. parabuchneri strains, isolated from a well-flavoured Caciocavallo Pugliese cheese, to evaluate their contribution to the cheese biochemical characteristics. Using a “two step REP-PCR” protocol, L. paracasei subsp. paracasei strains were shown to sustain high viability during ripening, while the L. parabuchneri strains were not recovered. The inoculated cheese showed higher levels of free amino acids, as well as differences in the profiles of individual free amino acids in comparison with the control cheese. The addition of autochthonous Lactobacillus strains with interesting technological properties in Caciocavallo Pugliese cheese manufacturing could make it feasible to improve cheese processing, while still maintaining the sensory characteristics of this typical pasta-filata cheese.     

Effect of acidification on the activity of probiotics in yoghurt during cold storage

The viability of Lactobacillus acidophilus LAFTI^® L10, Bifidobacterium lactis LAFTI^® B94, and L. paracasei LAFTI^® L26 and their proteolytic activities were assessed in yoghurt at different termination pH of 4.45, 4.50, 4.55, and 4.60 in the presence of L. delbrueckii ssp. bulgaricus Lb1466 and Streptococcus thermophilus St1342 during 28 days of storage at 4 °C. All strains achieved the recommended level of 6.00 log cfu g⁻¹ of the product with L. acidophilus LAFTI^® L10 and L. paracasei LAFTI^® L26 exceeding the number to 8.00 and 7.00 log cfu g⁻¹, respectively. Lactobacilli strains showed a good cellular stability maintaining constant concentration throughout storage period regardless of termination pH. On the other hand, the cell counts of B. lactis LAFTI^® B94 decreased by one log cycle at the end of storage. The presence of probiotic organisms enhanced proteolysis significantly in comparison with the control batch containing L. delbrueckii ssp. bulgaricus Lb1466 and S. thermophilus St1342 only. The proteolytic activity varied due to termination pH, but also appeared to be strain related. The increased proteolysis improved survival of L. delbrueckii ssp. bulgaricus Lb1466 during storage resulting in lowering of pH and production of higher levels of organic acids, which might have caused the low cell counts for B. lactis LAFTI^® B94.     

Influence of probiotic bacteria on the proteolysis profile of a semi-hard cheese

Two probiotic strains, Lactobacillus acidophilus and Lactobacillus paracasei subsp. paracasei, were used as adjunct cultures in semi-hard cheesemaking experiments, in order to study their influence on proteolysis during ripening. Cheeses with and without probiotic bacteria were manufactured. The population of probiotics remained above 10⁷ cfu g⁻¹ during all ripening, and they did not influence primary proteolysis. However, L. acidophilus produced a significant increase in the level of low molecular weight nitrogen compounds and individual free amino acids; the amino acid profiles were also different. Multivariate analysis of peptide profiles showed that samples were grouped mainly by ripening time, although the impact of probiotics was also noticeable. L. acidophilus showed a clear influence on secondary proteolysis, while a minor effect of L. paracasei was evidenced at the end of the ripening. These results showed that the tested strains influenced distinctly proteolysis of cheeses, probably as a consequence of their different proteolytic systems and their activity via the alimentary matrix (cheese).     

Mesophilic lactobacilli in Fiore Sardo cheese: PCR-identification and evolution during cheese ripening

The mesophilic lactobacilli colonizing Fiore Sardo ewe's milk cheese were characterized. They seemed to be the dominant non-starter lactic acid bacteria composing its natural microflora, with a viable cell number varying from 10⁵ CFU g⁻¹ (1-day-old cheese) to 10⁸ CFU g⁻¹ (30-day-old cheese) and then slowly decreasing up to 10⁴ CFU g⁻¹ after 7 months’ ripening. Considering the relevance of mesophilic lactobacilli in affecting the cheese ripening, a PCR-based taxonomic identification of the Lactobacillus species isolated was performed. Cheese samples were collected from 3 farms and 457 isolates from cheeses at different ripening times were analysed with species-specific primers for L. plantarum, L. casei group, L. paracasei, L. casei, L. rhamnosus, L. pentosus, L. paraplantarum, L. curvatus, L. graminis and L. sake. L. plantarum and L. paracasei were the most frequently detected species. Moreover, the development and the evolution during ripening of the facultatively heterofermentative Lactobacillus species (FHL) were different in the three batches of cheese.     

A combined morphologic and molecular approach for characterizing fungal microflora from a traditional Italian cheese (Fossa cheese)

Phenotypic and genotypic methods were used to identify filamentous fungi that characterize traditional Italian Fossa cheese and its ripening environment. After ageing for 60 days at a dairy, it was ripened for an additional three months in a pit. In the fully ripened cheese, moulds ranged from 3 to 3.4 log cfu g^?1 and Penicillium was the prevalent species. Pit environmental fungi ranged from 530 to 750 cfu m^?3 (air) and from 130 to 340 cfu cm^?2 (surfaces). The dominant pit strains were Alternaria spp., Aspergillus spp., Cladosporium spp. and Penicillium spp. Phylogenetic analyses of 18S rRNA gene and ITS1-5.8S-ITS2 regions highlighted Penicillium camemberti, Aspergillus nidulans and Aspergillus versicolor as traceable species occurring in both the cheese and pit environment, suggesting their involvement in the development of typical Fossa cheese characteristics. This approach may be used for the identification of microflora on other cheese varieties to better understand the fungal contribution in cheese ripening.     

Heat resistance of Lactobacillus paracasei isolated from semi-hard cheese made of pasteurised milk

Nine strains of non-starter Lactobacillus paracasei isolated from semi-hard cheese made of pasteurised milk were selected for their anticlostridial activity. Resistance to thermisation (60 °C, 5 min) and pasteurisation (73 °C, 15 s) was investigated using a submerged-coil apparatus. MRS broth-grown cultures of all nine strains survived thermisation in buffer. The level of resistance to thermisation was strain dependent and lower for freshly grown cells (stationary phase cells) than for resting cells (freshly grown cells kept diluted 10-fold in MRS broth at 17 °C for 6 days). None of the nine Lb. paracasei strains survived or recovered after pasteurisation in buffer when grown in MRS broth, while seven of the nine strains survived pasteurisation in UHT whole milk when grown in milk. Identity of the strains was successfully confirmed during the experiments using repetitive-PCR analysis. The potential of Lb. paracasei strains to survive pasteurisation of cheese milk was demonstrated.     

Effect of combinations of high-pressure treatment and bacteriocin-producing lactic acid bacteria on the survival of Listeria monocytogenes in raw milk cheese

The combined effect of high-pressure (HP) treatment and bacteriocin-producing lactic acid bacteria (BP-LAB) on the survival of Listeria monocytogenes Scott A in cheeses made from raw milk that was inoculated with the pathogen at 4.80 log cfu mL⁻¹, a commercial starter and one of seven strains of BP-LAB was investigated. On day 3, the counts of L. monocytogenes were 7.03 log cfu g⁻¹ in a control cheese (without BP-LAB, not HP treated), 6.06–6.74 log cfu g⁻¹ in cheeses with BP-LAB, 6.13 log cfu g⁻¹ in a cheese without BP-LAB and treated on day 2 at 300 MPa, 2.01 log cfu g⁻¹ in a cheese without BP-LAB and treated on day 2 at 500 MPa, 3.83–5.43 log cfu g⁻¹ in cheeses with BP-LAB and treated on day 2 at 300 MPa, and 1.81 log cfu g⁻¹ or less in cheeses with BP-LAB and treated on day 2 at 500 MPa. HP treatment was more effective on day 51 than on day 2.     

Fermentation of carbohydrates from cheese sources by non-starter lactic acid bacteria isolated from semi-hard Danish cheese

Carbohydrate fermentation of 45 isolates of non-starter lactic acid bacteria from Danish semi-hard cheeses was studied using BioScreen C equipment. Thirty-nine of the isolates were identified as Lactobacillus paracasei/casei/rhamnosus, 2 as Lb. curvatus and 4 as a new species, Lb. danicus, using ITS-PCR. A specially designed carbohydrate-restricted medium supplemented with one of nine carbohydrates was used to evaluate potential carbohydrate sources in cheese–milk-fat globule membrane (MFGM), glycomacropeptide (GMP), or lysed cells. Lb. paracasei strains grew well on the carbohydrates from MFGM, GMP and bacterial cell wall peptidoglycan. The highest growth rates were observed on N-acetylglucosamine (NAG) (0.32–0.56 h⁻¹) and the lowest on ribose (0.12–0.23 h⁻¹, if ribose was fermented at all). Lb. danicus strains grew better on carbohydrates from lysed bacterial cells than on carbohydrates from MFGM or GMP, and it was the only species with a shorter lag-phase on ribose or NAG after being pre-incubated on ribose.     

Effect of high-pressure treatment on microbiology,proteolysis, lipolysis and levels of flavour compounds in mature blue-veined cheese

The effect of high-pressure (HP) treatment (400 MPa, 600 MPa) on ripening of mature 42-day-old Irish blue-veined cheese was studied. Counts of non-starter lactic acid bacteria, lactococci, yeasts, moulds, enterococci and total aerobic bacteria significantly decreased due to HP, with moulds being most sensitive and 600 MPa the most effective treatment. The levels of pH 4.6-soluble nitrogen and (12%) trichloroacetic acid-soluble nitrogen increased immediately after both HP treatments; however, after 28 days of storage, values were lower in HP-treated cheeses than in the control cheese. Urea-polyacrylamide gel electrophoresis showed increased breakdown of β-casein due to HP treatment at both 400 MPa and 600 MPa. Levels of free fatty acids were lower in HP-treated cheese than in the control, but not significantly so, and no significant changes could be observed in the level of flavour compounds of blue-veined cheese. Overall, HP treatment of blue-veined cheese reduced microbiological activity and decelerated proteolysis, with no statistically significant effects on development of flavour compounds.Industrial relevanceHigh-pressure treatment has been studied for the past 100 years; nevertheless, it was not applied in dairy industry, until recently, for a cheese spread. In this study, HP-induced inactivation of microbes and enzymes, which could arrest the ripening of high-quality mature (i.e., ripened) Irish farmhouse blue-veined cheese and thus extend shelf-life at optimal quality, was examined.     

Effect of dairy farm and milk refrigeration on microbiological and microstructural characteristics of matured Serra da Estrela cheese

This work was aimed at enumerating the viable microorganisms in ripened Serra da Estrela cheeses, manufactured from both refrigerated and non-refrigerated milk, in various dairies located throughout the demarcated region. Scanning electron microscopy was used to analyze the microstructure, and thus aid in understanding possible differences in their microbiological profile. The cheeses were allowed to ripen under controlled conditions, and sampled at 60, 90, 120, 150 and 180 d following manufacture. Viable numbers of lactic acid bacteria, staphylococci, Enterobacteriaceae and yeasts were obtained following standard plate counting on a number of selective media. Lactococcus was the most abundant genus (above 10⁸ cfu g⁻¹ of cheese) up to 120 d of ripening. No significant microstructural differences were observed in cheeses manufactured in different dairies over the ripening process. However, microstructural differences were apparent between cheeses manufactured with refrigerated versus non-refrigerated milk.     

Lysis of starters in UF cheeses: Behaviour of mesophilic lactococci and thermophilic lactobacilli

The objective of this work was to study the autolytic behaviour of strains of mesophilic (Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris) and thermophilic lactic acid bacteria (Lactobacillus helveticus, Lb. delbrueckii subsp. lactis and Streptococcus thermophilus) in UF cheese. Cheeses were made from a UF-retentate (milk concentrated by a factor of 6) of microfiltered milk (0.8 μm pore size membrane) using the following starter systems: (1) single inocula of autolytic strains of L. lactis (US3, AM2 or AM1), non-autolytic strains of L. lactis (AM2-C or CNRZ-144), (2) a co-inocula of strains of Lb. helveticus (ITG-LH1, CNRZ-32 or CNRZ-303), Lb. delbrueckii subsp. lactis (ITG-LL14 or ITG-LL51) with the same strain of S. thermophilus CNRZ-1358. Cell viability was monitored over a 28 day ripening period by enumeration on selective media. Degree of lysis was determined by the measurement of the intracellular marker lactate dehydrogenase (LDH) activity, and also by immunodetection of intracellular proteins with species specific antibodies. In UF cheeses, lysis of autolytic strains of L. lactis was significantly delayed, showing release of intracellular components after 21 days of ripening. No lysis was observed for non-autolytic L. lactis strains or for S. thermophilus. Lysis of thermophilic lactobacilli (Lb. helveticus, Lb. delbrueckii), was observed from the start of ripening, but the onset and the level of lysis observed was strain and species dependent.     

A comparison of the chemical,microbiological and sensory characteristics of bovine and ovine Halloumi cheese

Commercial samples of fresh and mature Halloumi cheeses made from ovine or bovine milk were studied in order to establish their chemical, microbiological and sensory characteristics. Significant differences were observed between the two types of Halloumi cheese both when fresh and mature. The free volatile fatty acid (FVFA) content of the cheeses increased with maturation from 483 to 1356 mg kg⁻¹ for the ovine product, but lower values (380–1248 mg kg⁻¹) were found in the bovine cheese. During maturation for 40 days, Enterococcus faecium, which dominated the microflora of fresh ovine cheese, was replaced by lactobacilli, including a new species, Lactobacillus cypricasei, which was not found in the bovine samples. Fewer than 100 cfu g⁻¹ lactic acid bacteria (LAB) were present in the fresh bovine cheeses, but a microflora dominated by lactobacilli developed with time. Yeast counts in the mature ovine and bovine cheeses reached 2.3–2.8×10⁵ cfu g⁻¹ and, as some of the yeasts were proteolytic and/or lipolytic, it was assumed that they were having a positive impact of the flavour of the cheeses. The sensory panel distinguished significant differences in texture and flavour between the fresh and mature samples of both ovine and bovine cheeses and, overall, there was a significant preference for the ovine brand.     

Accelerated ripening of Caciocavallo Pugliese cheese with attenuated adjuncts of selected nonstarter lactobacilli

The nonstarter lactic acid bacteria Lactobacillus plantarum CC3M8, Lactobacillus paracasei CC3M35, and Lactobacillus casei LC01, previously isolated from aged Caciocavallo Pugliese cheese or used in cheesemaking, were used as adjunct cultures (AC) or attenuated (by sonication treatment) adjunct cultures (AAC) for the manufacture of Caciocavallo Pugliese cheese on an industrial scale. Preliminary studies on the kinetics of growth and acidification and activities of several enzymes of AAC were characterized in vitro. As shown by the fluorescence determination of live versus dead or damaged cells and other phenotype features, attenuation resulted in a portion of the cells being damaged and a portion of the cells being capable of growing with time. Compared with the control cheese (without adjunct cultures) and the cheese with AAC, the addition of AC resulted in a lower pH after manufacture, which altered the gross composition of the cheese. As shown by plate count and confirmed by random amplification of polymorphic DNA-PCR, the 3 species of nonstarter lactobacilli persisted during ripening but the number of cultivable cells varied between AC and AAC. Slight differences were found between cheeses regarding primary proteolysis. The major differences between cheeses were the accumulation of free amino acids and the activity levels of several enzymes, which were highest in the Caciocavallo Pugliese cheeses made with the addition of AAC. As shown by triangle test, the sensory properties of the cheese made with AAC at 45d did not differ from those of the control Caciocavallo Pugliese cheese at 60d of ripening. In contrast, the cheese made with AC at 45d differed from both the Caciocavallo Pugliese cheese without adjuncts and the cheese made with AAC. Attenuated adjunct cultures are suitable for accelerating the ripening of Caciocavallo Pugliese cheese without modifying the main features of the traditional cheese.     

Antimicrobial activity of pediocin-producing Lactococcus lactis on Listeria monocytogenes,Staphylococcus aureus and Escherichia coli O157:H7 in cheese

The antimicrobial activity of two pediocin-producing transformants obtained from wild strains of Lactococcus lactis on the survival of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 during cheese ripening was investigated. Cheeses were manufactured from milk inoculated with the three pathogens, each at approximately 6 log cfu mL⁻¹. Pediococcus acidilactici 347 (Ped⁺), Lc. lactis ESI 153, Lc. lactis ESI 515 (Nis⁺) and their respective pediocin-producing transformants Lc. lactis CL1 (Ped⁺) and Lc. lactis CL2 (Nis⁺, Ped⁺) were added at 1% as adjuncts to the starter culture. After 30 d, L. monocytogenes, S. aureus and E. coli O157:H7 counts were 5.30, 5.16 and 4.14 log cfu g⁻¹ in control cheese made without adjunct culture. On day 30, pediocin-producing derivatives Lc. lactis CL1 and Lc. lactis CL2 lowered L. monocytogenes counts by 2.97 and 1.64 log units, S. aureus by 0.98 and 0.40 log units, and E. coli O157:H7 by 0.84 and 1.69 log units with respect to control cheese. All cheeses made with nisin-producing LAB exhibited bacteriocin activity throughout ripening. Pediocin activity was only detected throughout the whole ripening period in cheese with Lc. lactis CL1. Because of the antimicrobial activity of pediocin PA-1, its production in situ by strains of LAB growing efficiently in milk would extend the application of this bacteriocin in cheese manufacture.     

Ripening of Cheddar cheese made from blends of raw and pasteurised milk

Cheddar cheeses were made from pasteurised milk (P), raw milk (R) or pasteurised milk to which 10 (PR10), 5 (PR5) or 1 (PR1) % of raw milk had been added. Non-starter lactic acid bacteria (NSLAB) were not detectable in P cheese in the first month of ripening, at which stage PR1, PR5, PR10 and R cheeses had 10⁴, 10⁵, 10⁶ and 10⁷ cfu NSLAB g⁻¹, respectively. After ripening for 4 months, the number of NSLAB was 1–2 log cycles lower in P cheese than in all other cheeses. Urea–polyacrylamide gel electrophoretograms of water-soluble and insoluble fractions of cheeses and reverse-phase HPLC chromatograms of 70% (v/v) ethanol-soluble as well as -insoluble fractions of WSF were essentially similar in all cheeses. The concentration of amino acids were pro rata the number of NSLAB and were the highest in R cheese and the lowest in P cheese throughout ripening. Free fatty acids and most of the fatty acid esters in 4-month old cheeses were higher in PR1, PR5, PR10 and R cheeses than in P cheese. Commercial graders awarded the highest flavour scores to 4-month-old PR1 cheeses and the lowest to P or R cheese. An expert panel of sensory assessors awarded increasingly higher scores for fruity/sweet and pungent aroma as the level of raw milk increased. The trend for aroma intensity and perceived maturity was R>PR10>PP5>PR1>P. The NSLAB from raw milk appeared to influence the ripening and quality of Cheddar cheese.     

Identification of cultivable lactic acid bacteria isolated from Algerian raw goat's milk and evaluation of their technological properties

One hundred and fifty-eight strains of lactic acid bacteria isolated from Algerian raw goat's milk were identified and technologically characterized. Five genera were found: Lactobacillus (50.63%), Lactococcus (25.94%), Streptococcus (14.56%), Leuconostoc (7.59%) and Pediococcus (1.26%). The predominant species were Lactococcus lactis (32 strains), Streptococcus thermophilus (23 strains), Lactobacillus bulgaricus (19 strains), Lb. helveticus (16 strains) and Lb. plantarum (14 strains).Approximately 39% of the lactic acid bacteria isolated produced more than 0.6% lactic acid (w/v) after 18 h of incubation, and belonged to the Lactococcus and Lactobacillus genera. The highest proteolytic activity was approximately 3 mg tyrosine l⁻¹ for mesophilic strains and nearly 5 mg tyrosine l⁻¹ for thermophilic lactobacilli after 72 h. High aromatic activity (more than 0.8 mg diacetyl l⁻¹ after 16 h) was detected in 14% of the strains.Nine strains were used to make dairy products (a yoghurt-like product and Edam-type cheese) on a pilot scale in the laboratory. The best-liked organoleptic characteristics were noted in a yoghurt produced with a mixed culture made up of S. thermophilus (strain 16TMC+) and Lb. helveticus (strain 20TMC) and in a cheese made with a starter composed of Lc. lactis subsp. lactis (strain 10MCM) and L. lactis subsp. lactis (V.P. +) (strain 19MCM).     

Effect of temperature on growth and metabolism of probiotic bacteria in milk

The growth and metabolism of six probiotic strains with documented health effects were studied in ultra-high temperature (UHT) treated milk supplemented with 0.5% (w/v) tryptone or 0.75% (w/v) fructose at different temperatures. The probiotic strains were Lactobacillus acidophilus La5, Lb. acidophilus 1748, Lb. johnsonii LA1, Lb. rhamnosus GG, Lb. reuteri SD 2112 and Bifidobacterium animalis BB12. Fermentation was followed for 48 h at 20, 30, 37 and 45 °C and the samples were analysed for pH, log cfu mL⁻¹, volatile compounds, organic acids and carbon dioxide. All six probiotic strains showed very different profiles of metabolites during fermentation, however, the two Lb. acidophilus strains were the most alike. All strains, except Lb. reuteri SD 2112, showed viable cell numbers above 6.5 log cfu mL⁻¹ after 48 h fermentation at 30, 37 and 45 °C. The probiotic strains produced different amounts of metabolic products according to temperature and fermentation time illustrating the importance of controlling these parameters.     

Survival of Listeria monocytogenes in Galotyri,a traditional Greek soft acid-curd cheese,stored aerobically at 4°C and 12°C

Galotyri is a traditional Greek soft acid-curd cheese, which is made from ewes’ or goats’ milk and is consumed fresh. Because cheese processing may allow Listeria monocytogenes post-process contamination, this study evaluated survival of the pathogen in fresh cheese during storage. Portions (0.5 kg) of two commercial types (<2% salt) of Galotyri, one artisan (pH 4.0±0.1) and the other industrial (pH 3.8±0.1), were inoculated with ca. 3 or 7 log cfu g⁻¹ of a five-strain cocktail of L. monocytogenes and stored aerobically at 4°C and 12°C. After 3 days, average declines of pathogen's populations (PALCAM agar) were 1.3–1.6 and 3.7–4.6 log cfu g⁻¹ in cheese samples for the low and high inocula, respectively. These declines were independent (P>0.05) of the cheese type or the storage temperature. From day 3, however, declines shifted to small or minimal to result in 1.4–1.8 log cfu g⁻¹ of survivors at 28 days of storage of all cheeses at 4°C, indicating a strong “tailing” independent of initial level of contamination. Low (1.2–1.7 log cfu g⁻¹) survival of L. monocytogenes also occurred in cheeses at 12°C for 14 days, which were prone to surface yeast spoilage. When ca. 3 log cfu g⁻¹ of L. monocytogenes were inoculated in laboratory scale prepared Galotyri of pH ≅4.4 and ≅3% salt, the pathogen died off at 14 and 21 days at 12°C and 4°C, respectively, in artisan type cheeses fermented with the natural starter. In contrast, the pathogen survived for 28 days in cheeses fermented with the industrial starter. These results indicate that L. monocytogenes cannot grow but may survive during retail storage of Galotyri despite its low pH of or slightly below 4.0. Although contamination of Galotyri with L. monocytogenes may be expected low (<100 cfu g⁻¹) in practice, that long-term survival of the pathogen in commercial cheeses was shown to be unaffected by the artificial contamination level (3 or 7 logs) and the storage temperature (4°C or 12°C), which should be a concern.