Characterization ofListeriastrains isolated from soft and semi-soft cheeses

Two hundred strains ofListeria monoctyogenespreviously isolated from 19 soft and semi-soft cheeses by enrichment were characterized by serotyping and pulsed-field gel electrophoresis (PFGE). Strains of serogroup 1/2 predominated, with 33.5% of all strains belonging to serovar 1/2a, 58.5% to serovar 1/2b and 5% to serovar 1/2c. By using a PFGE method, 16 different clonal types were obtained. All 10 isolates ofL. monocytogenesrecovered from one white mould cheese were serovar 1/2a, but displayed two different PFGE profiles. Another 10L. monocytogenesisolates from white mould cheese recovered after a 48-h enrichment broth procedure revealed two clonal types, only one of which could be detected after 5 additional days of incubation in the same enrichment broth. Cross-contamination in dairy plants and/or retail stores may play an important role in the incidence and diversity ofL. monocytogenesclonal types recovered from soft and semi-soft cheeses. Therefore, it is necessary to characterize several isolates from the same sample when conducting epidemiological investigations.     

Selected characteristics of micrococci isolated from Spanish dry fermented sausages

The flora development of two batches of Spanish dry sausages has been studied. Lactic acid bacteria and organisms growing on Mannitol—Salt agar increased during the first five days, remaining at constant levels afterwards. From Mannitol—Salt agar, 629 strains were isolated. The maximum recovery of Gram-positive, catalase-positive cocci was between 5 and 15 days of ripening and after 20 days no micrococci were detected. All isolates of nitrate reducing micrococci (42 strains) showed an acceptable growth at temperatures (19–25°C) and water activities (0.93–0.95) of the sausages during the fermentation step. From the quantitative determination of the nitrate reduction ability, six strains were selected as potential starters for industrial sausage production.     

Anticlostridial activity of Lactobacillus isolated from semi-hard cheeses

Non-starter lactic acid bacteria grow to high numbers in semi-hard cheeses during ripening, and may suppress harmful bacteria. In this study, about 400 Lactobacillus isolates from Danish semi-hard cheeses were identified to species level using internal transcribed spacer-polymerase chain reaction (ITS-PCR) analysis. The majority of isolates belonged to the Lb. paracasei complex and were classified into approximately 135 types using pulsed field gel electrophoresis (PFGE). Lactobacillus isolates representing all the different PFGE types were screened, using an agar well diffusion assay, for antimicrobial activity against 15 single-strain Clostridium cultures. Almost half of the isolates possessed anticlostridial activity, and 10% possessed a broad and consistent activity. Nine strains were further investigated for properties of importance for use as mixed cultures in cheese and silage. The results showed that anticlostridial non-starter Lactobacillus growing in high quality semi-hard cheeses could be useful as protective adjunct cultures against the growth of Clostridium.     

Spoilage potentials and antimicrobial resistance of Pseudomonas spp. isolated from cheeses

Pseudomonas spp. are aerobic, gram-negative bacteria that are recognized as major food spoilage microorganisms. A total of 32 (22.9%) Pseudomonas spp. from 140 homemade white cheese samples collected from the open-air public bazaar were isolated and characterized. The aim of the present study was to investigate the biochemical characteristics, the production of extracellular enzymes, slime and β-lactamase, and antimicrobial susceptibility of Pseudomonas spp. isolated from cheeses. The identified isolates including Pseudomonas pseudoalcaligenes, Pseudomonas alcaligenes, Pseudomonas aeruginosa, Pseudomonas fluorescens biovar V, and P. pseudoalcaligenes ssp. citrulli were found to produce extracellular enzymes, respectively: protease and lecithinase production (100%), and lipase activity (85.7, 42.9, 100, and 100%, and nonlipolytic, respectively). The isolates did not produce slime and had no detectable β-lactamase activity. The antimicrobial susceptibility of the isolates was tested using the disk diffusion method. Pseudomonas spp. had the highest resistance to penicillin G (100%), then sulphamethoxazole/trimethoprim (28.1%). However, all Pseudomonas spp. isolates were 100% susceptible to ceftazidime, ciprofloxacin, amikacin, gentamicin, and imipenem. Multidrug-resistance patterns were not observed among these isolates. In this study, Pseudomonas spp., exhibiting spoilage features, were isolated mainly from cheeses. Isolation of this organism from processed milk highlights the need to improve the hygienic practices. All of the stages in the milk processing chain during manufacturing have to be under control to achieve the quality and safety of dairy products.     

Nonstarter lactobacilli isolated from soft and semihard Argentinean cheeses: genetic characterization and resistance to biological barriers

Nonstarter lactic acid bacteria isolated from Argentinean cheeses were identified and characterized by focusing on their resistance to biological barriers, along with other physiological features of potential interest, in the search for future probiotic organisms. Lactobacilli were enumerated and isolated from semihard and soft cheeses made with multistrain Streptococcus thermophilus starters. Lactobacilli counts in 1-week-old cheeses were between 10(5) and 10(7) CFU/g and then reached 10(7) CFU/ g in all 1-month samples, while streptococci were always above 10(9) CFU/g. A total number of 22 lactobacilli isolates were retained, identified, and characterized by in vitro tests. Species identity was determined by carbohydrate metabolism and species-specific PCR assays. Genetic diversity was explored by random amplified polymorphic DNA (RAPD) PCR analysis. The Lactobacillus strains were assigned to the species L. casei, L. plantarum, L. rhamnosus, L. curvatus, L. fermentum, and L. perolens. All the strains studied tolerated 25 ppm of lysozyme, and most of them showed resistance to 0.3% bile. After incubation in gastric solution (pH 2.0), counts decreased by several log units, ranging from 3.2 to 7.0. The strains were able to grow in the presence of bile salts, but only three isolates were capable of deconjugation. The nonstarter lactobacilli that were assayed fermented the prebiotic substrates (especially lactulose and inulin). Some strains showed high cell hydrophobicity and beta-galactosidase activity, as well as inhibitory activity against pathogenic bacteria. It was concluded that most of the lactobacilli isolated in this study demonstrated resistance to biological barriers and physiological characteristics compatible with probiotic properties, which make them suitable for further research in in vivo studies aimed at identifying new probiotic organisms.     

Inhibition of yeasts isolated from traditional Turkish cheeses by Lactobacillus spp.

Antifungal activity of some Lactobacillus strains was determined. Yeasts were isolated and identified as Saccharomyces cerevisiae (10 of 17) and one each of Candida pseudotropicalis, C. krusei, C. lipolytica, C. lusitaniae, C. ciferrii, Torulopsis glabrata and Rhodotorula rubra . Lactobacillus strains were found to have fairly strong antifungal activity against S. cerevisiae . Of the 19 bacteria tested, L. plantarum Lp 21 seemed to have the most inhibitory effect against all of the S. cerevisiae strains except for S. cerevisiae 13. We suggest that L. plantarum Lp 21 can be used with starter cultures for cheese production.     

Characterization of autochthonal yeasts isolated from Spanish soft raw ewe milk protected designation of origin cheeses for technological application

The yeasts involved in the ripening process of artisanal soft raw ewe milk Protected Designation of Origin (PDO) Torta del Casar and Queso de la Serena cheeses produced in Extremadura, Spain, were isolated throughout their ripening process, strain typed, and characterized for some important technological properties. A total of 508 yeast isolates were obtained and identified by inter-single sequence repeat anchored PCR amplification analysis and subsequent sequencing of the internal transcribed spacer ITS1/ITS2 5.8S rRNA. A total of 19 yeast species representing 8 genera were identified. Debaryomyces hansenii, Pichia kudriavzevii, Kluyveromyces lactis, and Yarrowia lipolytica were the predominant species. We selected 157 isolates, by genotyping and origin, for technological characterization. The evaluation of yeast isolates' growth under stress conditions of cheese ripening showed that 87 presented better performance. Among them, 71 isolates were not able to catabolize tyrosine to produce a brown pigment. Principal component analysis of the biochemical features of these isolates showed that 9 strains stood out, 3 K. lactis strains (2287, 2725, and 1507), 2 Pichia jadinii (1731 and 433), 2 Yarrowia alimentaria (1204 and 2150), Y. lipolytica 2495 and P. kudriavzevii 373. These strains displayed strong extracellular proteolytic activity on skim milk agar as well as an adequate enzymatic profile (strong aminopeptidase and weak protease activity), suggesting their great potential for cheese proteolysis. Extracellular lipolytic activity was mainly restricted to Yarrowia spp. isolates and weakly present in P. kudriavzevii 373 and K. lactis 2725, although enzymatic characterization by API-ZYM (bioMérieux SA) evidenced that all may contribute, at least in part, to the lipolysis process. Moreover, these strains were able to assimilate lactose, galactose, and glucose at NaCl concentrations higher than that usually found in cheese. However, lactate and citrate assimilation were limited to Y. lipolytica 2495, P. kudriavzevii 373, and P. jadinii 433, and may contribute to the alkalinizing process relevant to biochemical processes that take place in the last stages of ripening. By contrast, K. lactis strains showed acidifying capacity and β-galactosidase activity and may take part in the initial stages of ripening, together with lactic acid bacteria. Thus, considering the technological characteristics studied, the 9 selected strains presented biochemical features well suited to their potential use as adjunct cultures, alone or in combination with autochthonous starter bacteria in the cheesemaking process, to overcome the heterogeneity of these PDO cheeses, preserving their unique sensory characteristics.     

Histamine formation by enterococci isolated from home-made goat cheeses

A survey was made of the histamine-producing capability of enterococci isolated from goat cheeses. The strains, 130 Streptococcus faecium and 106 S. faecalis, were grown in Trypticase Soy Broth Histidine medium (TSBH) at 35 degrees C for 24 h and the histamine formed was determined by fluorometry. Forty-one (31.5%) of the S. faecium strains and 2 (1.9%) of the S. faecalis strains produced histamine. The largest amount detected was 4.0 micrograms histamine/ml TSBH. Compared with the amounts of histamine produced by some Gram-negative bacteria, the histamine production by enterococci seems to be low. Under the present conditions the enterococci seem to have no relevance from a histamine intoxication point of view.     

Phenotypic and genetic diversity of enterococci isolated from Italian cheeses

In the present study, 124 enterococcal strains, isolated from traditional Italian cow, goat and buffalo cheeses, were characterized using phenotypic features and randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). The RAPD-PCR profiles obtained with four primers and five different amplification conditions were compared by numerical analysis and allowed an inter- and intraspecific differentiation of the isolates. Whole cell protein analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used as a reference method for species identification. The strains were identified as Enterococcus faecalis (82 strains), E. faecium (27 strains), E. durans (nine strains), E. gallinarum (four strains) and E. hirae (two strains). Species recognition by means of RAPD-PCR was in agreement with the SDS-PAGE results except for eight strains of E. faecium that clustered in separated groups. On the other hand, phenotypic identification based on carbohydrate fermentation profiles, using the rapid ID 32 STREP galleries, gave different results from SDS-PAGE in 12.1% of the cases. The majority of the strains had weak acidifying and proteolytic activities in milk. One E. faecium strain showed vanA (vancomycin resistance) genotype while four strains showed a beta-haemolytic reaction on human blood. Several strains showed antagonistic activity towards indicator strains of Listeria innocua, Clostridium tyrobutyricam and Propionibacterium freudenreichii subsp. shermanii.     

Characterization of enterococci isolated from homemade Bulgarian cheeses and katuk

A collection of 107 lactic acid bacteria (LAB) isolates was obtained from traditional Bulgarian dairy products??homemade cheeses and katuk samples, produced from heat-treated cow, goat, ewe and buffalo milk without the addition of any bacterial starter culture. The samples were collected from mountain region of Rhodope (south part of Bulgaria), Tracian valley and mountain region of Stara Planina (west part of Bulgaria). These LAB produced bacteriocin-like inhibitory substances (BLIS) and proteinases. Preliminary strain determination was performed according to their fermentation ability using API 50CHL and API 20 Strep. Most of the characterized strains (58) belong to genus Enterococcus; 21, 20 and 11 strains were identified as Lactobacillus sp., Streptococcus sp. and Lactococcus sp., respectively. Isolated enterococcal strains were characterized using phenotypic features as well as by DNA typing. The strains were identified as Enterococcus faecium (34), Enterococcus durans (22) and Enterococcus faecalis (2). The proteolytic activity varied from 0.094 to 0.455?mM/L Gly into the group of E. faecium and from 0.109 to 0.487?mM/L Gly into the group of E. durans strains, while both E. faecalis strains showed relatively high proteolytic activity. The samples obtained after 3?h of hydrolysis of ??-casein by E. faecalis B1 strain were further used for mass spectrometry analysis, and 31 peptides were identified.     

Formation of cadaverine, histamine, putrescine and tyramine by bacteria isolated from meat, fermented sausages and cheeses

Enterobacteriaceae (EB, n = 149), Lactobacillus (LB, n = 162) and Leuconostoc sp. (LC, n = 89) and enterococci (EC, n = 137), isolated from raw meat (n = 65), fermented sausages (n = 50) and cheese (n = 55) samples, were cultivated in a broth containing precursor amino acids (each 3 g/l). After incubation, the liquid culture was chemically analysed for cadaverine (CAD), putrescine (PUT), histamine (HIS) and tyramine (TYR) formation at pH 5.2 and at pH 6.7. The majority of EB isolates (147 of 149) was capable of forming >100 mg/l of either CAD or PUT. Among the most frequently isolated species Hafnia alvei and Serratia liquefaciens, formation of >100 mg/l HIS occurred, but with low prevalence (1.6 and 6.5%, respectively). Twelve of 149 isolates (8%) were able to produce more than 10 mg/l HIS. One hundred forty-two isolates (95.3%) produced less than 10 mg/l TYR, and 7 isolates (4.7%) 10 mg/l to a maximum of 35.3 mg/l TYR. For LB + LC, one isolate (Leuconostoc mesenteroides ssp. mesenteroides) formed >100 mg/l PUT and one >100 mg/l CAD (of all 251 LB + LC isolates 0.4% each). Formation of >100 mg/l HIS and TYR was detected in 3.6 and 19% of the LB + LC isolates, respectively. For the EC isolates, maximum levels for PUT, CAD and HIS were 25.4 mg/l, 6.0 mg/l and 15.7 mg/l, respectively. TYR was formed in quantities of 100–1000 mg/l by 47.9% of EC faecalis (n = 75), and 59.7% of EC faecium (n = 62) isolates. More than 1000 mg/l TYR were formed by 50.7 and 35.5% of the isolates, respectively. A low initial pH of 5.2 compared to the initial pH of 6.7 favoured tyramine production by lactic acid bacteria, but was associated with lower CAD yield by EB. A considerable intra-species variability in amine formation was observed.     

Phenotypic and genotypic characterization of lactobacilli isolated from Spanish goat cheeses

Lactic acid bacteria from 18 Spanish goat cheeses produced by seven dairies were isolated to evaluate the genetic diversity of this bacterial community. 136 representative isolates were characterized by phenotyping and Randomly Amplified Polymorphic DNA (RAPD-PCR) analysis. Ten species were identified with predominance of Lactobacillus paracasei subsp. paracasei. The presence of L. curvatus, L. pentosus, L. cellobiosus and L. rhamnosus has not hitherto been reported in Spanish goat cheeses. A high degree of genetic diversity was found for L. paracasei subsp. paracasei, L. curvatus and L. plantarum. Some of the identified strains displayed strong acidifying and proteolytic capacities.     

Characterization and probiotic potential of Lactobacillus plantarum strains isolated from cheeses

Ninety-eight Lactobacillus plantarum strains isolated from Italian and Argentinean cheeses were evaluated for probiotic potential. After a preliminary subtractive screening based on the presence of msa and bsh genes, 27 strains were characterized. In general, the selected strains showed high resistance to lysozyme, good adaptation to simulated gastric juice, and a moderate to low bile tolerance. The capacity to agglutinate yeast cells in a mannose-specific manner, as well as the cell surface hydrophobicity was found to be variable among strains. Very high β-galactosidase activity was shown by a considerable number of the tested strains, whereas variable prebiotic utilization ability was observed. Only tetracycline resistance was observed in two highly resistant strains which harbored the tetM gene, whereas none of the strains showed β-glucuronidase activity or was capable of inhibiting pathogens. Three strains (Lp790, Lp813, and Lp998) were tested by in vivo trials. A considerable heterogeneity was found among a number of L. plantarum strains screened in this study, leading to the design of multiple cultures to cooperatively link strains showing the widest range of useful traits. Among the selected strains, Lp790, Lp813, and Lp998 showed the best probiotic potential and would be promising candidates for inclusion as starter cultures for the manufacture of probiotic fermented foods.     

Formation of conjugated linoleic acid by a Lactobacillus plantarum strain isolated from an artisanal cheese: Evaluation in miniature cheeses

Among 129 lactic acid bacteria previously isolated from raw-milk starter-free cheeses manufactured in Galicia (NW Spain), two strains of Lactobacillus plantarum were definitely recognised as producers of conjugated linoleic acid (CLA). Gas chromatography analysis identified cis-9, trans-11 C18:2 as the predominant CLA isomer formed in MRS broth supplemented with linoleic acid. A centrifugation-based model for the manufacture of miniature cheeses was used to evaluate the formation of CLA by Lb. plantarum L200, the highest producer of CLA in MRS broth. The miniature cheeses made with the addition of the L200 strain showed significantly (P < 0.05) higher contents of cis-9, trans-11 CLA than those of the control cheeses (1.09 versus 0.69 percentage of total fatty acids, respectively). These results suggest that Lb. plantarum L200 strain could be used as an adjunct culture to slightly increase the concentrations of CLA in short-ripened cows' milk cheeses.     

Antimicrobial susceptibility and beta-lactamase testing of staphylococci isolated from dairy herds

Evaluation of 722 staphylococcal isolates from seven dairy herds revealed marked herd differences in antimicrobial susceptibility patterns. A low overall resistance to penicillin was noted for Staphylococcus aureus, although isolates from three herds had greater than or equal to 16% penicillin resistance. Other staphylococcal species demonstrated a higher resistance to penicillin than did S. aureus. The predominant staphylococci other than S. aureus were S. hyicus and S. chromogenes for six of the seven herds while S. simulans and S. epidermidis predominated in one herd. beta-Lactamase testing using a commercially available chromogenic cephalosporin was an accurate predictor of penicillin resistance for S. aureus. Staphylococcus hyicus, S. chromogenes, and S. epidermidis that were penicillin resistant by susceptibility testing demonstrated negative beta-lactamase results of 35, 11, and 9%, respectively. Retesting of these isolates after exposure to penicillin resulted in positive beta-lactamase results, indicating induction of beta-lactamase. These results indicate the variability in antimicrobial susceptibility patterns for staphylococci and suggest the importance of epidemiologic knowledge of a dairy herd to guide initial therapy.     

Characterization of Lactobacillus helveticus strains isolated from cheeses by distribution studies of insertion sequences

A collection of 38 Lactobacillus helveticus strains, isolated from a number of different artisan Italian cheeses, and 4 reference strains were studied with respect to the presence of insertion sequences and their distribution and abundance. The mobile genetic element ISLh1, that contains one open reading frame coding for a putative transposase of the IS982 family, was used for DNA fingerprinting, together with IS1201 and ISL2, previously isolated from L. helveticus. The number of insertion sequences per strain and the size of DNA restriction fragments containing them, was variable and allowed the discrimination at the strain-level. The genomic distribution of the three unrelated insertion sequences showed significant correlations and allowed the differentiation of the strains also with regard to the specific ecological niche of origin of the isolates. Consequently, insertion sequences comparison may be useful in determining the history of a group of strains known to be related because of identity and offers a further parameter for evaluating the population polymorphism in L. helveticus.     

Aggregation and hydrophobicity properties of 6 dairy propionibacteria strains isolated from homemade Turkish cheeses

In the present study, 6 dairy Propionibacterium strains were assessed with regard to their hydrophobic characteristics and their autoaggregation and coaggregation abilities since these traits have been shown to be indicative of adherence in other microorganisms. Aggregation assays and bacterial adhesion to hydrocarbons demonstrated significant differences in cell surface properties among the tested propionibacteria strains. Almost all strains appeared relatively hydrophilic, which showed low affinity for p-xylene. Four of the tested strains showed the strong adhesion to ethyl acetate, a basic solvent, in comparison with microbial adhesion to chloroform, an acidic solvent, which demonstrated the particularity of propionibacteria to have an important electron donor and acidic character. Also, these strains simultaneously showed affinity to 3 hydrocarbons, suggesting a high complexity of the cell surface. All propionibacteria strains tested showed autoaggregation and coaggregation ability with the Escherichia coli ATTC 11229, but the results were strain-specific and dependent on incubation conditions. Anaerobic incubation conditions were determined as the best condition for aggregation abilities of propionibacteria strains. A relationship was obtained between aggregation abilities (auto- and coaggregation) and a correlation between adhesion to hydrocarbon (chloroform) and autoaggregation was possible. Our results indicate that the ability to autoaggregation together with cell surface hydrophobicity and coaggregation abilities with E. coli strain can be used for preliminary screening in order to identify potentially probiotic bacteria suitable for human or animal use. PRACTICAL APPLICATION: Autoaggregation, cell surface hydrophobicity, and coaggregation abilities with E. coli of the selected dairy propionibacteria strains could be used as probiotic in foods after in vivo studies.     

Identification and preliminary characterization of strains of enterococci and micrococci isolated from Arzúa raw cows'-milk cheese

152 enterococcal isolates and 88 micrococcal isolates obtained from secondary microflora of Arzúa raw cows'-milk cheese at various stages of ripening were characterized. The most frequent Enterococcus specie was E. faecalis. The most frequent Micrococcus specie was M. varians, followed by M. luteus and M. lylae; 27 micrococcal isolates could not be assigned to species. Although most strains of micrococci did not cause appreciable acidification of milk, the proportions of micrococcal isolates displaying proteolytic and lipolytic activities were higher than those for enterococcal isolates. The biochemical and enzymatic characteristics of isolates from both groups displayed considerable variability, even within the same species. In screening microorganisms of this type for suitability as components of cheese starter cultures, it is therefore necessary, as has been pointed out by previous authors, to work with single strains.