Hydrophilic and hydrophobic peptides produced in cheese by wild Lactococcus lactis strains

AIMS: To study the production of hydrophilic and hydrophobic peptides in cheese by 32 wild Lactococcus lactis strains of different RAPD patterns and to compare them with the peptides produced by lactococcal cells incubated with whole casein. METHOD AND RESULTS: Chromatograms of peptides from cheeses made using each strain as single starter culture were divided into five regions, and strains were classified in three groups by hierarchical cluster analysis of region areas. Thirty out of the 32 wild L. lactis strains produced higher levels of hydrophobic peptides in cheese than on whole casein. CONCLUSIONS: Cheese was a more favourable substrate than whole casein for hydrophobic peptide formation by L. lactis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: New strains of lactococci should be screened for bitterness under cheese conditions, as the formation of hydrophobic peptides may be underestimated in assays with casein as substrate.     

Genetic diversity of lactococci and enterococci isolated from home-made Pecorino Sardo ewes' milk cheese

AIMS: To assess the intraspecific genetic diversity of lactococci and enterococci isolated from 24-h, 1- and 2-month-old home-made Pecorino Sardo ewes' milk cheese. METHODS AND RESULTS: Two molecular techniques, plasmid profiling and pulsed-field gel electrophoresis, were used in order to type the isolates at strain level. The present study revealed that the lactococcal and enterococcal microbial populations of home-made Pecorino Sardo cheese were complex, not only 24 h after manufacture, but also after 1 and 2 months of ripening. The genetic diversity at subspecies level ranged from 58 to 80% during the three periods examined. The study also showed that the strains that dominated in the first stage of ripening were not necessarily predominant in the later periods. A high number of strains isolated at 24 h were still present in the mature cheese, but many of the genotypes were only found in the cheese after 1 or 2 months. CONCLUSIONS: The results showed a high intraspecific genetic diversity in the natural microbial population colonizing home-made Pecorino Sardo cheese. Two molecular techniques are necessary for a thorough and precise typing at strain level in order to better distinguish between closely related isolates and between isolates that probably belong to the same clonal lineage. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic complexity observed in the present study is of particular relevance in the preservation of the natural microflora of traditional Protected Designation of Origin raw milk cheeses, as well as in the selection of new starter strains for the dairy industry.     

Diversity among lactococci isolated from ewes' raw milk and cheese

P. GAYA, M. BABÍN, M. MEDINA and M. NUÑEZ.1999.The technological and genetic characteristics of lactococci present in ewes' raw milk and 1-d-old ewes' raw milk cheeses sampled over a 1-year period were investigated. The proportion of lactic acid bacteria isolates from milk samples able to decrease milk pH by more than 1·25 units after 6 h incubation at 30 °C reached 14·5% in spring vs 10·7% in summer, 8·3% in autumn and 3·0% in winter. In 1-d-old cheese samples, the proportion of lactic acid bacteria able to lower milk pH by more than 1·25 units increased up to 32·3% in spring vs 23·4% in summer, 8·0% in autumn and 10·3% in winter. Fast acid-producing lactic acid bacteria mainly belonged to the genus Lactococcus . Using polymerase chain reaction protocols, fast acid-producing lactococci were grouped as 61  Lactococcus lactis subsp. lactis , 13  L. lactis subsp. cremoris and 14  L. lactis subsp. lactis biovar diacetylactis. Randomly amplified polymorphic DNA (RAPD) fingerprinting of fast acid-producing lactococci, using two primers, resulted in 21 different RAPD patterns for L. lactis subsp. lactis isolates, nine RAPD patterns for L. lactis subsp. cremoris isolates and three RAPD patterns for L. lactis subsp. lactis biovar diacetylactis isolates. Up to 19 different RAPD patterns were found for L. lactis isolates from cheeses made in a particular month.     

Genotypic and phenotypic characterization of the dynamics of the lactic acid bacterial population of adjunct-containing Cheddar cheese manufactured from raw and microfiltered pasteurised milk

AIMS: This study investigates the dynamics of the microflora, particularly the lactobacilli, in Cheddar cheese manufactured from raw and microfiltered milk containing different adjunct cultures. METHODS AND RESULTS: Sixteen cheeses - raw milk, adjunct and control cheeses - were manufactured in four trials. Lactobacilli were identified by PCR methods in one trial, and by phenotypic typing for all trials. Numbers of lactobacilli were significantly different at day 1 and 3 months in the control and adjunct-containing cheeses. In the raw milk cheeses, Lactobacillus paracasei was detected throughout ripening, Lact. curvatus at the end, and Lact. plantarum at day 1 only. Lactobacillus strain diversity decreased from raw, control to adjunct cheeses. Enteroccoci and coliform numbers further differentiated raw cheeses from the others. Lactococcal starter numbers also differed in the three cheese types and differences were observed within adjunct cheeses. Although adjunct lactobacilli dominated in the cheese to which they were added, strains with similar phenotypic profiles were also detected on occasions in some of the control cheeses. CONCLUSIONS: The addition of adjunct lactobacilli modified the growth kinetics of both adventitious lactobacilli and starter lactococci during ripening. Appropriate strain tracking is necessary to monitor changes in the population profiles of control and experimental cheeses in trials utilizing adjunct cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Investigations of the role of adjunct strain(s) in cheeses may be complicated by the interactions between the adjunct and the other cheese strains, and effective strain monitoring by genotypic or phenotypic methods is essential if valid comparisons are to be made.     

Caseinolysis in cheese by Enterobacteriaceae strains of dairy origin

AIMS: To study the effect of Enterobacteriaceae strains of dairy origin on caseins under cheese manufacture and ripening conditions. METHODS AND RESULTS: Strains belonging to the genera Enterobacter, Escherichia, Hafnia and Serratia were isolated from fresh raw milk cheeses. Residual caseins in cheeses made from milk individually inoculated with 10 strains of Enterobacteriaceae were determined by capillary electrophoresis. Hierarchical cluster analysis of strains based on data of residual caseins grouped together strains from the same genus, excepting Hafnia strains, which were separated into two groups. Serratia was the most proteolytic genus in our study. Preferences for degradation of casein fractions differed among the four genera studied. CONCLUSIONS: Enterobacteriaceae strains posses proteolytic systems active on all casein fractions under cheese manufacture and ripening conditions. The effects on caseins were similar for strains belonging to the same genus. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Enterobacteriaceae in cheeses may affect proteolysis during ripening. Assays of Enterobacteriaceae proteolytic activity on milk agar plates may underestimate their caseinolytic activity in cheese.     

Resident lactic acid bacteria in raw milk Canestrato Pugliese cheese

AIMS: Investigation of the autochthonous lactic acid bacteria (LAB) population of the raw milk protected designation of origin Canestrato Pugliese cheese using phenotypic and genotypic methodologies. METHODS AND RESULTS: Thirty phenotypic assays and three molecular techniques (restriction fragment length polymorphism, partial sequencing of the 16S rRNA gene and recA multiplex PCR assay) were applied to the identification of 304 isolates from raw milk Canestrato Pugliese cheese. As a result, 168 of 207 isolates identified were ascribed to genus Enterococcus, 25 to Lactobacillus, 13 to Lactococcus and one to Leuconostoc. More in details among the lactobacilli, the species Lactobacillus brevis and Lactobacillus plantarum were predominant, including 13 and 10 isolates respectively, whereas among the lactococci, Lactococcus lactis subsp.cremoris [corrected] was the species more frequently detected (seven isolates). CONCLUSIONS: Except for the enterococci, phenotypic tests were not reliable enough for the identification of the isolates, if not combined to the genotype-based molecular techniques. The polyphasic approach utilized allowed 10 different LAB species to be detected; thus suggesting the appreciable LAB diversity of the autochthonous microbial population of the Canestrato Pugliese cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: A comprehensive study of the resident raw milk Canestrato Pugliese cheese microbial population has been undertaken.     

Technologically important properties of lactic acid bacteria isolates from Beyaz cheese made from raw ewes'' milk

AIMS: The aim of the study was to characterize isolates of lactic acid bacteria from Beyaz cheese and to study some of their technologically important properties. METHODS AND RESULTS: Seventy-seven lactic acid bacteria isolated from Beyaz, a Turkish white-brined cheese made from raw ewes' milk without any starter, were classified by phenotypic criteria and sodium dodecyl sulphate-polyacrylamide gel electrophoresis of whole-cell proteins. Whole cells of Lactococcus lactis subsp. lactis and enterococci showed lipolytic and proteolytic activities. Strains were found that differed in terms of their acidifying and caseinolytic activity. Most of the enterococci isolates showed tyrosine decarboxylase activity; moreover, lactobacilli exhibited weak antibacterial activities against foodborne pathogens. CONCLUSIONS: Strains of lactic acid bacteria with interesting biotechnologically important properties may be found in Beyaz cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactic acid bacteria isolates with interesting biotechnological profiles may influence the quality and variety of dairy products, if they are used as starters, and their cheese-making characteristics seem promising.     

Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese

Growth and survival of two enterotoxigenic strains of Staphylococcus aureus were studied during manufacture and ripening of eight batches of raw ewes' milk Manchego cheese. Only 2-3 generations of Staph. aureus occurred in the vat and during pressing. The death rate of Staph. aureus (mean decrease in log cfu/g/week of ripening) from day 1 to day 60 was 0.421 in cheese made with 1% Streptococcus lactis starter and 0.404 in cheese made without starter. Thermostable nuclease was produced in the vat by growing Staph. aureus cells; it was inactivated by rennet during the first 24 h and synthesized again by surviving cells of Staph. aureus from day 1 to day 60. Staphylococcal enterotoxins A, B, C and D were not detected in any batches of cheese, even though Staph. aureus counts exceeded 10(7) cfu/g.     

Inactivation of Staphylococcus aureus in raw milk cheese by combinations of high-pressure treatments and bacteriocin-producing lactic acid bacteria

AIMS: To investigate the combined effect of high-pressure treatments (HPT) and milk inoculation with bacteriocin-producing lactic acid bacteria (BP-LAB) on the survival of Staphylococcus aureus during ripening of raw milk cheese. METHODS AND RESULTS: Cheeses were manufactured from raw milk artificially contaminated with S. aureus at ca 5 log CFU ml(-1), a commercial starter culture and one of seven strains of BP-LAB, added as adjuncts at 0.1%. HPT of cheeses were performed on days 2 or 50 at 300 MPa (10 degrees C, 10 min) or 500 MPa (10 degrees C, 5 min). On day 3, S. aureus counts were 6.46 log CFU g(-1) in control cheese. Milk inoculation with different BP-LAB lowered S. aureus counts on day 3 when compared with control cheese by up to 0.46 log CFU g(-1), HPT at 300 MPa on day 2 by 0.45 log CFU g(-1) and HPT at 500 MPa on day 2 by 2.43 log CFU g(-1). Combinations of BP-LAB with HPT at 300 and 500 MPa on day 2 lowered S. aureus counts on day 3 by up to 1.02 and 4.00 log CFU g(-1) respectively. CONCLUSIONS: The combined effect of milk inoculation with some of the BP-LAB tested and HPT of cheese on S. aureus inactivation was synergistic. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of HPT at lower pressures with BP-LAB inoculation is a feasible system to improve cheese safety in case of deleterious effects on cheese quality caused by HPT at higher pressures.     

Bacterial dynamics in a raw cow's milk Caciotta cheese manufactured with aqueous extract of Cynara cardunculus dried flowers

Aims: To investigate the bacterial dynamics of a Caciotta cheese traditionally manufactured in the Montefeltro area (Central Italy) with raw cow’s milk and an aqueous extract of dried flowers from Cynara cardunculus as a coagulating agent. Methods and Results: Conventional methods and a combined PCR‐DGGE approach, relying on culture‐dependent and ‐independent analyses, were used to investigate the cheese bacterial community, with a special focus on lactic acid bacteria. A heterogeneous population, including enterococci, lactococci, lactobacilli, food spoilage and other banal micro‐organisms, was found. Significance and Impact of the Study: The study contributed to highlighting the influence of different technological parameters on bacterial dynamics of a raw milk Caciotta cheese coagulated with vegetable rennet. Conclusions: None of the species found in the vegetable rennet became dominant during the cheese‐making and a prevailing role of the adventitions microbita coming from the raw milk and the dairy environment was highlighted.     

Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese

Growth and survival of two enterotoxigenic strains of Staphylococcus aureus were studied during manufacture and ripening of eight batches of raw ewes' milk Manchego cheese. Only 2–3 generations of Staph. aureus occurred in the vat and during pressing. The death rate of Staph. aureus (mean decrease in log cfu/g/week of ripening) from day 1 to day 60 was 0.421 in cheese made with 1% Streptococcus lactis starter and 0.404 in cheese made without starter. Thermostable nuclease was produced in the vat by growing Staph. aureus cells; it was inactivated by rennet during the first 24 h and synthesized again by surviving cells of Staph. aureus from day 1 to day 60. Staphylococcal enterotoxins A, B, C and D were not detected in any batches of cheese, even though Staph. aureus counts exceeded 10⁷ cfu/g.     

The use of cadmium resistance on the phage-resistance plasmid pNP40 facilitates selection for its horizontal transfer to industrial dairy starter lactococci

AIMS: To facilitate the horizontal transfer and selection of phage-resistance plasmids in industrial lactococci. METHODS AND RESULTS: Cadmium-resistance properties similar to those previously identified in Lactococcus were linked to the well-known phage-resistance plasmid pNP40. This finding was exploited to facilitate delivery of the plasmid to an industrial cheese starter Lactococcus lactis DPC4268. Additionally, 25 different cadmium-sensitive cheese starter lactococci were also identified as potential recipients for the phage-resistance plasmid pNP40, and also the plasmids pAH90/pAH82 which also encode cadmium resistance. All three plasmids were successfully conjugated to strain DPC4268. Cheddar cheese was manufactured in industry with the pNP40 phage-resistant transconjugant. SIGNIFICANCE AND IMPACT OF THE STUDY: Food-grade enhancement of phage resistance in industrial starter strains has been made simpler by the use of this selection, especially since the majority of potential recipient starter strains analysed were cadmium sensitive.     

Enterobacteriaceae, coliforms, faecal coliforms and salmonellas in raw ewes' milk

Thirty-nine samples of freshly drawn ewes' milk collected at 13 farms, and 120 samples of raw ewes' milk collected on arrival at a dairy were examined. Farm samples had geometric mean counts of 4.4 X 10(2) Enterobacteriaceae/ml, 3.9 X 10(2) coliforms/ml and 2.0 X 10(2) faecal coliforms/ml, whereas the respective mean counts were 6.2 X 10(3)/ml, 5.4 X 10(3)/ml and 1.3 X 10(3)/ml for dairy samples. Salmonellas were not detected by enrichment procedures in any of the 159 samples examined. Escherichia coli (47.5% strains), Enterobacter cloacae (17.7%), Ent. agglomerans (11.3%), Hafnia alvei (6.5%) and Klebsiella oxytoca (6.0%) were the predominant species in 434 Enterobacteriaceae strains isolated from farm samples. Levels and species of Enterobacteriaceae found in the present work in raw ewes' milk imply a considerable risk of early blowing in cheese-making from unpasteurized milk.     

Growth and survival of E. coli O157:H7 during the manufacture and ripening of a smear-ripened cheese produced from raw milk

AIMS: The behaviour of Escherichia coli O157:H7 was studied during the manufacture and ripening of a smear-ripened cheese produced from raw milk. METHODS AND RESULTS: Cheese was manufactured on a laboratory scale using milk (20 l) inoculated with E. coli O157:H7, and enumeration was carried out using CT-SMAC. From an initial level of 1.52 +/- 0.03 log cfu ml-1 in the milk (34 +/- 2 cfu ml-1), the numbers increased to 3.4 +/- 0.05 log cfu g-1 in the cheese at day 1. During ripening, the numbers decreased to <1 cfu g-1 and <10 cfu g-1 in the rind and core, respectively, after 21 days, although viable cells were detected by enrichment after 90 days. The presence of E. coli O157:H7 in the cheese was confirmed by latex agglutination and by multiplex PCR. CONCLUSION: The results indicate that the manufacturing procedure encouraged substantial growth of E. coli O157:H7 to levels that permitted survival during ripening and extended storage. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of low numbers of E. coli O157:H7 in milk, destined for raw milk cheese manufacture, could constitute a threat to the consumer.     

Effect of heat treatment on the proteolytic activity of mesophilic bacteria isolated from goats' milk cheese

Strains of mesophilic lactococci and lactobacilli isolated from goats' milk cheese were grown to maximum density in milk at 30°C, pH 6·5. They were subsequently cooled to 12°C and then heated at 50°, 52° and 54°C (holding time, 15 s). The micro-organisms tested were Lactococcus lactis subsp. lactis IFPL 60, IFPL 22 and IFPL 359, Lactobacillus casei subsp. casei IFPL 731 and Lactobacillus plantarum IFPL 3, isolated from raw goats' milk cheese. The heated cells presented lower viability and acidification capacity than unheated cells. After heat treatment at 50°C, all the test strains effected practically no reduction in pH of milk (6 h), except for Lactococcus lactis subsp. lactis IFPL 60, which reduced pH to 5·9 as compared to 4·9 attained by the unheated controls. After treatment, proteolytic, aminopeptidase and dipeptidase activities of cell-free extracts decreased to a lesser extent than the number of viable cells with acidifying ability. The results suggest that these strains, if treated at 50°C, may be suitable as extra sources of important ripening enzymes in cheese making.     

Genotypic and phenotypic heterogeneity among lactococci isolated from traditional Pecorino Sardo cheese

Twenty-nine Lactococcus lactis isolates from one traditional 24 h-old Pecorino Sardo cheese were characterized phenotypically, technologically and genotypically in order to assess the biodiversity within this wild microbial population. Two DNA-based techniques, plasmid profiling and PFGE, were used for the genetic typing of the isolates. All 29 isolates were characterized at strain level and eight different genotypes were recognized. In addition, by combining the results from plasmid profile analysis and PFGE, it was possible to identify closely related isolates probably belonging to the same clonal lineage. The dominant biotype was identified in the 24 h-old cheese, as were the strains believed to act as starters for the curd. Atypical lactococci, able to grow in 6.5% NaCl, were isolated. The results suggest that wild bacterial populations should be preserved in order to protect the traditional raw milk cheeses, and to select new starter strains for the dairy industry.     

Effect of a lactic starter culture on the growth and protease activity of Aeromonas hydrophila

Lactococcus lactis subsp. lactis strain Lac 388 (isolated from goats' cheese made without starter) had inhibitory activity against three strains of Aeromonas hydrophila either by the plating method or by the associative culture approach (broth, skim milk and ewes' milk). Low pH was only partially responsible for the antagonism. Inhibition due to hydrogen peroxide and nutrient depletion was excluded. This inhibitory compound(s) was not destroyed by proteolytic enzymes. In mixed cultures the strains of Aer. hydrophila did not produce detectable amounts of protease.     

Enterobacteriaceae, coliforms, faecal coliforms and salmonellas in raw ewes'milk

Thirty-nine samples of freshly drawn ewes' milk collected at 13 farms, and 120 samples of raw ewes' milk collected on arrival at a dairy were examined. Farm samples had geometric mean counts of 4.4 × 10² Enterobacteriaceae/ml, 3.9 × 10² coliforms/ml and 2.0 × 10² faecal coliforms/ml, whereas the respective mean counts were 6.2 × 10³/ml, 5.4 × 10³/ml and 1.3 × 10³/ml for dairy samples. Salmonellas were not detected by enrichment procedures in any of the 159 samples examined. Escherichia coli (47.5% strains), Enterobacter cloacae (17.7%), Ent. agglomerans (11.3%), Hafnia alvei (6.5%) and Klebsiella oxytoca (6.0%) were the predominant species in 434 Enterobacteriaceae strains isolated from farm samples. Levels and species of Enterobacteriaceae found in the present work in raw ewes' milk imply a considerable risk of early blowing in cheese-making from unpasteurized milk.     

The microstructure and distribution of micro-organisms within mature Serra cheese

The distribution of micro-organisms in mature Serra, a traditional Portuguese cheese made from unpasteurised ewes' milk without added starter culture, was examined by light microscopy and electron microscopy. Four populations of micro-organisms were recognized according to their position within the cheese: (i) those present as apparently axenic colonies within the curd matrix; (ii) bacteria growing along curd junctions; (iii) yeasts and bacteria present in the smear on the surface of the cheese and (iv) bacteria found in cracks which penetrated the outer part of the cheese from the rind. Two types of crystals were observed, together with contaminants of vegetable origin and somatic cells originating from the milk.