Brining time effect on physicochemical and microbiological parameters in Idiazábal cheese.

Physicochemical and microbiological parameters were compared for three brining times (12, 24 and 36 h) for fresh, young, semihard and hard Idiazabal cheese. Longer brining time produced higher salt, dry matter and salt-moisture ratio and lower water activity values for all types of cheese according to ripening time, while non-significant changes were observed for pH. In fresh cheese (1-15 days ripening), non-significant differences for microbiological counts in relation to brining time were observed, except for moulds. In young and hard cheeses, Lactobacillus and Leuconostoc showed lower counts with longer brining times. In contrast, Micrococaceae, yeast and moulds were stimulated by higher salt content in matured cheeses. In addition. this work has proved that there are lower water activity values and lower microbiological counts in longer-matured Idiazabal cheeses. For the different brining and ripening times, positive correlations were observed among most of the microbial groups studied, but a different behavior was established for Enterococcus, Clostridium tyrobutyricum, yeast and moulds.     

Microbiological study of semi-hard goat's milk cheese (Majorero)

The development of microbial flora in industrially produced semi-hard cheese made from pasteurized goat's milk was studied during manufacture and over a 90-day ripening period.
Estimates of total count, streptococci, lactobacilli, leuconostocs, coliforms, micrococci and staphylococci, Staphylococcus aureus and yeasts and moulds were carried out at various stages of the ripening process; streptococci and lactobacilli were identified by species.
Initially, the total count increased rapidly, primarily as a result of the growth of mesophilic lactic streptococci mainly Streptococcus lactis and Strep, cremoris. Subsequently, both these counts stabilized or decreased. Lactobacilli increased, and by the end of the ripening period were the predominant microorganisms. Most common were Lactobacillus casei var. casei. , especially at the end of storage; L. casei var. rhamnosus , L. casei var. plantarum and L. cellobiosus were also isolated. Leuconostocs were not found in any of the cheeses, and hence no eye formation took place. Coliforms, enterococci, yeasts and moulds remained below 10²–10³ c.f.u. g⁻¹. Maximum levels of micrococci and staphylococci were found after 15–30 days of ripening and decreased gradually towards the end of the ripening period. Neither the milk curd, nor cheese contained Staph. aureus.     

Potential use of presumptive enterococci and staphylococci as indicators of sanitary condition in plants making hard Italian-type cheese

Raw milk, pasteurized milk, unripened cheese (1 day old), and partially ripened cheese (3 months) from 42 milk lots at a plant making hard Italian-type cheese were analyzed for presumptive enterococci using kanamycin esculin azide agar pour plates. Fully ripened (> or =10 months) cheeses, derived from other milk lots, were also tested. Numbers of presumptive staphylococci (Baird-Parker agar [B-P]) were determined in the partially and fully ripened cheeses. Presumptive enterococci were ubiquitous in raw milk, usually at levels of 2.1 to 3.0 log CFU/ml. Enterococci were detected in 11 (26%) of 42 pasteurized milk samples. Enterococci and staphylococci were detected in 39 (93%) and 6 (14%) of unripened cheeses and in 33 (80%) and 4 (10%) of partially ripened cheeses, respectively. Only eight and five samples of enterococci-positive unripened and partially ripened cheese, respectively, were made from pasteurized milk in which presumptive enterococci were detected. Of 42 samples of fully ripened cheese, 35 (83%) and 8 (19%), respectively, contained presumptive enterococci and staphylococci. Results suggest either that low numbers of presumptive enterococci survive pasteurization and cheese ripening or that contamination of cheese by enterococci occurs after pasteurization. Biochemical testing confirmed 63% of presumptive enterococci isolates. None of the 20 presumptive staphylococci isolates produced colonies typical of Staphylococcus aureus on B-P agar; the isolates were identified as 1 Staphylococcus epidermidis, 1 Staphylococcus xylosus, 2 Staphylococcus saprophyticus, 1 Staphylococcus warneri, 5 Kocuria spp., and 10 unidentified gram-positive, catalase-positive cocci. Three staphylococci isolates decreased in numbers by more than 3.0 log CFU/ml in 9.9 ml of skim milk heated 30 min in a 62.8 degrees C water bath. This finding suggests that most presumptive staphylococci detected may have been prepasteurization contaminants. Unless specificity of the kanamycin esculin azide and B-P media is improved, use of presumptive enterococci and staphylococci as indicators of postpasteurization sanitation in plants making hard Italian-type cheese cannot be recommended.     

Effects of Penicillium roqueforti and whey cheese on gross composition,microbiology and proteolysis of mould‐ripened Civil cheese during ripening

Four different types of mould‐ripened Civil cheese were manufactured. A defined (nontoxigenic) strain of a Penicillium roqueforti (SC 509) was used as the secondary starter with and without addition of the whey cheese (Lor); in parallel, secondary starter‐free counterparts were manufactured. Chemical composition, microbiology and proteolysis were studied during the ripening. The incorporation of whey cheese in the manufacture of mould‐ripened Civil cheese altered the gross composition and adversely affected proteolysis in the cheeses. The inoculated P. roqueforti moulds appeared to grow slowly on those cheeses, and little proteolysis was evident in all cheese treatments during the first 90 days of ripening. However, sharp increases in the soluble nitrogen fractions were observed in all cheeses after 90 days. Microbiological analysis showed that the microbial counts in the cheeses were at high levels at the beginning of ripening, while their counts decreased approximately 1–2 log cfu/g towards the end of ripening.     

Occurrence of moulds associated with ovine raw milk and cheeses of the Spanish region of Castilla La Mancha

The distribution of mould species was examined at several points of the processing chain in a Manchego cheese plant and associated dairy farms. Geotrichum and Fusarium were the genera most frequently isolated from milk samples as well as in cheeses ripened for one month, evidencing a direct transfer from raw milk. Conversely, the mycobiota of long‐ripened cheeses consisted mainly of Penicillium species, which gained entry to the cheese through the air of ripening rooms. This study contributes to the understanding of the dynamics of fungal populations in semihard and hard cheeses, highlighting that airborne transfer from the stables could have a direct impact on their quality.     

Stability of the biodiversity of the surface consortia of Gubbeen, a red-smear cheese

A total of 1,052 bacteria and 828 yeasts were isolated from the surface flora of 6 batches of Gubbeen cheese made in 1996-1997 and 2002-2003. Stability of the microflora was evaluated over time and also during ripening at 4, 10, and 16 d (batches 4, 5, and 6) or at 4, 16, 23, and 37 d (batches 1, 2, and 3). Bacteria were identified using pulsed-field gel electrophoresis, repetitive extragenic palindromic-PCR, and 16S rRNA gene sequencing, and yeasts were identified by Fourier transform infrared spectroscopy. The bacteria included at least 17 species, of which the most common were Staphylococcus saprophyticus (316 isolates), Corynebacterium casei (248 isolates), Brevibacterium aurantiacum (187 isolates), Corynebacterium variabile (146 isolates), Microbacterium gubbeenense (55 isolates), Staphylococcus equorum/cohnii (31 isolates), and Psychrobacter spp. (26 isolates). The most common yeasts were Debaryomyces hansenii (624 isolates), Candida catenulata (135 isolates), and Candida lusitaniae (62 isolates). In all batches of cheese except batch 2, a progression of bacteria was observed, with staphylococci dominating the early stages of ripening and coryneforms the later stages. No progression of yeast was found. Pulsed-field gel electrophoresis showed that several different strains of the 5 important species of bacteria were present, but generally only one predominated. The commercial strains used for smearing the cheese were recovered, but only in very small numbers early in ripening. Four species, B. aurantiacum, C. casei, C. variabile, and Staph. saprophyticus, were found on all batches of cheese, but their relative importance varied considerably. The results imply that significant variation occurs in the surface microflora of cheese.     

Effect of natamycin-containing coating on the evolution of biochemical and microbiological parameters during the ripening and storage of ovine hard-Gruyère-type cheese

The effect of a coating containing natamycin on the ripening course of the hard-Gruyère-type cheese Graviera Kritis was assessed. A single treatment at an early stage of ripening was carried out; samples from natamycin-treated (NT) and control cheeses (CTR) were then taken throughout a 12 month ripening and storage period. Coating gave a statistically significant reduction in the yeasts and moulds counts in the cheese rind. It did not influence the counts and the evolution of the thermophilic bacteria related to starter or of the propionic acid bacteria, nor did it affect the associated aminopeptidase activities. Gross composition of NT cheeses did not differ significantly from that of the control cheeses; the same was also true for proteolysis. Natamycin in the cheese rind after the removal of coating was lower than 0.1 mg dm⁻² at all stages of ripening and no migration to the cheese interior was observed.     

Microbial dynamics during the ripening of a mixed cow and goat milk cheese manufactured using frozen goat milk curd

To overcome the seasonal shortage of goat milk in mixed milk cheese manufacture, pasteurized goat milk curd and high-pressure-treated raw goat milk curd manufactured in the spring were held at −24°C for 4 mo, thawed, and mixed with fresh cow milk curd for the manufacture of experimental cheeses. Control cheeses were made from a mixture of pasteurized cow and goat milk. The microbiota of experimental and control cheeses was studied using culture-dependent and culture-independent techniques. Bacterial enumeration by classical methods showed lactic acid bacteria to be the dominant population in both control and experimental cheeses. In total, 681 isolates were grouped by partial amplified rDNA restriction analysis (ARDRA) into 4 groups and identified by 16S rRNA gene sequencing as Lactococcus lactis ssp. lactis (563 isolates), Leuconostoc pseudomesenteroides (72 isolates), Lactobacillus spp. (34 isolates), and Lc. lactis ssp. cremoris (12 isolates). Temporal temperature gradient gel electrophoresis (TTGE) analysis of cheese showed (1) the predominance of Lc. lactis in all cheeses; (2) the presence of Leu. pseudomesenteroides population in all cheeses from d 15 onward; (3) the presence of a Lactobacillus plantarum population in control cheese until d 15 and in experimental cheeses throughout the ripening period. Due to the most diverse and complete set of peptidases present in the genus Lactobacillus, the prevalence of this population in experimental cheeses could give rise to differences in cheese flavor between experimental and control cheeses.     

Physicochemical,microbiological, textural and sensory changes during the ripening of pasteurised goat milk cheese made with plant coagulant (Cynara scolymus)

This study describes the changes that occur during the ripening of cheeses made with a plant coagulant derived from artichoke flowers (Cynara scolymus). The results indicate that the physicochemical composition during ripening evolves similarly to other cheeses. The texture and sensory features of the cheeses during ripening evolved differently from that observed for other goat cheeses. Although it is common for a bitter taste to develop during the ripening of cheeses elaborated with plant coagulants, bitterness was scored very low in the cheeses made with artichoke, so that these cheeses could be suitable for marketing.     

The fate of indigenous microbiota, starter cultures, Escherichia coli, Listeria innocua and Staphylococcus aureus in Danish raw milk and cheeses determined by pyrosequencing and quantitative real time (qRT)-PCR

The purpose of this work was to study the bacterial communities in raw milk and in Danish raw milk cheeses using pyrosequencing of tagged amplicons of the V3 and V4 regions of the 16S rDNA and cDNA. Furthermore, the effects of acidification and ripening starter cultures, cooking temperatures and rate of acidification on survival of added Escherichia coli, Listeria innocua and Staphylococcus aureus in cheeses at different stages of ripening were studied by pyrosequencing and quantitative real time (qRT)-PCR. A high diversity of bacterial species was detected in raw milk. Lactococcus lactis, Streptococcus thermophilus, Lactobacillus casei and Lactobacillus rhamnosus were the main bacteria detected in raw milk and cheeses. Bacteria belonging to the genera Brevibacterium, Staphylococcus, Escherichia, Weissella, Leuconostoc, Pediococcus were also detected in both 16S rDNA and cDNA obtained from raw milk and cheeses. E. coli, which was added to milk used for production of some cheeses, was detected in both DNA and RNA extracted from cheeses at different stages of ripening showing the highest percentage of the total sequence reads at 7 days of ripening and decreased again in the later ripening stages. Growth of E. coli in cheeses appeared to be affected by the cooking temperature and the rate of acidification but not by the ripening starter cultures applied or the indigenous microbiota of raw milk. Growth of L. innocua and S. aureus added to milks was inhibited in all cheeses at different stages of ripening. The use of 16S rRNA gene pyrosequencing and qRT-PCR allows a deeper understanding of the behavior of indigenous microbiota, starter cultures and pathogenic bacteria in raw milk and cheeses.     

Properties and antimicrobial activity of the smear surface cheese coryneform bacterium <Emphasis Type="Italic">Brevibacterium linens</Emphasis>

Surface microorganisms contribute to the ripening of some low-moisture cheese varieties and the composition of the surface microflora is dynamic. Brevibacterium linens is an important surface microorganism that is present in the smear of surface-ripened cheeses and is commonly regarded as the organism primarily responsible for the characteristic taste, aroma, and color of surface cheese. The enzymology and biochemical characteristics of B. linens influence the ripening and final characteristics of smear surface-ripened cheeses. Proteolytic, peptidolytic, esterolytic, and lipolytic activities are of particular importance in the ripening process. Because of its putative importance to the ripening in smear-ripened cheeses, B. linens is the best studied component of the microflora, although in comparision with other dairy-related microorganisms, it is poorly characterized. B. linens produces antimicrobial substances that inhibit the growth of many food poisoning bacteria as well as several yeast and moulds. Some inhibitory substances produced by this species were identified as bacteriocins. Bacteriocins could appear as potential agents to be applied in food conservation systems in order to provide microbiologically stable foods. This article describes the properties of B. linens and discusses about the potential of this species to produce bacteriocins and other antimicrobial substances, which are important for production of high quality cheese.     

Identification of micrococcaceae isolated from goat''s milk and cheese in the Poitou-Charentes region

One hundred and ninety strains of coagulase-negative staphylococci were isolated from goat's milk, whey and cheese at various stages of manufacture.

Sixteen different coagulase negative Staphylococci (CNS) species were recovered, 3 of which were predominant: Staphylococcus simulans, Staphylococcus epidermidis and Staphylococcus xylosus.

The prevalent species were recovered at least at two different stages of cheese manufacturing, suggesting a better adaptation to the environment. After 15 days of ripening, the cheeses showed lower counts of Micrococcaceae.     

Staphylococcus aureus Growth and Toxin Production in Imitation Cheeses

The ability of eleven imitation (or substitute) cheeses to support the growth and toxin production of Staphylococcus aureus at 26°C was evaluated. All established enterotoxin serotypes were tested by inoculating suspensions of the requisite strains into 100-g samples of cheese (about 30 staphylococci/g). Water activity (a_w) of the cheese samples ranged from 0.942–0.973; pH values ranged from 5.33–6.14. Seven cheeses supported extensive growth of S. aureus; one or more serotypes of enterotoxin were produced in six cheeses. Enterotoxin in the cheese was detected in 4 days at 3 × 10⁶ staphylococci/g. However, the ability of some cheeses to support growth and toxin production of S. aureus could not be correlated with pH, a_w, or product formulation.     

Evolution of Free Amino Acids during the Ripening of Cheddar Cheese Containing Added Lactobacilli Strains

Cheddar cheese was produced with different lactobacilli strains added to accelerate ripening. The concentration of proteolytic products was determined as free amino acids in the water-soluble fraction at two, four, seven and nine months of aging and at two different maturation temperatures (6°C, 15°C). All amino acids increased during ripening and were higher in the Lactobacillus- added cheeses than in the control cheese, and higher in cheeses ripened at 15°C than at 6°C. Glutamic acid, leucine, phenylalanine, valine and lysine were generally in higher proportion in all cheeses. The cheeses with added L. casei-casei L2A were classified as having a “strong Cheddar cheese” flavor after only seven months of ripening at 6°C.     

The microflora of Tilsit cheese. Part 2. Development of a surface smear starter culture

Single strains of bacteria isolated from the surface of commercial Tilsit cheeses were screened for their ability to produce typical Tilsit flavour and colour and for fast growth in milk. Three milk based model systems were developed for screening. Shake liquid milk cultures were suitable to determine production of colour and volatile flavour compounds. Milk agar plates were used to study synergistic and antagonistic effects between isolates. With mini cheeses in centrifuge bottles, cheese conditions were simulated under sterile conditions. Volatile aroma production and pigmentation of the surface flora were studied with this system. Additional growth studies in other growth media with various combinations of strains revealed some of the possible roles of surface bacteria. Brevibacterium linens promoted growth of yellow coryneform bacteria. A pigmented Arthrobacter strain was responsible for the production of a yellow coloured watersoluble pigment, a precursor for the typical red-brown colour of Tilsit cheese. In mixed culture with pigmented or non-pigmented strains of B. linens, the yellow colour turned into red-brown. A proteolytic Staphylococcus strain seemed to be important for the initiation of surface starter growth. Staphylococci showed fast growth at pH 5.5 and below. They also promoted growth of the yellow Arthrobacter strain. Based on these results, a defined surface starter was developed consisting of 5 strains. The yeast Debaryomyces hansenii was used for deacidification of the cheese rind. A combination of a non-pigmented, proteolytic B. linens, a yellow Arhrobacter strain, a cream-coloured coryneform bacterium, and a proteolytic Staphylococcus sciuri were used for cheese ripening. Experimental cheeses were produced on a 10 kg scale. The defined starter grew fast on the cheese surfaces, and produced the typical taste and flavour and colour of Tilsit cheese.     

Microbiological and biochemical changes in herby cheese during ripening

The aim of this study was to determine and compare the microbiological, biochemical and sensory characteristics of herby cheese made with two different methods. In the first method (M1), milk and herbs were pasteurized at 65°C for 30 min, and Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris were added as starter culture at an inoculum ratio of 1.5%. In the second method (M2), the conventional cheesemaking was applied. Microbiological and biochemical changes were monitored throughout the ripening period of 90 days. Samples were taken from cheeses on days 1, 15, 30, 60, and 90. At the end of ripening, sensory characteristics of cheeses manufactured with both methods were evaluated. The obtained results suggested that most changes in pH, titratable acidity, and dry matter contents of cheese varieties were not found to differ statistically significant, but the difference in salt content was significant (P < 0.01). Total aerobic count, lactic acid bacteria, Staphylococcus aureus, coliforms, moulds, yeasts, proteolytic and lipolytic microorganism counts were lower in M1 cheese samples than those of M2 cheese samples (P < 0.01). The numbers of psychrotrophic microorganism in both cheese types were not found to differ significantly. Moreover, the results suggested that there were significant differences (P < 0.01) in the degrees of proteolysis and lipolysis of the cheese varieties. High proteolysis and lipolysis rates were monitored in the traditional cheese samples. However, there were no significant differences between the sensory characteristics of cheese samples.     

Occurrence of foodborne pathogens in Irish farmhouse cheese

Food safety is a critical factor in the production of farmhouse cheese. In Ireland the varieties of farmhouse cheese produced reflect a much broader range than those produced commercially and some of these cheese varieties are associated with greater microbiological risk. These include cheese produced from unpasteurised milk and soft ripened cheese such as mould or smear-ripened cheeses which have high pH and relatively short ripening times. The aim of this study was to determine the microbiological quality of farmhouse cheeses in Ireland. Three hundred and fifty one cheese samples, from 15 cheese producers, were analysed for microbiological quality on a monthly basis throughout the year. The analyses included enumeration of Escherichia coli, Staphylococcus aureus and Listeria monocytogenes (using the relevant agars) and enrichment for L. monocytogenes. The cheeses selected were produced from ovine, caprine and bovine milk. Both unpasteurised and pasteurised milk cheeses were sampled and these included hard, semi-hard and soft cheeses, internal/external mould-ripened and smear-ripened cheeses and the cheeses represented different geographic regions. Of the cheeses tested, 94% were free of L. monocytogenes, all were within the EU limits for E. coli and only one cheese variety had S. aureus levels above the recommended numbers for the first 6 months of the year. Due to a modified production process the numbers were within the guidelines for the second six months. The results indicate that Irish farmhouse cheeses are of a high microbiological quality.     

Enterotoxin-producing Staphylococcus aureus genotype B as a major contaminant in Swiss raw milk cheese

The objective of this study was to characterize Staphylococcus aureus isolates from Swiss raw milk cheeses that had been found to be contaminated with coagulase-positive staphylococci and to estimate the frequency of the various genotypes, in particular the mastitis-associated Staph. aureus genotype B (GTB). The isolates were also tested for staphylococcal enterotoxin (SE) genes and other virulence factors. From 623 coagulase-positive staphylococci isolated from 78 contaminated raw milk cheeses, 609 were found to be Staphylococcus aureus. Genotyping of all Staph. aureus isolates was performed by PCR amplification of the 16S–23S rRNA intergenic spacer region, as this method was used previously to differentiate between mastitis subtypes associated with their clinical outcome. In total, 20 different genotypes were obtained and the 5 most frequently occurring genotypes were distributed in 6.4% or more of the samples. The enterotoxin-producing Staph. aureus GTB, known for its high contagiousness and increased pathogenicity in Swiss mastitis herds, was found to be the most abundant subtype at the sample level (71.8%) as well as among the isolates (62.0%). A subset of 107 isolates of the different genotypes were analyzed for the presence of SE genes and revealed 9 different SE gene patterns, with sed being most frequently detected and 26% being PCR-negative for SE genes. Almost all isolates of the major contaminant GTB contained the SE gene pattern sed, sej, ser, with half of them additionally carrying sea. Production of SE in vitro was consistent with the SE genes detected in most of the cases; however, some isolated GTB did not produce SEA. Staphylococcus aureus Protein A (spa) typing revealed 30 different subtypes and most GTB isolates belonged to the bovine spa type t2953; GTB/t2953 was linked among other subtypes to SE production in cheese and staphylococcal intoxication cases. Furthermore, 1 of the 623 isolates was a methicillin-resistant Staph. aureus, which was an seh-carrying Staph. aureus spa type tbl 0635 (non-GTB). We conclude that control and reduction of enterotoxigenic Staph. aureus GTB in dairy herds in Switzerland will not only prevent economic losses at the farm level but also improve the safety of raw milk cheeses; distribution of methicillin-resistant Staph. aureus via raw milk cheese is of less concern.     

Cultures for the ripening of smear cheeses

Data on typical surface microflora of smeared semi-soft, soft and acid curd cheeses and the minimal composition of suitable surface starter cultures are reviewed. Cultures for semi-soft cheeses should contain Debaryomyces hansenii, Staphylococcus equorum, Corynebacterium casei, Microbacterium gubbeenense (or Arthrobacter nicotianae), and Brevibacterium linens. Apart from D. hansenii, soft cheese surface cultures should contain Geotrichum candidum, which is responsible for the typical appearance and aroma development. M. gubbeenense or A. nicotianae and B. linens are essential for soft cheese ripening, but C. casei is not. S. equorum, not regularly found on the surface of commercial soft cheeses, accelerated deacidification and smear development. Cultures for acid curd cheeses, produced from quarg, should contain Kluyveromyces marxianus and Candida krusei. Staphylococci seem to be essential for ripening. S. equorum can replace the non-food-grade S. saprophyticus that is always present on commercial acid curd cheeses. Suitable corynebacteria for spraying of cheeses are B. linens and C. variabile.     

Characterisation of proteolysis profile of Argentinean sheep cheeses made by two different production methods

BACKGROUND: In this work the proteolysis profiles of Argentinean sheep cheeses made by two different production methods were studied in order to develop products with typical and defined features. Cheeses with a starter of Streptococcus thermophilus, curd cut to corn grain size, washed and heated to 43 °C (S cheeses) and cheeses with a mixed starter of Streptococcus thermophilus, Lactobacillus helveticus and Lactobacillus bulgaricus, curd cut to rice grain size, unwashed and heated to 47 °C (L cheeses) were manufactured. The cheeses were ripened at 12 °C and 80% relative humidity for 180 days and samples were taken throughout this period. RESULTS: Gross composition and primary proteolysis were similar for both types of cheeses. Streptococci counts diminished from 10⁹ to 10⁷ colony‐forming units g^?1 during ripening in both S and L cheeses. Lactobacilli counts in L cheeses decreased during ripening and disappeared at 180 days. L cheeses had significantly lower pH values and showed higher peptidolysis than S cheeses. Triangle sensory evaluation indicated important differences between the two types of cheeses. CONCLUSION: S cheeses had a low proteolysis level and a soft flavour, making them appropriate for consumption after a short ripening time. L cheeses had a higher proteolysis level and more intense sensory characteristics, making them appropriate for consumption after a longer ripening time. Copyright © 2009 Society of Chemical Industry