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.     

Aspects of enzymology and biochemical properties of Brevibacterium linens relevant to cheese ripening: a review.

Brevibacterium linens is a major surface microorganism that is present in the smear of surface-ripened cheeses. 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, which are of particular importance in the ripening process, are discussed in detail. This review also describes the production of volatile compounds, especially sulfur-containing ones, by B. linens, which are thought to be important in respect to the flavor of smear surface-ripened cheeses. The unique orange-colored carotenoids and the factors effecting their production by B. linens are also presented. The catabolism of aromatic amino acids, bacteriocin production, plasmids, and miscellaneous biochemical and physiological properties (peptidoglycan type, antibiotic resistance, insecticide degradation, and biotechnological applications) of B. linens are discussed. The problem associated with the current taxonomical classification of B. linens strains caused by strain variation is evaluated. Finally, the application of B. linens cell extracts or its proteolytic enzymes as cheese ripening accelerants for semi-hard or hard cheese varieties is considered.     

The microflora of Tilsit cheese. Part 1. Variability of the smear flora

The microflora of 25 Tilsit cheeses from 2 cheese plants was analysed. Debaryomyces hansenii was found to be the predominant yeast in all stages of ripening. 75–95% of the bacterial flora consisted of coryneform bacteria. Several of the isolates were identified as Arthrobacter. Brevibacterium linens was found at 0-15%. In all cheeses tested, 5-15% of total cell counts were made up by staphylococci. They were determined as being not Staphylococcus aureus or other pathogenic staphylococci since all isolates were negative with respect to thermo-nuclease, clumping, coagulase, and hemagglutination. Most of the isolates were hemolysis negative. By genetical analysis, several selected isolates were classified as Staphylococcus equorum, one isolate as S. sciuri. Contamination of cheeses with Fusarium moulds indicated the influence of the smearing strategy on spreading of undesirable microorganisms. In plant A, old cheeses were smeared first, then young cheeses were smeared with the same smear liquid. Fusarium contamination could be detected in all stages of ripening. In plant B, young cheeses (0-3 weeks) were smeared with a commercial surface starter cocktail. In all cheeses of this age, problems with Penicillium contaminations were observed. Older cheeses (>3 weeks) were smeared according to the strategy applied in plant A. Consequently, Fusarium moulds were detected in cheeses 4-8 weeks of age.     

First isolation of Brevibacterium sp. pigments in the rind of an industrial red‐smear‐ripened soft cheese

The smear‐ripened soft cheeses are characterised by a surface orange‐red‐brown colour, which has a microbial origin. For a long time, this colouration was mainly imputed to Brevibacterium linens. However, the latest published works, based on molecular biology, have shown a minor role for this bacterium. This study shows the results obtained with an industrial cheese named Vieux‐Pané, which is characterised by the presence of carotenoids from Brevibacterium linens group at its surface. This demonstrates that, under certain conditions, the Brevibacterium linens group (Brevibacterium linens and Brevibacterium aurantiacum sp. nov.) is able to produce pigments and to colour cheeses effectively.     

Cheese yeasts

Numerous traditionally aged cheeses are surface ripened and develop a biofilm, known as the cheese rind, on their surfaces. The rind of such cheeses comprises a complex community of bacterial and fungal species that are jointly responsible for the typical characteristics of the various cheese varieties. Surface ripening starts directly after brining with the rapid colonization of the cheese surface by yeasts. The initially dominant yeasts are acid and salt-tolerant and are capable of metabolizing the lactate produced by the starter lactic acid bacteria and of producing NH₃ from amino acids. Both processes cause the pH of the cheese surface to rise dramatically. This so-called deacidification process enables the establishment of a salt-tolerant, Gram-positive bacterial community that is less acid-tolerant. Over the past decade, knowledge of yeast diversity in cheeses has increased considerably. The yeast species with the highest prevalence on surface-ripened cheeses are Debaryomyces hansenii and Geotrichum candidum, but up to 30 species can be found. In the cheese core, only lactose-fermenting yeasts, such as Kluyveromyces marxianus, are expected to grow. Yeasts are recognized as having an indispensable impact on the development of cheese flavour and texture because of their deacidifying, proteolytic, and/or lipolytic activity. Yeasts are used not only in the production of surface-ripened cheeses but also as adjunct cultures in the vat milk in order to modify ripening behaviour and flavour of the cheese. However, yeasts may also be responsible for spoilage of cheese, causing early blowing, off-flavour, brown discolouration, and other visible alterations of cheese.     

Integrated Roles of Lactate,Ammonia, and Calcium in Texture Development of Mold Surface-Ripened Cheese

Pure cultures of Penicillium caseicolum and Geotrichum candidum each rapidly metabolized lactic acid from an acidified milk-agar medium. Penicillium caseicolum rapidly produced ammonia on milk agar (within 1 wk) but G. candidum elaborated ammonia only in older cultures (>3 wk). Brevibacterium linens showed little metabolic activity on milk agar systems because of inhibition by low pH or lack of proteases or essential nutrients. Analysis of samples (center, corner, surface locations) from wheels of a lot of ripening Brie cheese (227 g each) held at 17°C over 57 d also showed metabolic depletion of lactic acid and the production of ammonia by the surface microflora. During ripening, calcium concentrations became elevated in surface locations and diminished in interior locations of Brie wheels. An integrated model is proposed that incorporates a pH mediation of systems for calcium migration and insolubilization, proteolytic enzyme activity, and casein solubilization whose combined activities control texture development in mold surface-ripened cheeses.     

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.     

The quality and safety of washed-rind cheeses with a focus on antilisterial protection

The surface microbiota of washed-rind cheeses is variable and complex. The processes involved in the production of these cheeses are not fully understood and pathogenic bacteria have been occasionally reported in the smear on the surface of the cheeses. This review describes the prevalent factors that determine development of the complex consortia of microorganisms in the smear of washed-rind cheeses, with a focus on factors that encourage or suppress growth of Listeria monocytogenes. The natural microflora in smears have been shown to exert antilisterial activities (e.g., bacteriocins), which can be utilized to develop commercial starter and smear cultures. The potential of bacteriophage to control pathogens is also being researched. A more detailed knowledge of the microbial ecology of washed-rind cheeses will reveal opportunities to maintain and improve cheese safety and quality.     

Influence of traditional brine washing of smear Taleggio cheese on the surface spreading of Listeria innocua

The influence of a traditional procedure of washing of smear Taleggio cheese on surface spreading of Listeria innocua was studied. This practice is carried out during ripening to remove molds, to select the surface microflora, and to control the ripening process. One cheese, both of 2 (i) and 4 (ii) weeks of ripening, was surface-inoculated with approximately 3 log CFU of L. innocua per entire cheese surface. The inoculated cheeses and others of the same age were weekly washed with brine solution. Listeria was spread both on the surface of the inoculated cheese and on the other cheeses, and it was also found in the brines and on the wooden boxes where the cheeses were ripened. The time of ripening when contamination occurs influenced the behavior of Listeria. At the moment of contamination, the smear surface microflora of (i) cheese was approximately 2 log CFU/g higher than of (ii) cheese. Listeria inoculated on 2-week-ripened cheese was able to colonize the entire surface of the cheese and to cross-contaminate the other cheeses. On the contrary, Listeria inoculated on a 4-week-ripened cheese was partially spread on the surface of the originally inoculated cheese, and the transfer of contamination by the washing procedure was restrained. Because a random distribution of Listeria on cheese surface was observed, the importance of the mode of sampling was discussed. Because of the lack of critical control points during ripening of Taleggio cheese, the Listeria hazard needs to be controlled by taking appropriate control measures to break off the contamination cycle (cheese --> brine --> wooden boxes --> cheese).     

HPLC analysis of the pigments produced by the microflora isolated from the ‘Protected Designation of Origin’ French red-smear soft cheeses Munster,Epoisses, Reblochon and Livarot

Red-smear ripened soft cheeses are characterized by their orange-red color which originates from the carotenoids and other pigments produced by ripening bacteria. A total of 114 different bacterial strains, belonging to Brevibacterium linens group, Micrococcaceae and coryneform bacteria, were isolated from four French red-smear ripened soft cheeses with “protected designation of origin” (i.e., Livarot, Reblochon, Munster, and Epoisses). Among the 114 strains, 67 were selected for their orange or yellow color and their methanolic extracts were analysed by HPLC. All orange bacteria showed the same HPLC profile typical of B. linens, which is known to produce the three aromatic carotenoids, isorenieratene, 3-hydroxyisorenieratene, and 3,3′-dihydroxyisorenieratene. Yellow bacteria produced four different chromatographic profiles. Their analysis (retention time and absorption spectrum) revealed strong similarities among three chromatographic profiles and led us to propose that they represent different isomers of the same compound. This study contributes to the characterization of pigments synthesized by the microflora of French red-smear ripened soft cheeses as part of the effort to identify these valuable microorganisms.     

The occurrence,growth, and biocontrol of Listeria monocytogenes in fresh and surface-ripened soft and semisoft cheeses

Listeria monocytogenes continues to pose a food safety risk in ready-to-eat foods, including fresh and soft/semisoft cheeses. Despite L. monocytogenes being detected regularly along the cheese production continuum, variations in cheese style and intrinsic/extrinsic factors throughout the production process (e.g., pH, water activity, and temperature) affect the potential for L. monocytogenes survival and growth. As novel preservation strategies against the growth of L. monocytogenes in susceptible cheeses, researchers have investigated the use of various biocontrol strategies, including bacteriocins and bacteriocin-producing cultures, bacteriophages, and competition with native microbiota. Bacteriocins produced by lactic acid bacteria (LAB) are of particular interest to the dairy industry since they are often effective against Gram-positive organisms such as L. monocytogenes, and because many LAB are granted Generally Regarded as Safe (GRAS) status by global food safety authorities. Similarly, bacteriophages are also considered a safe form of biocontrol since they have high specificity for their target bacterium. Both bacteriocins and bacteriophages have shown success in reducing L. monocytogenes populations in cheeses in the short term, but regrowth of surviving cells can commonly occur in the finished cheeses. Competition with native microbiota, not mediated by bacteriocin production, has also shown potential to inhibit the growth of L. monocytogenes in cheeses, but the mechanisms are still unclear. Here, we have reviewed the current knowledge on the growth of L. monocytogenes in fresh and surface-ripened soft and semisoft cheeses, as well as the various methods used for biocontrol of this common foodborne pathogen.     

The surface microflora dynamics of bacterial smear-ripened Tilsit cheese determined by T-RFLP DNA population fingerprint analysis

Three Tilsit-type smear-ripened cheeses were manufactured: one was treated with an undefined smear starter mix, the other two were treated with a different defined smear starter mix. The composition of the surface microflora over 8 weeks of ripening was analyzed using Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis. During the whole period of ripening starter strains were found to be present; additionally, strains apparently acquired from the environment were found. Most of the strains reached a maximum level after 2–4 weeks but were not observed after 8 weeks, except for the Corynebacterium species, which remained as the dominant bacterial genus on the surface of the fully ripened cheese. This study constitutes the first and successful application of an ALFexpress™ automatic sequence analyzer to T-RFLP analysis. It was found to be an excellent tool for rapid and specific culture-independent assessment of population composition and dynamics of a cheese system.     

Production of bacteriocins by <Emphasis Type="Italic">Enterococcus</Emphasis> spp. isolated from traditional,Iranian, raw milk cheeses,and detection of their encoding genes

Strong bacteriocins, or bacteriocins with a wide range of activity against pathogens and spoilage microorganisms, are actively sought for use as natural food preservatives. This work reports the inhibitory activity of 96 enterococcal isolates from two Iranian, raw milk cheeses against five indicator organisms (including Listeria innocua). Forty-eight isolates inhibited at least one indicator in spot agar assays. Of these, 20 isolates corresponding to 15 different strains were shown to produce bacteriocin-like substances in liquid cultures. PCR analysis revealed the genes coding for enterocins (enterococcal bacteriocins) A, B, P or X, or their combinations, in all but one of these 15 strains. In addition, the gene coding for enterocin 31 was detected in two strains. No amplification was obtained in one strain when using specific primers for all 13 bacteriocin genes sought. Three different enterocin genes were identified in most strains and four in one strain. Although the concomitant production of bacteriocins is still to be verified, producers of multiple enterocins could be of great technological potential as protective cultures in the cheese industry.     

The color of Brevibacterium linens depends on the yeast used for cheese deacidification

The color of smear cheeses (Muenster) is traditionally thought to be due to the bacterial flora, e.g., Brevibacterium linens. This study was carried out to evaluate indirect effects of yeast on the color of B. linens. A 60% cheese medium was desacidified with Debaryomyces hansenii or Kluyveromyces marxianus until pH 5.8 was reached. After inactivation of the yeast and addition of agar-NaCl, B. linens was inoculated on the medium surface and incubated at 12°C from d 2 to 28. For each bacterial biofilm, color was evaluated by L*C*h° (brightness, chroma, hue angle) spectrocolorimetry. After d 14 (D. hansenii deacidification) and d 21 (K. marxianus desacidification), the color level (as a function of all 3 factors) of B. linens biofilms became maximal and remained so until d 28. Debaryomyces hansenii 304 (LGMPA) was less efficient for deacidification than K. marxianus Laf5. However, color intensity (function of chroma only) was higher when D. hansenii was used. The yeast used had an effect on the composition of the cheese medium in relation to production and consumption of metabolites during deacidification. The results concerning color are discussed with respect to this cheese medium composition.     

Microbial diversity and dynamics of Pecorino di Filiano PDO,a traditional cheese of Basilicata region (Southern Italy)

The evolution of microbial populations of ‘Pecorino di Filiano’ (PF) cheese was investigated during ripening in natural cave and storeroom. 62.5% of isolates grow at 45 and 15 °C and 77.7% showed high salt concentrations tolerance. Brevibacterium linens was dominant in surface samples. Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus paracasei subsp. paracasei were more frequently isolated both surface and core samples, while Leuconostoc lactis and Leuconostoc mesenteroides subsp. mesenteroides prevailed among Leuconostoc isolates. Our results suggest the importance of the ripening environment of cheeses and how a biological ecosystem affects and produces the typical features of artisanal products.     

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.     

Microbial succession of Debaryomyces hansenii strains during the production of Danish surfaced-ripened cheeses

Surface-ripened cheeses of the Danbo type were analyzed for the presence of yeasts with special emphasis on Debaryomyces hansenii. Samples were taken from pasteurized milk, brine, and inoculation slurries and from cheese surfaces during ripening at a Danish dairy. D. hansenii was found to be the dominant yeast species throughout the ripening period, whereas other yeast species such as Trichosporon spp., Rhodotorula spp., and Candida spp. were found in minor concentrations during early stages of cheese ripening. Mitochondrial DNA RFLP was used to show that several strains of D. hansenii were present from the onset of ripening. Thereafter, a microbial succession among the strains took place during the ripening. After 3 d of ripening, only one strain was found. This particular strain was found to be dominant in 16 additional batches of surface-ripened cheeses. We investigated the cause of the observed microbial succession by determining the variation in strains with regard to their ability to grow on lactate and at different pH and NaCl concentrations. The strains were shown to vary in their ability to grow on lactate. In a full factorial design at three levels with factor levels close to the actual levels on the cheese surface, differences in pH and NaCl tolerances were observed. The dominant strain was found to be better adapted than other strains to the environmental conditions existing in surface-ripened cheeses during production [e.g., lactate as the main carbon source, pH 5.5 to 6.0 and NaCl concentrations of 7 to 10% (wt/vol)].     

Lysis of Lactobacillus casei 5Mn 373 accelerates Grana cheese ripening

The influence of the adjunct of a peptidolytic Lactobacillus casei strain on Grana cheese ripening was studied. Strain erythromycin resistance enabled the monitoring of its growth and death kinetics during cheese maturation. Cell lysis was estimated by the dosage of intracellular X-prolyl-dipeptidyl aminopetidase in cheese extracts. L. casei growth reached a maximum level after the second month of cheese ripening when the initial added cell level was 5×10⁵ cfu/g cheese, while L. casei counts decreased from the beginning of the ripening period when the initial added cell level was 4.5×10⁷ cfu/g cheese. The maximum death rate occurred two months after the maximal cell growth, and bacterial lysis was observed approximately two months later. The characteristic amino acid pattern of control Grana cheese was obtained for all of the mature experimental cheeses independently of the inoculum size, and more rapidly when higher amounts of inocula were used due to L. casei cell lysis. The adjunct of the L. casei strain to cheese-milk substantially shortened the ripening time with no negative effects on cheese-making, chemical gross composition or flavour in the mature experimental cheeses compared to the control.