Characterization of yeasts involved in the ripening of Pecorino Crotonese cheese

The aims of this work were to identify and characterize for some important technological properties the yeast species present throughout the ripening process of Pecorino Crotonese, a traditional cheese produced in a well defined area of Southern Italy. In particular, the strain technological properties considered include fermentation/assimilation of galactose and lactose, assimilation of lactate and citrate in the presence of different NaCl concentrations, hydrolysis of butter fat, skim milk, gelatine and casein, production of brown pigments in cheese agar and ability to produce biogenic amines. High yeast levels were recorded in cheese samples already after 5 h of brining (about 5 log cfu/g) and these concentration remained constant during ripening. The yeast isolates belonged to restrict number of yeast species. While Kluyveromyces lactis and Saccharomyces cerevisiae were isolated prevalently in the first stages of Pecorino Crotonese production, Yarrowia lipolytica and Debaryomyces hansenii dominated during the later stages of maturation. Otherwise, the latter two were very NaCl resistant species. In fact, D. hansenii strains conserved the ability to assimilate lactose and galactose in the presence of 10% NaCl, while almost all the strains of Y. lipolytica isolated assimilated citrate and lactate up to 7.5% NaCl. Y. lipolytica isolates evidenced also the highest proteolytic and lipolytic activities and the capability to catabolize tyrosine producing brown pigment. In addition they resulted in the highest aminobiogenic potential decarboxylating ornithine, phenylalanine, tyrosine and lysine. However, they were not able to produce histamine, biogenic amine produced by three strains of D. hansenii.     

The growth, properties and interactions of yeasts and bacteria associated with the maturation of Camembert and blue-veined cheeses

The growth of yeasts and bacteria were monitored during the maturation of Camembert and blue-veined cheese produced in Australia. Yeasts were prominent throughout maturation, growing to 10(5)-10(9)/g, depending on the manufacturer. Debaryomyces hansenii predominated, but there were lesser, inconsistent contributions from Yarrowia lipolytica. Of the non-lactic acid bacteria, Acinetobacter species were significant during the maturation of Camembert but not blue-veined cheeses, and grew to 10(6)-10(8) cfu/g. Staphylococcus and Micrococcus species were consistently isolated from the cheeses with Staphylococcus xylosus growing to 10(5)-10(9) cfu/g, depending on the product. Lactic acid bacteria (10(7)-10(9) cfu/g) were present throughout maturation but were not identified. Interactions between the various yeasts and bacterial isolates were examined. Several strains of D. hansenii exhibited killer activity but not against Y. lipolytica. None of the yeasts were antagonistic towards the bacteria but some strains of D. hansenii enhanced the growth of Y. lipolytica and S. xylosus. The yeast and bacterial isolates exhibited various degrees of extracellular proteolytic and lipolytic activities.     

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.     

Safety assessment of dairy microorganisms: the hemiascomycetous yeasts

Hemiascomycetous yeasts constitute a class of unicellular fungi often associated with the food and drink processing industries. A number of species including Kluveromyces lactis, Debaryomyces hansenii, Yarrowia lipolytica, play a key role in the cheese-making process by providing aroma, affecting texture and/or permitting the growth of other microorgansisms. The large majority of yeast infections are due to a few opportunistic species presently classified within the genus Candida, and occur in immunocompromised patients. Recent advances in taxonomy have provided evidence for the incorrect classification of a number of yeasts and suggest that their association with the genus Candida should be reconsidered. Indeed, none of the most common pathogenic Candida species are found in cheese. Improved techniques, combined with more advanced analytical methods have brought to light several emerging pathogens, some of which are involved in cheese-making, for example D. hansenii and Y. lipolytica. Other emerging pathogens may also be found as rare occurrences in cheese. Problems in designation of these isolates are due in part to the still limited range of specific methods of identification and are exacerbated by lack of consensus concerning yeast taxonomy. These organisms cause rare infections in immunocompromised and hospitalized patients, which are generally mild and either self-limiting or easily treated. From studies with Saccharomyces cerevisiae, it seems that it is more the exposure to high doses of yeast than the identity of the species or strain that is associated with infection. As such yeasts in cheese cannot be considered to constitute a risk for healthy individuals.     

The role of autolysis of lactic acid bacteria in the ripening of cheese

The importance of autolysis of lactic acid bacteria in cheese ripening is evident from the literature. However, the mechanisms and the consequences still require investigation. The consequences of autolysis of mesophilic starters in Cheddar cheese are discussed and highlights from current physiological and genetic studies on starter autolysis are presented. The relative merits of measuring starter autolysis in cheese by viable starter cell densities, electron microscopic observations and assay of cell-free cytoplasmic enzymes are discussed for cheese studies using different starter strains and added phage to achieve different levels of autolysis. The balance of both the intact and autolysed starter cells in young curd appear to be important in cheese ripening. The intact cells are necessary for physiological reactions such as lactose fermentation and oxygen removal and possibly for a number of flavour reactions. In contrast, the main consequence of autolysed cells in cheese is to accelerate the peptidolytic reactions. The possible influences of autolysis of adventitious lactic acid bacteria during cheese ripening are discussed.     

Contribution of lactic acid bacteria to flavour compound formation in dairy products

Cheese flavour cannot be produced without starter bacteria. Lactic acid bacteria convert lactose to lactic acid and this together with their production of diacetyl and acetaldehyde are their main contributions to the flavour of cultured milks and fresh cheeses. In matured cheeses, the starter bacteria die out quickly and the rate at which they lyse and release their enzymes into the system has an influence on the rate at which free amino acids are formed. Rennet alone is mainly responsible for the formation of large, medium and small peptides but, without interaction with other enzymes, is capable of producing only methionine, histidine, glycine, serine and glutamic acid at quantifiable levels. Free amino acids in Cheddar cheese are mainly the result of microbial peptidase activity. These amino acids, together with the products of glycolysis, form substrates for secondary flora, the nature of which, in many cases, determines the cheese variety. They also form substrates for enzymes from the milk, e.g. the production of H₂S appears to be dependent on milk enzymes. Methionine, which is released by rennet, is further metabolized by starter enzymes with the production of methanethiol which plays a major role in cheese flavour possibly as a potentiator for other flavours. —Dicarbonyls, particularly methylglyoxal and diacetyl, and bacteria which can produce them, appear to play a crucial role in the formation of cheese flavour, both the desirable flavour of full-fat cheese and the meaty-brothy off-flavour of low-fat cheese. Although, theoretically, there are many compounds in cheese which could react purely chemically to form flavour compounds, these reactions are also mediated by enzymes in the cheese system and it seems unlikely that straight out chemical reactions play a major role in the production of cheese flavour. The role of the secondary flora is likely to be much more important than that of chemical reactions. Particularly in Cheddar and Emmental it has been shown that good quality cheeses have a low oxidation-reduction potential. This is more likely to be an indicator for the establishment of the anaerobic conditions required for the flavour forming reactions to proceed than an active causal agent of flavour formation. The function of glutathione is more likely to be as some sort of facilitator in enzyme reactions than as an agent for the reduction of oxidation-reduction potential. The ability of bacteria to accumulate glutathione from their media is likely to be one of the indicators of flavour generating capacity. Suitable selected strains of adjunct bacteria increase the rate and intensity of formation of Cheddar cheese flavour but unsuitable adjuncts can also cause off-flavours.     

Development of primers for detecting dominant yeasts in smear-ripened cheeses

PCR primers were developed for the specific detection of Clavispora lusitaniae, Debaryomyces hansenii var hansenii, Geotrichum candidum, Kluyveromyces lactis and K. marxianus and Yarrowia lipolytica, yeast species commonly found on the surface of smear cheese. Forty eight representative strains frequently found in smear cheeses or taxonomically related to the target yeasts were used as templates, to validate the designed primers. The specific and selective detection of these yeasts was effective in situ, in Livarot smear, without yeast isolation and culture and was comparable with data obtained with a conventional method. The primers described here have thus potential for PCR studies applied to cheese. It should also be possible to use some of these primers with other substrates.     

Evaluation of the ability of Yarrowia lipolytica to impart strain-dependent characteristics to cheese when used as a ripening adjunct

Four Yarrowia lipolytica strains were tested as cheese-ripening adjuncts with milk culture in cheese production to evaluate their effects on the microbiological and biochemical features of the cheeses. The Y. lipolytica strains were able to overcome the other naturally occurring yeasts and were compatible with lactic acid bacteria (LAB). Fourier transform infrared (FTIR) profiles and analysis of free fatty acids (FFAs) released during ripening showed that the strains induced a marked lipolysis and gave rise to different FFAs accumulation over time with respect to the control. Strain-dependent protein breakdown patterns were identified and these biochemical differences resulted in different cheese organoleptic characteristics.     

Starter strain related effects on the biochemical and sensory properties of Cheddar cheese

A detailed investigation was undertaken to determine the effects of four single starter strains, Lactococcus lactis subsp. lactis 303, Lc. lactis subsp. cremoris HP, Lc. lactis subsp. cremoris AM2, and Lactobacillus helveticus DPC4571 on the proteolytic, lipolytic and sensory characteristics of Cheddar cheese. Cheeses produced using the highly autolytic starters 4571 and AM2 positively impacted on flavour development, whereas cheeses produced from the poorly autolytic starters 303 and HP developed off-flavours. Starter selection impacted significantly on the proteolytic and sensory characteristics of the resulting Cheddar cheeses. It appeared that the autolytic and/or lipolytic properties of starter strains also influenced lipolysis, however lipolysis appeared to be limited due to a possible lack of availability or access to suitable milk fat substrates over ripening. The impact of lipolysis on the sensory characteristics of Cheddar cheese was unclear, possibly due to minimal differences in the extent of lipolysis between the cheeses at the end of ripening. As anticipated seasonal milk supply influenced both proteolysis and lipolysis in Cheddar cheese. The contribution of non-starter lactic acid bacteria towards proteolysis and lipolysis over the first 8 months of Cheddar cheese ripening was negligible.     

The isolation and identification of yeasts obtained during the manufacture and ripening of Cheddar cheese

Sources of yeast, which may contaminate the curd during the manufacture of Cheddar cheese, were examined in a single cheese factory. A total of 77 yeast species present in the factory environment, manufacturing and ripening of Cheddar cheese were identified according to cellular long-chain fatty acid analysis and verified with conventional identification techniques. Product line samples were taken at critical control points in the manufacturing process and analysed after incubation at 25°C for 96 h. The progression of yeast species during cheese-making and ripening was monitored after renneting and at subsequent 48-h intervals. Dominant species wereDebaryomyces hanseniiandCryptococcus albidus, whileYarrowia lipolytica, Rhodotorula minuta, Torulaspora delbrueckii, Rhodotorula glutinisandKluyveromyces marxianuswere present at low numbers. The results obtained showed that yeasts were present in all cheese samples examined, at quantities ranging from 9×10²to 1·4×10⁷cfu g⁻¹.     

Sensory properties of Cheddar cheese: changes during maturation

The aroma, flavour and texture of 16 samples of commercial Cheddar cheese have been profiled after ripening at 10 °C for 3, 4, 6, 8, 10 and 12 months. Systematic changes in sensory character have been studied and the main changes during maturation identified. Although sensory character changed slowly during ripening, assessment early in the maturation period was an unreliable estimate of ultimate sensory character. Progressive changes in Cheddar aroma and flavour, creamy flavour, acid flavour and mouth-coating character were noted during ripening. Changes in minor components of aroma and flavour were also observed but, on average, were small. Two samples eventually developed marked rancid character and another became excessively bitter. The relation between gross composition of the cheese and sensory properties was investigated. In the early stages of ripening, the ratings for Cheddar flavour and mouth-coating character were associated with the salt content of the cheese and with the concentration of fat in dry matter. However, as the cheese matured these associations weakened.     

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)].     

Occurrence and characterization of yeasts isolated from artisanal Fiore Sardo cheese

The occurrence of yeast microflora in artisanal Fiore Sardo cheese during ripening was studied. Mean yeast counts ranged from 2.64+/-1 log(10) cfu ml(-1) in milk to 0.65+/-1 log(10) cfu g(-1) in 9 months cheese, with the higher counts observed in 48-h-old cheese. Strains belonging to the prevalent species Debaryomyces hansenii, Kluyveromyces lactis, Geotrichum candidum, Candida zeylanoides and Candida lambica were selected for technological and genotypic characterization. All D. hansenii strains fermented glucose and assimilated lactate, a high percentage assimilated citrate and only a few showed proteolytic and lipolytic activity. All K. lactis strains were able to both assimilate and ferment lactose, to assimilate lactate and to exhibit proteolytic activity on casein. G. candidum assimilated lactate and some strains showed proteolytic and lipolytic activity. C. zeylanoides showed lipolytic activity on tweens and the majority of strains assimilated citrate. C. lambica fermented glucose and assimilated lactate. Considering their diffusion and technological characteristics, an important role for K. lactis and G. candidum in the early stages of the ripening process and for D. hansenii after the first month of ripening can be suggested. RAPD-PCR analysis with M13 primer grouped the isolates in well-separated clusters with their type strains and confirmed the previous phenotypic identification. The high intraspecific homogeneity observed in tested strains could be explained by their isolation from a common substrate and from neighbouring geographical areas. This preliminary study allowed us to isolate autochthon yeast strains showing particular properties which can contribute to the production of typical cheese taste and flavour.     

Effects of pectic fat mimetics and transglutaminase on the regularity of protein and fat degradation and flavour compounds in Cheddar cheese during ripening

The effect of simultaneous transglutaminase (TGase) treatment with pectic fat mimetics (PFM) addition on regularity of protein and fat degradation and flavour compound variations in Cheddar cheese during ripening was investigated. In the early stages of fermentation, the cheese with 20% PFM and 15 U/L TGase had a higher amino acid content. In the middle stage of fermentation, cheese with high concentration of TGase hydrolysed more proteins. Cheese supplemented with PFM and TGase showed increased content of long-chain polyunsaturated fatty acids, especially linoleic acid (C18:2). In addition, PFM and TGase compensated the flavour deficiencies of low-fat cheeses and had positive effects on volatile compounds such as alcohols, acids and methyl ketones. Overall, the characteristics and flavours of Cheddar cheese with the addition of PFM and TGase were superior to the control group, which could provide a theoretical basis for the application of PFM and TGase in cheese production.     

Isolation and characterisation of exopolysaccharide-producing Weissella and Lactobacillus and their application as adjunct cultures in Cheddar cheese

This study characterised exopolysaccharide-producing lactic acid bacteria and examined their potential for use in Cheddar cheese manufacture. Two strains were chosen for incorporation as adjunct cultures in Cheddar cheese manufacture: namely, the homopolysaccharide-producers Weissella cibaria MG1 and Lactobacillus reuteri cc2. These strains both produce dextrans with molecular masses ranging from 10⁵ to 10⁷ Da. Both strains were used in the production of miniature Cheddar cheeses that employed a conventional commercial cheese starter culture Lactococcus lactis R604. A cheese was also included that used purified dextran as an ingredient. The W. cibaria strain survived in cheese with levels increasing by 1.5 log cycles over the ripening period. All experimental cheeses (adjunct or exopolysaccharide ingredient) had higher moisture levels compared with the control cheese made using starter alone. Inclusion of the adjunct strains had no detectable negative effects on cheeses in terms of proteolysis.     

Utilisation of a cell‐free extract of lactic acid bacteria entrapped in yeast to enhance flavour development in Cheddar cheese

A lactococcal cell‐free extract (CFE) was successfully entrapped in freeze‐dried attenuated yeast. The entrapment process involved passive diffusion of enzymes from the CFE into the yeast during hydration. The entrapped CFE was subsequently added during Cheddar cheese production and its impact on a range of ripening parameters compared to added attenuated yeast or CFE alone. Statistically significant differences were evident for secondary proteolysis, sensory attributes and volatiles, which were related to enhanced enzymatic and metabolic activity from the attenuated yeast and entrapped CFE. This study highlights the potential of attenuated yeast as a vector to augment flavour development in Cheddar cheese.     

Ammonia production and its possible role as a mediator of communication for Debaryomyces hansenii and other cheese-relevant yeast species

Ammonia production by yeasts may contribute to an increase in pH during the ripening of surface-ripened cheeses. The increase in pH has a stimulatory effect on the growth of secondary bacterial flora. Ammonia production of single colonies of Debaryomyces hansenii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Geotrichum candidum was determined on glycerol medium (GM) agar and cheese agar. The ammonia production was found to vary, especially among yeast species, but also within strains of D. hansenii. In addition, variations in ammonia production were found between GM agar and cheese agar. Ammonia production was positively correlated to pH measured around colonies, which suggests ammonia production as an additional technological parameter for selection of secondary starter cultures for cheese ripening. Furthermore, ammonia appeared to act as a signaling molecule in D. hansenii as reported for other yeasts. On GM agar and cheese agar, D. hansenii showed ammonia production oriented toward neighboring colonies when colonies were grown close to other colonies of the same species; however, the time to oriented ammonia production differed among strains and media. In addition, an increase of ammonia production was determined for double colonies compared with single colonies of D. hansenii on GM agar. In general, similar levels of ammonia production were determined for both single and double colonies of D. hansenii on cheese agar.     

Influence of the starter culture on the microbiological and sensory characteristics of ewe's cheese

Changes which take place in the sensory characteristics of cheeses during ripening are influenced by different factors, involving rennet, starter culture and adventitious contamination of the cheese by non-starter lactic acid bacteria. The objective of this work was to study the influence of the starter on sensory and microbiological ewe's cheese properties during ripening time. Four batches (two with starter added and two without) were manufactured. Milk and cheeses at different stages of ripening were analysed. Cheeses manufactured without adding starter showed a significantly higher level of mesophilic aerobic microflora, lactobacilli, facultatively heterofermentative lactobacilli and enterococci (indigenous microflora) than cheeses manufactured with starter. This study has also shown that adding or not adding starter affects the flavour profile of the cheese. Cheeses with starter added showed greater intensity of the following attributes: refreshing, astringent, sweet; and received lower scores on bitterness. With respect to texture, the said cheeses develop a more homogenous texture and greater elasticity throughout ripening.