Blue Cheese

Chapter 37

Blue Cheese Mette Dines Cantor*, Tatjana van den Tempel**, Tine Kronborg Hansen*, Ylva Ardö† *Chr. Hansen A/S, Hoersholm, Denmark **DSM Food Specialities, Delft, The Netherlands † University of Copenhagen, Frederiksberg, Denmark

INTRODUCTION Blue, or blue-veined cheeses, are characterized by growth of the mold, Penicillium roqueforti, providing the typical appearance and flavor. Blue cheese is produced in many countries all over the world, where local types of Blue cheeses have been developed, each with different characteristics (Table 37.1) and involving different manufacturing methods (Fig. 37.1). The best-known varieties today, worldwide, are considered to be Gorgonzola, Roquefort, Stilton, and Danablu, all of which have been granted the status of Protected Designation of Origin/Protected Geographical Indication (PDO/PGI), together with a number of other European Blue cheeses. Blue cheeses have probably been produced for a long time, either deliberately or by accident, before they were described in writing. Gorgonzola was the first Blue cheese to be mentioned in the literature, in 879, and Roquefort was described in customs papers in 1070; however, in the 8th century, chronicles from monasteries mention the transport of Roquefort across the Alps (Kloster, 1980). Stilton, being a bit younger, was not mentioned until the 17th century. In Denmark, the production of Danablu and Mycella, Blue cheeses from cows’ milk, started in the 1870s. In 1916, a method for homogenizing the cream was invented and used for the production of Danablu, making the cheese as white as the traditional Roquefort made from sheep’s milk. In addition, homogenization affects ripening by accelerating lipolysis. As Blue cheeses are becoming more and more popular, there has been an increased interest in scientific characterization of cheeses of various types and origins. Apart from the aforementioned cheeses, the last decade has seen research on not so well-known types, such as Argentinian cheeses (Wolf et al., 2011), Turkish cheeses, moldy Civil (Cakmakci et al., 2012) and Kuflu (Hayaloglu et al., 2008; Hayaloglu and Kirbag, 2007), Czech Niva cheese (Komprda et al., 2008), and Polish Rokpol (Chrzanowska et al., 2003). Monitoring ripening and final quality with various in-

strumental methods, such as spectroscopy (Kulmyrzaev et al., 2008) and analysis of volatiles (Trihaas, 2005), has also been investigated, which will aid in optimizing the overall quality of the cheeses. In addition, the health aspects of Blue cheeses haven been discussed, in general views by Petyaev and Bashmakov (2012), who hypothesize that cheese consumption might explain the “French paradox,” that is, the resistance to cardiovascular diseases despite a high intake of saturated fat. This has been investigated by the detection of andrastins that are active against cancer cells and a potent inhibitor of a major enzyme of cholesterol biosynthesis, farnesyltransferase (Nielsen et al., 2005), and in a peptidomic study of (bioactive) peptides from Valdeon cheese present before and after simulated gastrointestinal passage (Sánchez-Rivera et al., 2014). This chapter aims to review knowledge on different aspects of Blue cheese ripening, emphasizing changes in the microenvironment, the microorganisms that contribute to ripening, and various biochemical changes, that is, lipolysis, proteolysis, and aroma formation. Finally, thoughts on the selection of appropriate starter and mold cultures, as well as new, possible adjunct cultures, will be discussed.

MICROENVIRONMENT IN BLUE CHEESE The microenvironment in Blue cheese is, in general, heterogeneous with pronounced gradients of pH, salt, and water activity (aw), among other parameters. The ripening temperature is typically between 8 and 15°C, depending upon the variety. Furthermore, there are considerable structural differences within these cheeses, which affect the level and distribution of O2, CO2, and NaCl. These parameters and the changes that occur during the course of ripening have a great impact on the growth, spatial distribution, and biochemical activity of the various microorganisms present in the cheese, and thereby the quality of the final product. Therefore, knowledge of the levels encountered at different ripening times and preferably at different points in the

Cheese: Chemistry, Physics and Microbiology. http://dx.doi.org/10.1016/B978-0-12-417012-4.00037-5 Copyright © 2017 Elsevier Ltd. All rights reserved.

929

930 S ECTION | II  Diversity of Cheese

TABLE 37.1 A Few Examples of Blue Cheeses and the Range in Values of Reported Gross Composition Name

Origin a

Type of Milk Used for Production

% Moisture

% Fat

% Protein

% NaCl

Reference

Cabrales

Spain

Raw cows’ milk, seasonal addition of raw sheep’s or goats’ milk

35.4–41.6

33.8–38.2

20.4–23.6

1.8–3.4

2, 15

Chetwynd

Ireland

Pasteurized cows’ milk

49.2–50.2

26

19.3–20.8

3.2–3.8

4

Danablua

Denmark

Thermized cows’ milk

42.7–47.3

29–31

18.5–23.9

3–3.9

1, 4, 9, 11

Gamonedo

Spain

Raw cows’, goats’, and ewes’ milk

33–40.4

29.2–32.3

23.3–27.5

3.1–4.9

3

Gorgonzolaa

Italy

Pasteurized cows’ milk

42.2–49.6

29.6–31

19–22.9

1.6–2.9

4, 5, 9

Kopanisti

Greece

Raw cows’, goats’, or ewes’ milk or a mixture of these

44.6–69.4

13–30

14.2–27

1–4.7

12

Kvibille Ädel

Sweden

Pasteurized cows’ milk

43

29

21

3–4

6

Picon BejesTresvisoa

Spain

Raw cows’ milk or raw cows’ and raw ewes’ milk

36.9–41.5; 40.4–45.1

36.7–40.4; 30.6–34.1

20.3–23.1; 20.8–23.8

1.8–2.1; 3.2–4.4

7, 8

Roqueforta

France

Raw ewes’ milk

42–44

29

20

4.1

10, 11

Stiltona

Great Britain

Pasteurized cows’ milk

37–41.6

32–35.2

21–28.7

2.2–2.7

1, 4, 9

Rokpol

Poland

Pasteurized cows’ milk

41.5–44.3

34–35.9

18.5–19.1

2.7–3

13

Civila

Turkey

Raw skimmed cows’ milk

45.2–57.3

1–6.2

30.1–43.5

2.9–7.5

14

b

a

1, Madkor et al. (1987a); 2, Marcos et al. (1983); 3, González de Llano et al. (1992); 4, Zarmpoutis et al. (1997); 5, Gobbetti et al. (1997); 6, Ardö, personal communication; 7, Prieto et al. (1999); 8, Prieto et al. (2000); 9, Muir et al. (1995); 10, Masui and Yamada (1996); 11, de Boer and Kuik (1987); 12, Kaminarides (1986); 13, Chrzanowska et al. (2003); 14, Cakmakci et al. (2012); 15, Flórez et al. (2006b). a Cheeses with PDO/PGI. b Gamonedo cheeses are smoked for 3–4 weeks.

cheese, for example, core vs. rind, is important to construct realistic model systems, understand the population dynamics, and aid in optimizing and monitoring cheese quality. The initial pH of Blue cheese ranges from approximately pH 4.6–4.7 in Stilton, Danablu, and Mycella (Hansen et al., 2001; Madkor et al., 1987a) to pH 5.03–5.30 in Cabrales, Gorgonzola, Picon Bejes-Tresviso, and Valdeon (Alonso et al., 1987; Diezhandino et al., 2015; Gobbetti et al., 1997; Prieto et al., 1999, 2000). The conversion of lactose to lactic acid by the lactic acid bacteria (LAB) of the primary starter culture is facilitated by the manufacturing procedure; the curd is very moist when placed in hoops and no pressure is applied during whey drainage (2–3 days), giving the LAB access to large amounts of lactose. Lactose is quickly converted to lactate and galactose, up to 75% decrease was measured after 15 days of ripening in Gamonedo and Valdeon cheeses (Diezhandino et al., 2015; González de Llano et al., 1992), and in 1-day-old Picón Bejes-Tresviso cheese 0.9% lactose was measured, after which it could no longer be detected (Prieto et al., 2000). During ripening, the pH in Mycella increases in the core to 6.5 and in the surface layer to 5.9 (Hansen et al., 2001). Similar values were found in Danablu, as depicted in the partial least squares plot in Fig. 37.2 (Hansen, 2001) and for other variants of Blue cheeses (Seratlic et al., 2011; Zarmpoutis et al., 1997). However, higher pH values up to pH 7 have been reported as well (Cakmakci et al., 2012; Chrzanowska et al., 2003; Gobbetti et al., 1997; González de Llano

et al., 1992; Wolf et al., 2011; Zarmpoutis et al., 1996, 1997). The pH of the interior rises more rapidly than on the surface (Gobbetti et al., 1997; Hansen et al., 2001; Seratlic et al., 2011), as the level of NaCl is lower and therefore allows a faster and earlier growth of the mold cultures. The rise in pH is due to the metabolism of lactic acid to CO2 by yeasts and molds and the increased proteolysis leading to production of NH3 from amino acids (Godinho and Fox, 1982; Hayaloglu et al., 2008; Zarmpoutis et al., 1996, 1997). Salting done by immersing the cheeses in brine, applying dry salt to the cheese surface, or mixing the curd with salt before molding, is an important step in the manufacture of most Blue cheeses. The first two methods create an NaCl gradient from the surface of the cheese to the core, which equilibrates slowly during ripening (Fig. 37.2) (Gobbetti et al., 1997; Godinho and Fox, 1981b; Hansen et al., 2001), whereas the last method ensures a more even NaCl distribution from the beginning of ripening. The overall NaCl content in ripe Blue cheese ranges from 2% to 5% (Table 37.1; Cakmakci et al., 2012; Chrzanowska et al., 2003; Diezhandino et al., 2015; Hayaloglu et al., 2008; Wolf et al., 2011; Zarmpoutis et al., 1996). The high content of salt is due to a fairly long salting period for these cheeses, for example, 2 days brine-salting for Danablu, the high moisture content, and the loose structure of the cheese matrix. The diffusion of NaCl into the cheese core is faster in the piercing channels and in areas with fissures, creating an even more inhomogeneous salt

Blue Cheese Chapter | 37

931

FIGURE 37.1  An outline of (A) the possible steps in the manufacture of different varieties of Blue cheese, and (B) the steps involved in the production of Gorgonzola.

distribution. The NaCl concentration measured in Danablu cheeses after 8 weeks of maturation was approximately 2.0% (w/w) in the core and 4.0% (w/w) in the surface layer, corresponding to an NaCl in water of 7.5 and 10.0%, respectively (Hansen, 2001).

The concentration of NaCl, the level of lipolysis and proteolysis, especially the increase in low molecular weight peptides, will significantly affect the aw in Blue cheeses (Marcos, 1993). Furthermore, the fat content affects cheese structure, and thereby the diffusion coefficient of NaCl and

932 S ECTION | II  Diversity of Cheese

FIGURE 37.2  Partial least squares (PLS) contour plots of pH, NaCl, and aw in Danablu 50+ after 1 and 5 weeks of ripening. The contour plots show the gradients from the core to the surface of the cheese, corresponding to the gray area on the cheese depicted (Nielsen et al., 2005). Data were visualized by PLS regression using SIMCA-P, ver. 3.01 (UMETRI AB, Sweden).

Blue Cheese Chapter | 37

the equilibration of aw throughout the cheese (cf. Chapter 13, Volume 1). In Danablu, the highest aw, c.0.98, is found in the interior after 1 week of ripening, whereas the value for the exterior region ranges from 0.85 to 0.90 (Fig. 37.2). After 5 weeks, aw for both the interior and exterior regions of Danablu is usually in the range 0.91–0.94 (Fig. 37.2). Similar values have been found for Picón Bejes-Tresviso, Mycella, Cabrales, and Valdeon cheeses (Diezhandino et al., 2015; Flórez et al., 2006b; Hansen et al., 2001; Prieto et al., 1999, 2000). It is well known that the growth of fungi is affected by the gaseous composition, that is, in cheese, the concentration of O2 and CO2. The level of O2 has been shown to decrease rapidly throughout the cheese; in Danablu, after 1 week of ripening, a 50% decrease was found 4 mm under the rind, whereas after 13 weeks, O2 was completely absent, except for the outer 0.25 mm (van den Tempel et al., 2002). This anaerobic environment was already evident after 3 weeks of ripening, except from small areas in the cheese, probably in fissures, with 3% oxygen saturation, that is, 3% of the 21% O2 in air. The results are in accordance with observations in white mold cheese (Boddy and Wimpenny, 1992), but lower than the values found in Roquefort (Thom and Currie, 1913), where oxygen was measured in the gas phase of the cheese. P. roqueforti is well-adapted to growth inside Blue cheese where a low level of O2 is combined with a high level of CO2 (20%–40%), as this does not significantly affect growth (Taniwaki et al., 2001; van den Tempel and Nielsen, 2000).

MICROORGANISMS THAT CONTRIBUTE TO RIPENING OF BLUE CHEESE Several microorganisms make up the complex microbiota of Blue cheeses, contributing at different time points and levels to the ripening. The primary and secondary starter culture, LAB and P. roqueforti, respectively, are the most important, but yeast and nonstarter lactic acid bacteria (NSLAB), even though they are not added to the cheese milk deliberately, most probably affect the ripening as well. It should be noted that some varieties of Blue cheeses, such as Valdeon, Kuflu, and Civil, are ripened naturally, that is, there is no addition of cultures in the manufacturing process. The aforementioned groups of microorganisms are, however, present in both naturally ripened Blue cheeses and Blue cheeses with added cultures. Traditionally, the population dynamics and diversity of cheese microbiota have been investigated by culturing methods, which might not be selective enough, and do not enumerate viable, but nonculturable microorganisms. Molecular methods, or culture-independent methods, are gaining more popularity, being less laborious, cheaper, and faster, but they too have some limitations. The methods complement each other, and combining them improves the

933

overall characterization of the ripening events. Population dynamics have been thoroughly investigated in Cabrales cheese, showing the strengths of a combined approach for LAB, yeast, and fungi (Alegria et al., 2011; Flórez et al., 2006a; Flórez and Mayo, 2006b). The main microbial groups, and their characteristics, will be described in the following sections, together with recent findings on the population growth dynamics and spatial distribution in the heterogeneous cheese matrix.

Lactic Acid Bacteria Mesophilic and thermophilic LAB are used as the primary starter culture for the production of different varieties of Blue cheese. A mesophilic, undefined mixed culture will typically contain lactic acid-producing Lactococcus lactis (subspecies lactis and cremoris) and sometimes also citrate-positive strains of Lactococcus lactis subsp. lactis and Leuconostoc species, which produce CO2 and open up the structure to facilitate the penetration of air and development of the mold. The thermophilic starters used in Blue cheese usually contain Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. For naturally ripened cheeses, such as Cabrales, the LAB originates from the milk and the dairy environment (Flórez and Mayo, 2006a,b). The most important role of the LAB starter culture is to acidify the milk by metabolizing lactose to lactate. In general, the numbers of LAB (lactococci and lactobacilli) and the evolution in both naturally ripened cheeses and cheeses with added primary starter cultures are very similar. In the cheese interior (core), the LAB counts decrease slowly from about 109 cfu g−1 after salting to 107–108 cfu g−1 at the end of maturation. For the whole period, the population in the core is dominated by lactococci. The number on the surface after salting is 108–1010 cfu g−1 and remains almost stable at the end of maturation (Chrzanowska et al., 2003; Devoyod et al., 1968; Flórez et al., 2006a; Gobbetti et al., 1997; González de Llano et al., 1992; Hansen et al., 2001; Nuñez, 1978; Ordonez et al., 1980). In Cabrales and Stilton cheese, the diversity and microbial succession during ripening have been investigated by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) (Alegria et al., 2011; Ercolini et al., 2003; Flórez and Mayo, 2006a,b) and compared with classical plating techniques. In conclusion, the results correlated quite well; with PCR-DGGE, a quick overview was obtained which could then be investigated in more detail, either with specific primers or with selective plating. In Stilton, the analysis has been taken a bit further, looking at the spatial distribution of different species by fluorescence in situ hybridization (FISH) experiments (Ercolini et al., 2003). Using FISH, different colonies of bacteria can be localized in the blue veins, the white core, or the crust. Lactococci dominated the core, making up

934 S ECTION | II  Diversity of Cheese

70%, with 15% Leuconostoc and 15% unidentified cocci of the detected colonies. Lactococci was also found inside the veins in mixed colonies, and unidentified cocci in large colonies grew along the veins, and a bit further away, Lactobacilli. The crust section had unidentified cocci, like the ones in the veins, and a mixture of Lactobacilli and Leuconostoc. This study not only highlighted the heterogeneity of the distribution of the microbiota, but also provided information on the type of microorganism present, the colony size, how these spread, and indications for possible interactions.

Penicillium roqueforti P. roqueforti has previously been known under other names, but in 1980 several species, including Penicillium stilton, Penicillium italicum, Penicillium gorgonzola, Penicillium glaucum, Penicillium bioruge, Penicillium suavolens, and Penicillium aromaticum were found to belong to the same species and collected under the taxon P. roqueforti (Pitt, 1979; Stolk et al., 1990). Taxonomically, P. roqueforti is classified under the genus Penicillium Link, the subgenus Penicillium, and the species roqueforti Thom (Pitt and Hocking, 1997). Identification of P. roqueforti is based on the micro- and macromorphology, and one or more of the following methods: molecular analysis, volatile profile, or analysis of extrolites (Visagie et al., 2014). The different methods used to determine the identity of P. roqueforti are listed in Table 37.2. Conidia of P. roqueforti may be added directly to the cheese milk, sprayed on the curd, or colonize the cheeses naturally. The addition of conidia is crucial for the quality of the Blue cheese varieties made from pasteurized milk. P. roqueforti can assimilate all the main carbohydrates that

occur in cheese, that is, lactose, glucose, and galactose, utilize lactate and citrate, and grow without difficulty at the pH values and temperatures encountered during ripening of Blue cheese (Cerning et al., 1987; Vivier et al., 1992). P. roqueforti is the Penicillium species with the highest tolerance to low levels of O2 (Pitt and Hocking, 1997). It has been demonstrated that the rate of growth of P. roqueforti is not significantly affected in the range 4%–21% O2 (Taniwaki et al., 2001; Thom and Currie, 1913; van den Tempel and Nielsen, 2000), but growth seems to be affected by interactions between the levels of O2 and CO2. P. roqueforti grows in the presence of 25% CO2 (van den Tempel and Nielsen, 2000) and O2 at levels of 0.3%–21%. Taniwaki (1995) found that growth and sporulation of P. roqueforti occur at 20% CO2 in an atmosphere with 0.5% O2. P. roqueforti grows in fissures and piercing channels in the cheese. The color of the mold varies from white through several green nuances to brownish, depending upon the type and age of the strain. The growth rate of P. roqueforti is strongly affected by increasing concentration of NaCl. The effect of aw on growth, sporulation, and germination of four strains of P. roqueforti was investigated in laboratory media containing added NaCl at concentrations corresponding to aw in the range 0.99–0.92 (0%–13% NaCl, w/w). The growth of most strains was stimulated by 3.5% NaCl, corresponding to an aw of 0.98 (Hansen, 2001). Similar results have been reported by other authors (Godinho and Fox, 1981a; Lopéz-Díaz et al., 1996b; Valik et al., 1998). Higher concentrations of NaCl cause a decrease in the growth rate, for example, a 92% reduction at an aw of 0.92 compared with the optimum growth rate at an aw of 0.98 (Hansen, 2001). Concerning sporulation, an optimum was observed at an aw of 0.98 for three of the four strains of P. roqueforti examined; the fourth strain showed an optimum at an aw of 0.96.

TABLE 37.2 Methods Used for the Taxonomical Classification of Penicillium roqueforti Methods

Analysis

Classical methods based on phenotypic classification

Micro- and macromorphology, growth rate on specific media, Samson et al. (1977, 1995), Pitt (1979), Pitt and assimilation of carbohydrates and acids, growth on different Hocking (1997), Visagie et al. (2014) nitrogen sources, resistance to preservatives and chemicals.

Reference

Profiles of secondary metabolites

Production of secondary metabolites under specific and controlled conditions (assayed by TLC, HPLC, and GC).

Frisvad (1982), Lund et al. (1995), Boysen et al. (1996)

Volatile metabolites

Production of volatile metabolites and the specific profile (assayed by GC and MS).

Larsen and Frisvad (1995a,b)

PCR-based methods

RAPD ITS-PCR rDNA-RFLP AFLP; β-tubulin (BenA), calmodulin (CaM), RNA polymerase II second largest subunit (RPB2)

Geisen et al. (2001), Glas and Donaldson (1995), Boysen et al. (1996, 2000), Boysen (1999), Flórez et al. (2007), Visagie et al. (2014)

Image analysis

Based on the same criteria as the classical methods, but instead of visual analysis of the macromorphology, digital image analysis and multivariate data analyses are used.

Dörge et al. (2000)

AFLP, Amplified fragment length polymorphism; GC, gas chromatography; HPLC, high-performance liquid chromatography; ITS-PCR, internal transcribed spacer-polymerase chain reaction; MS, mass spectroscopy; RAPD, random amplified polymorphic DNA; RFLP, restriction fragment length polymorphism; TLC, thin-layer chromatography.

Blue Cheese Chapter | 37

Sporulation was strongly inhibited at an aw of 0.94 for the three salt-sensitive strains, whereas the NaCl-tolerant strain still showed a pronounced sporulation at an aw of 0.94, but not at an aw of 0.92 (Hansen, 2001). Germination of P. roqueforti conidia is stimulated by 1%–3% NaCl for most strains, but differences in NaCl tolerance have been observed (Godinho and Fox, 1981a; Lopéz-Díaz et al., 1996b). Below an aw of 0.96, the rate of germination decreases with decreasing aw, and it was observed that NaCl inhibits the rate of swelling of the conidia as well as the further development of the germ tube. The germination rate was also affected by the microenvironment in which the conidia were produced, that is, conidia produced and harvested at an aw of 0.92 germinated faster at an aw of 0.99 than conidia produced at a higher aw (Hansen, 2001). The aw in the core of Blue cheeses after salting is optimal for germination and growth, and the concentration of NaCl is in the range where P. roqueforti is stimulated (Godinho and Fox, 1981a). During the first 3 weeks of ripening, the NaCl concentration in the core increases to a level that induces sporulation and reduces the germination rate and growth of mycelia. These changes affect the appearance of the cheese as the blue-green color is due to the conidia, and also prevents growth of a thick mycelium in fissures and piercing channels. A thick mycelium feels like rubber in the mouth and is therefore undesirable in Blue cheese. Due to the NaCl gradient, the development of P. roqueforti occurs from the interior to the exterior part of the cheese. The conidia in the exterior part of the cheese will germinate with a significantly prolonged lag-phase and a slow development of hyphae compared with conidia in the interior part. This difference in the rate of germination will only persist until the concentration of NaCl in the exterior part is close to the concentration in the interior part. Concerning the further growth of P. roqueforti, the aw values determined in the surface layer of Danablu and Mycella, for example, indicate that mycelial growth will not occur in the surface layer, which might be of importance for possible differences in enzymatic activity of conidia and mycelium. The population and growth dynamics of P. roqueforti in cheese have been investigated, both by classical culturing and by molecular methods (Alegria et al., 2011; Flórez et al., 2007; Flórez and Mayo, 2006b; Le Dréan et al., 2010), and as for the LAB, the two methods complement each other. For molds, however, classical culturing offers the advantage of selective counting of morphologically different isolates, whereas similar looking, but different species, will only be identified by the molecular methods (Alegria et al., 2011).

Yeast The natural occurrence of yeast in dairy products, such as cheese, is linked to unique physical and chemical

935

properties, which select the growth and prevalence of specific yeast species. Yeasts form a substantial part of the microbiota of surface-ripened cheeses (Bockelmann and Hoppe-Seyler, 2001; Eliskases-Lechner and Ginzinger, 1995; Mounier et al., 2008), white-mold cheeses (Schmidt and Lenoir, 1980a,b), and Blue cheeses (de Boer and Kuik, 1987; Gobbetti et al., 1997; González de Llano et al., 1992; Roostita and Fleet, 1996a; van den Tempel and Jakobsen, 1998). It is not unusual to find yeast counts as high as 107–108 cfu g−1 (Diezhandino et al., 2015; Fleet, 1990; Gkatzionis et al., 2014; Tzanetakis et al., 1998; Viljoen and Greyling, 1995), indicating that the presence of yeast has significant impact on cheese ripening and flavor development (Gkatzionis et al., 2013; Price et al., 2014; Sørensen et al., 2011). Yeasts seem to originate from the raw milk and, for brine salted cheeses, from the brine. Investigations have shown that yeasts can be detected only at low levels (<10 cfu g−1) in Danablu cheese before brine-salting (van den Tempel, 2000). Changes in the sensory properties of cheese do not become apparent until the yeasts have grown to populations of 105–106 cfu g−1 (Fleet, 1992). Despite the frequent occurrence of yeasts in Blue cheeses, they seem generally not to cause defects, except from yeasty off-flavor and brown discoloration on the surface which is mainly linked to some yeast species, such as Yarrowia lipolytica (Fleet, 1990; Jakobsen and Narvhus, 1996; Nichol and Harden, 1993; Viljoen and Greyling, 1995; Weichhold et al., 1988; Williams and Withers, 2007).

Origin of Yeasts in Blue Cheese Raw Milk Traditionally, raw milk was used for the manufacture of Blue cheese varieties, which is still the case for cheeses such as the traditional Spanish Cabrales cheese (Flórez and Mayo, 2006a). Several yeast species have been identified in raw milk and are mainly linked to the ability to assimilate important milk components, such as lactose, galactose, citrate, and lactic acid. The predominant yeasts found in raw milk from four Danablu dairies in Denmark included Debaryomyces hansenii (Candida famata), Candida catenulata, Candida lipolytica, Candida krusei, and Trichosporon cutaneum. Yeast populations exceeded 101–104 cfu mL−1 (van den Tempel and Jakobsen, 1998). Other authors have also described the presence of D. hansenii (C. famata) in raw milk in Australia (Fleet, 1990; Fleet and Mian, 1987). German investigations demonstrated the occurrence of other yeast species, including Candida curvata and Saccharomyces spp. (Engel, 1986), and milk for Cabrales production contained Kluveromyces lactis, Pichia fermentas, Pichia norvegensis, and Rhodotorula mucilaginosa (Álvarez-Martín et al., 2007). Pasteurization (72°C × 15 s) and especially thermization (61°C × 15 s) allow the survival of some yeast species.

936 S ECTION | II  Diversity of Cheese

Fleet and Mian (1987) reported 103 cfu mL−1 in pasteurized milk, with C. famata as the predominant species, followed by Kluyveromyces marxianus, whereas thermization of milk for Danablu production allowed the survival of C. famata and C. lipolytica (van den Tempel, 2000). Before salting, the number and diversity of the yeast flora in the cheese will depend upon the heat treatment carried out, as well as the sanitary level in the manufacture of the cheese. Brine and the Dairy Environment Salting, especially brine-salting, is a source of yeasts (Devoyod and Sponem, 1970; Eliskases-Lechner and Ginzinger, 1995; Kaminarides and Laskos, 1992; van den Tempel and Jakobsen, 1998). The composition and environmental conditions of the brine vary from country to country and from dairy to dairy. In France, for example, the brines used for Blue cheese production are typically 19%–20% NaCl (w/v), pH 4–6, and 13–16°C (Seiler and Busse, 1990), whereas brines used in Denmark have a higher NaCl content (22%–23%), a higher temperature (19°C), and a pH of 4.5 (van den Tempel and Jakobsen, 1998). The environmental conditions in cheese brines select for salt-tolerant yeast species, mainly originating from the dairy environment, the brine, and the cheeses (Tudor and Board, 1993). Brines used for Danablu production may have a yeast population ranging from 104 to 106 cfu mL−1, depending upon the dairy (van den Tempel and Jakobsen, 1998). Despite distinct differences in the composition of the yeast flora among the dairies, D. hansenii (C. famata) was the predominant species in the brines, except from one dairy, where Candida globosa predominated. Several brines used for the production of soft surface ripened cheeses have shown the occurrence of 104–105 cfu mL−1, with C. famata as the predominant yeast species (Seiler and Busse, 1990). The frequent occurrence of C. famata in brines used for cheesemaking is explained by the high tolerance to salt (Devoyod and Sponem, 1970; Eliskases-Lechner and Ginzinger, 1995; Kaminarides and Laskos, 1992). Other species found were C. catenulata, C. lipolytica, Zygosaccharomyces spp., T. cutaneum, and Cryptococcus laurentii. The development of the yeast population during processing of Blue cheese has been analyzed by Viljoen et al. (2003), and in accordance with other studies (Fleet and Mian, 1987), the most abundant yeast isolated from the environment was D. hansenii.

Occurrence and Growth of Yeasts in Blue Cheeses Yeasts spontaneously develop during the manufacturing, ripening, and storage of Blue cheeses. Their occurrence is not unexpected because of a tolerance to low pH, elevated salt concentrations, and low storage temperatures (Fleet, 1990). Furthermore, high concentrations of lactate,

residual unfermented carbohydrates, and small amounts of citric and acetic acids will assist the growth and prevalence of particular species of yeasts (van den Tempel and Jakobsen, 1998). Blue cheese, such as Roquefort, made traditionally from raw milk may reach a population of 107–108 and 105– 106 cfu g−1 on the surface and in the interior, respectively, before brine-salting (Besancon et al., 1992). The same investigators showed that the yeast population on the surface significantly decreases (99%) after brine-salting, causing changes in the yeast population toward asporogenous yeast forms, C. famata in particular. These results confirm earlier investigations, which also showed a 90% reduction in the yeast flora and changes in yeast population, selecting for very salt-tolerant species, especially Candida spp. (Devoyod and Sponem, 1970; Galzin et al., 1970). The yeast flora in the interior of the cheese remains unaffected by salting during the early period of ripening, due to the slow diffusion of the salt from the surface to the interior of the cheese (Galzin et al., 1970; Hansen et al., 2001). Yeasts start to multiply on the surface of the cheese after a short adaptation period. There is an almost parallel development of the yeast population in the interior of the cheese, but with numbers 100-fold lower (Hansen et al., 2001; Viljoen et al., 2003). This can be explained by the low level of available oxygen and the high level of CO2, which reduce the growth of yeasts (van den Tempel and Nielsen, 2000). On the contrary, studies by Viljoen et al. (2003) showed that yeast representative of the interior of the cheeses had higher species diversity compared with the exterior, explained by the reduced salt concentration in the core allowing yeast with lower salt tolerance to grow. The spatial distribution of yeast was studied by Gkatzionis et al. (2014), showing that the mycobiota communities in Stilton cheese differed among the blue veins, the white core and the outer crust. The blue veins were dominated by K. lactis (70%) and smaller amounts of D. hansenii, whereas the white core and the outer crust consisted of the same species, but at different levels: K. lactis, D. hansenii, and Y. lipolytica, with D. hansenii dominating, showing an uneven distribution of two different morphological groups. D. hansenii is a very heterogeneous group of microorganisms which has also been demonstrated in interaction studies showing various effects of D. hansenii on other yeast species in the cheese matrix (Addis et al., 2001; Juszczyk et al., 2005a). All investigators seem to show the predominance of D. hansenii (C. famata) in Blue cheese, except the Greek variety, Kopanisti, in which T. cutaneum seems to dominate over D. hansenii (C. famata) (Kaminarides and Anifantakis, 1989). A survey of the literature on yeasts isolated from Blue cheeses demonstrates the great diversity of the yeast flora (Table 37.3).

Blue Cheese Chapter | 37

937

TABLE 37.3 Species of Yeast Isolated From Blue Cheeses Isolated Species

Type of Blue Cheese

Debaryomyces hansenii (Candida famata)

Roquefort1,3,4, Cabrales2,15, Gorgonzola3,9, Danablu3,10, Bleu d’Auvergne3, Bleu de Bresse3, Gamonedo5, Kopanisti6, Valdeón7, Australian Blue8,11, unknown brand of Blue cheese8, Hungarian blue12, Rokpol13, Stilton17

Kluyveromyces marxianus (Candida kefyr)

Roquefort1,3,4, Cabrales2, Gorgonzola3, Danablu3,10, Bleu d’Auvergne3, Bleu de Bresse3, Kopanisti6, Valdeón7, unknown brand of Blue cheese8, Rokpol13

Kluyveromyces lactis (Candida sphaerica)

Rokpol13, Cabrales15, Stilton17

Yarrowia lipolytica (Candida lipolytica)

Roquefort3, Gorgonzola3, Danablu3,10, Bleu d’Auvergne3, Bleu de Bresse3, Valdeón7, Australian Blue8,11, unknown brand of Blue cheese8, Hungarian blue12, Cabrales15, Stilton17

Pichia spp.

Roquefort1, Cabrales2, Gorgonzola9, Cabrales15

Cryptococcus laurentii

Danablu10, Gamonedo5, Valdeón7

Rhodotorula spp.

Kopanisti6, Valdeón7, Hungarian blue12, Cabrales15

Candida catenulata

Australian Blue8, unknown brand of Blue cheese8, Danablu10, Stilton17

Candida cabralensis

Cabrales16

Candida colliculosa

Valdeón7, unknown brand of Blue cheese8

Candida lambica

Roquefort3, Gorgonzola3, Danablu3,10, Bleu d’Auvergne3, Bleu de Bresse3

Candida rugosa

Cabrales2,15, Danablu10

Candida zeylonoides

Roquefort3, Gorgonzola3, Danablu3, Bleu d’Auvergne3, Bleu de Bresse3, Valdeón7, Australian Blue11

Geotrichum candidum

Roquefort3, Gorgonzola3, Danablu3, Bleu d’Auvergne3, Bleu de Bresse3, Valdeón7, Cabrales14

Saccharomyces cerevisiae

Roquefort3, Gorgonzola3, Danablu3, Bleu d’Auvergne3, Bleu de Bresse3, Kopanisti6

Trichosporon spp.

Kopanisti6, Danablu10, Stilton17

Cryptococcus albidus

Australian Blue8, unknown brand of Blue cheese8

Cryptococcus curvatus

Hungarian blue12, Cabrales15

Candida intermedia

Unknown brand of Blue cheese8, Rokpol13, Cabrales15

Candida norvegensius

Danablu10

Candida parapsilosis

Valdeón7

Candida tropicalis

Unknown brand of Blue cheese8

Candida mesenterica

Hungarian blue12

Zygosaccharomyces spp.

Danablu10, Cabrales15

1, Devoyod and Sponem (1970); 2, Nuñez et al. (1981); 3, de Boer and Kuik (1987); 4, Besancon et al. (1992); 5, González de Llano et al. (1992); 6, Kaminarides and Anifantakis (1989); 7, Lopéz-Díaz et al. (1995); 8, Roostita and Fleet (1996a); 9, Gobbetti et al. (1997); 10, van den Tempel and Jakobsen (1998); 11, Addis et al. (2001); 12, Vasdinyei and Deak (2003); 13, Chrzanowska et al. (2003); 14, Flórez et al. (2007); 15, Álvarez-Martín et al. (2007); 16, Flórez et al. (2010); 17, Gkatzionis et al. (2013).

In Blue cheeses, such as Danablu, the yeast flora develops from a heterogeneous population toward a more homogenous population as ripening progresses. On day 1 after salting, Danablu contains different species of yeasts, including C. famata, C. lipolytica, Zygosaccharomyces spp., Candida rugosa, and Candida sphaerica. After 28 days of ripening at 10°C, C. famata was the predominant yeast, reaching 6.2 × 106 and 1.4 × 108 cfu g−1 in the interior and on the surface of the cheese, respectively (van den Tempel and Jakobsen, 1998). Examination of Blue cheeses of different origin and age (>12 weeks) showed that D. hansenii, or the asporogenous form C. famata, dominated in all cheeses examined (Table 37.4).

Strong growth in the presence of salt, growth at a low temperature, and the ability to utilize lactate and citrate are likely the key determinants that encourage the predominance of D. hansenii (C. famata) in Blue cheeses (Addis et al., 2001; van den Tempel and Jakobsen, 2000). Another yeast species found in several Blue cheeses is Y. lipolytica (C. lipolytica). This yeast is characterized by the inability to ferment carbohydrates or assimilate nitrate, and strong lipolytic and proteolytic properties (Freitas et al., 1999; Juszczyk et al., 2005a; Roostita and Fleet, 1996a; van den Tempel and Jakobsen, 2000), and seems to be a more homogenous group than D. hansenii (van den Tempel, 2000). Y. lipolytica is not known to be salt

938 S ECTION | II  Diversity of Cheese

TABLE 37.4 Occurrence of Debaryomyces hansenii in Selected Blue Cheeses

Cheese

Surface (yeast g−1 cheese)

Interior (yeast g−1 cheese)

D. hansenii (%)

Roquefort

1.7 × 107

4.4 × 106

74

7

5

66

6

64

4

Petite Fourme Bleu D’Auvergue

2.1 × 10

7

1.7 × 10

5

2.0 × 10 5.5 × 10

Cambozola

2.7 × 10

2.6 × 10

70

Saint Agur

2.0 × 107

1.9 × 105

75

6

7.3 × 10

3

62

1.4 × 106

70

Gorgonzola

3.6 × 10

Fourme D’Ambert 2.1 × 107

Source: Modified from van den Tempel, T., 2000. The Role of Yeast in the Ripening of Blue Mould Cheese. PhD Thesis, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark.

tolerant which means that growth in Blue cheese will be reduced compared with D. hansenii, especially on the cheese surface (Addis et al., 2001). Strains of Y. lipolytica are able to grow at pH values from 4.0 to 6.0 at 10°C, the ripening temperature of Danablu (van den Tempel and Jakobsen, 2000), and have been shown to possess pronounced lipolytic and proteolytic activity contributing to the unique Blue cheese flavor in synergy with P. roqueforti (Gkatzionis et al., 2013; Price et al., 2014). Kluyveromyces subspecies marxianus or subspecies lactis are frequently found in Blue cheese. This species assimilates and ferments lactose and, due to gas production, could play an important role in the formation of the characteristic texture of Blue cheese (Fox and Law, 1991; Lenoir, 1984; Roostita and Fleet, 1996a). Furthermore, strains of Kluyveromyces are able to assimilate lactic and citric acids (Besancon et al., 1992; Fleet and Mian, 1987; Lenoir, 1984; Roostita and Fleet, 1996b), and produce a variety of flavor components in model cheese media (Leclercq-Perlat et al., 2004). A recent study by Price et al. (2014) indicates a positive impact of K. lactis on Blue cheese aroma due to the synergy with P. roqueforti at moderate inoculation levels. Earlier studies have, however, shown Kluyveromyces to exhibit a pronounced inhibitory effect on the growth of P. roqueforti (Hansen and Jakobsen, 1998; Kaminarides et al., 1992), emphasizing the high diversity within isolates. New species are also appearing: Flórez et al. (2010) described a new yeast species isolated from Cabrales cheese, Candida cabralensis, whose closest relatives are Pichia fermentans and Pichia membranefaciens, but it differentiates from these genetically and physiologically.

Nonstarter Lactic Acid Bacteria NSLAB are found in several cheese varieties during ripening, including Blue cheeses. As in many other cheeses,

they are commonly facultatively heterofermentative strains of Lactobacillus, that is, mainly of the Lactobacillus paracasei/casei complex and Lactobacillus plantarum. Other NSLAB found in Blue cheese are Lb. fermentum, Lb. brevis, Pediococcus spp., and Leuconostoc spp. (Fox et al., 1996; Gobbetti et al., 1997; González de Llano et al., 1992; Lopéz-Díaz et al., 2000; Martley and Crow, 1993). NSLAB grow in Blue cheese from 10–100 cfu g−1 after brining to about 107 cfu g−1 at the end of maturation, and are assumed to originate from the raw milk and the dairy plant environment (Alegria et al., 2011; Ercolini et al., 2003; Gobbetti et al., 1997; González de Llano et al., 1992; Nuñez and Medina, 1979). In Blue cheeses made from raw milk, high levels of Enterococcus spp. can sometimes be isolated (Alegria et al., 2011; Devoyod and Desmazeaud, 1971; Diezhandino et al., 2015; Flórez et al., 2006a,b; González de Llano et al., 1992; Lopéz-Díaz et al., 2000). It is, however, not known how normal variations in the composition of the NSLAB flora affect ripening and flavor of Blue cheese.

Contaminants Microorganisms other than P. roqueforti and LAB starter cultures can colonize and grow well on Blue cheeses, especially on the surface. Spoilage of cheese due to fungal growth is caused by formation of off-flavors (Sensidoni et al., 1994), mycotoxins, and possible discoloration of the cheese (Lund et al., 1998). The most important spoilage fungi of semisoft cheeses are Penicillium spp., mainly Penicillium commune (Flórez et al., 2007; Hayaloglu and Kirbag, 2007; Lund et al., 1995). Other species encountered are Penicillium nalgiovense, Penicillium verrucosum, Penicillium solitum, Penicillium chrysogenum, and Penicillium discolor (Filtenborg et al., 1996; Flórez et al., 2007; Hayaloglu and Kirbag, 2007; Lopéz-Díaz et al., 1995; Lund et al., 1995). Of special interest is the species, Penicillium caseifulvum, which is frequently found on Blue cheeses (Lund et al., 1998). P. caseifulvum has, to date, been found in various Blue cheese dairies in Denmark and France (Lund et al., 1998), where it was isolated from cheese curd (102 conidia g−1), brine (101–5 × 102 conidia g−1), and the surface of Danablu (102–103 conidia g−1). P. caseifulvum is sensitive to CO2 (van den Tempel and Nielsen, 2000), and therefore only grows on the surface of the cheese, where it can cause discoloration in the form of brown spots. Another contaminant frequently found in Blue cheese is Geotrichum candidum (de Boer and Kuik, 1987; Flórez et al., 2007; Hayaloglu and Kirbag, 2007; Lopéz-Díaz et al., 1995), which can cause considerable inhibition of the growth of Penicillium spp. (Nielsen et al., 1998; van den Tempel and Nielsen, 2000). G. candidum has been isolated from Danablu at levels of 102–103 cfu g−1, mainly from the

Blue Cheese Chapter | 37

interior of the cheese, as G. candidum is sensitive to salt at concentrations above 1% (Lecocq and Gueguen, 1994; Philip, 1985; van den Tempel and Nielsen, 2000). It should be noted that G. candidum is also seen as a positive asset to cheese ripening and considered as a starter culture (Ferreira et al., 2012).

MICROBIAL INTERACTIONS During the production and ripening of Blue cheese, interactions between the primary starter culture, P. roqueforti, and mold and yeast contaminants determine the maturation time, aroma, texture, and appearance of the final cheese. The mechanisms behind these interactions can be broadly divided into two groups: (1) antagonism, representing negative interactions caused by antimicrobial metabolites, competition for nutrients, or unfavorable changes to the microenvironment, and (2) synergism, representing positive interactions as mutual use and production of nutrients, changes to a more favorable microenvironment and atmosphere composition, degradation of antimicrobial compounds, physical attachments between microorganisms, and changes in microstructure. Microbial interactions have been deduced from examinations of Blue cheeses (Hansen et al., 2001; Hansen and Jakobsen, 1997; Kaminarides et al., 1992; van den Tempel and Jakobsen, 2000; van den Tempel and Nielsen, 2000), but there have been only a few detailed studies in this area. In the following sections, microbial interactions involving P. roqueforti will be described.

Penicillium roqueforti and Lactic Acid Bacteria Positive and negative interactions between LAB and Penicillium spp. have been described in the literature (Gripon et al., 1977; Gourama, 1997; Gourama and Bullerman, 1995; Hansen, 2001; Hansen and Jakobsen, 1997; Roy et al., 1996; Salvadori et al., 1974; Suzuki et al., 1991). However, only a limited number of investigations have been carried out to examine how several strains of LAB, both starter and nonstarter cultures, affect the growth and sporulation of different strains of P. roqueforti in model cheese systems. The results shown in Table 37.5 are based on screening of 20 strains of P. roqueforti and 15 strains of LAB in a model cheese system and a laboratory substrate (Hansen and Jakobsen, 1997). As shown in Table 37.5, both negative and positive interactions were demonstrated. Furthermore, the type of interactions observed was connected to the composition of the medium used. Positive interactions were stronger and found more frequently in the cheese agar than the laboratory medium. The interactions were found to be strain-specific for P. roqueforti, as well as for the LAB. The positive interactions were seen as faster growth and more

939

TABLE 37.5 Positive and Negative Interactions for Growth of Penicillium roqueforti in Laboratory Media and Cheese Agar for 300 Combinations of Strains of P. roqueforti and Lactic Acid Nacteria Cheese Agar

Laboratory Media

Positive interaction

136

22

Negative interaction

49

195

No sign of interaction

115

83

Source: Modified from Hansen, T.K., Jakobsen, M., 1997. Possible role of microbial interactions for growth and sporulation of Penicillium roqueforti in Danablu. Lait 77, 479–488.

pronounced sporulation of P. roqueforti. Furthermore, P. roqueforti developed a thicker and more velvet-like mycelium. Negative interactions were seen as a reduction in, or absence of growth of P. roqueforti. In the same way antifungal activity was found in LAB in laboratory medium in a study made on durum wheat. This study also showed that the interaction between LAB and the mold was strainspecific (Valerio et al., 2009). A synergistic effect in the casein breakdown between P. roqueforti and LAB has also been observed, indicated by the production of significantly higher amounts of nonprotein nitrogen and phosphotungstic acid-soluble nitrogen when the enzymes of the two types of organisms were incubated together than the amounts produced by only one of them (Ottogalli et al., 1974). The benefits obtainable by selecting the right combination of cultures are emphasized by results, showing that stimulation of the growth and sporulation of P. roqueforti were stronger at higher levels of NaCl. The levels of NaCl investigated corresponded to the concentration found in the surface layer of Blue cheese where the growth of P. roqueforti can be limited or even absent. The stimulation of mycelial growth and the sporulation of P. roqueforti obviously enhance maturation because both the conidia and the mycelium contribute to proteolysis and lipolysis in the cheese during ripening.

Penicillium roqueforti and Yeasts As for LAB and P. roqueforti, selecting the right combinations of yeast and P. roqueforti cultures may stimulate the growth and sporulation of P. roqueforti, and thereby enhance ripening and improve the appearance of the cheese in general. Interaction experiments carried out under environmental conditions similar to those in Blue cheese production have demonstrated radial growth of P. roqueforti to be stimulated by selected strains of D. hansenii (van den Tempel, 2000). The mechanism behind the observed positive interactions might be explained by a stimulation of P. roqueforti by D. hansenii caused by the release of nutrients on autolysis due

940 S ECTION | II  Diversity of Cheese

to low survival rates of yeasts at high levels of CO2 (Dixon and Kell, 1989; Ison and Gutteridge, 1987; Lumsden et al., 1986; van den Tempel and Nielsen, 2000). Conversely, studies by Juszczyk et al. (2005b) showed that strains of D. hansenii inhibited the vegetative growth of four commercial strains of P. roqueforti, especially at low aw. Gkatzionis et al. (2014) observed a delayed sporulation of P. roqueforti in coculture with D. hansenii, again indicating that interactions involving D. hansenii can be highly strainspecific, and emphasizing the need for careful selection of strains. Positive interactions between a strain of Saccharomyces cerevisiae (FB7) and strains of P. roqueforti have been demonstrated (Hansen et al., 2001; Hansen and Jakobsen, 2001). Measurement of radial growth and visual observations of sporulation showed that whole cell inocula of S. cerevisiae promoted faster growth, a thicker and more velvet-like mycelia, and a more intense blue color of the conidia. No interactions were seen when examining the supernatant or the disrupted cells of S. cerevisiae. The mechanism behind the positive fungal–yeast interaction was found to be correlated to a synergistic effect in the breakdown of casein, as shown by capillary electrophoresis. S. cerevisiae FB7 degraded casein, and coculturing with P. roqueforti resulted in a higher number and a different grouping of peptides. These findings have been confirmed in a large-scale production of Mycella cheese, showing that addition of S. cerevisiae FB7 to Mycella gave rise to a faster growth and sporulation of P. roqueforti, a softer cheese texture and a significantly higher relative concentration of aroma compounds (Hansen et al., 2001). Inhibition of P. roqueforti by Y. lipolytica has been observed in laboratory trials (Juszczyk et al., 2005b; van den Tempel and Jakobsen, 2000). The interactions were strainspecific for Y. lipolytica, as well as for P. roqueforti, but all strains of Y. lipolytica investigated were inhibitory toward mycelial growth and sporulation of P. roqueforti (van den Tempel, 2000). The inhibitory effect on sporulation was also observed by Gkatzionis et al. (2014), but at the same time a strong synergistic activity with P. roqueforti was evident, enhancing the production of ketones. Price et al. (2014) showed that the synergistic effect was dependent upon the yeast inoculum concentration, and a high level of Y. lipolytica could promote ripening in Stilton. Similar results were obtained with K. lactis at moderate inoculation levels (Price et al., 2014). Competition for nutrients seems to be the most frequently occurring mechanism of interaction between yeasts and molds in laboratory systems, but this does not exclude coexistence in more natural situations (Boddy and Wimpenny, 1992). The belief that competition for nutrients is the main interspecific mechanism between yeasts (e.g., Y. lipolytica and D. hansenii) and P. roqueforti is mainly based on investigations, showing (1) no inhibition of P. roqueforti

when using culture supernatant or disrupted cells, (2) the quick colonization of yeasts, for example, Y. lipolytica and D. hansenii, on cheese agar, (3) the quantitative relationship between the numbers of yeasts and inhibition of P. roqueforti, and (4) the inhibitory effect of Y. lipolytica and D. hansenii being absent or reduced by addition of nutrients (van den Tempel, 2000; van den Tempel and Jakobsen, 2000).

Penicillium roqueforti and Contaminants G. candidum has shown a growth potential similar to P. roqueforti in the absence of salt, indicating a possible overlap between the two species in the interior of the cheese during the initial ripening stage. Contamination of Blue cheese by G. candidum can cause inhibition of growth and sporulation of P. roqueforti resulting in “blind spots,” which affect the quality of the cheese significantly. This emphasizes the importance of good manufacturing practice in production of Blue cheese to prevent contamination by G. candidum. Studies by Tariq and Campell (1991) showed that G. candidum might compete by antibiosis, as volatile metabolites from arthrospore suspensions of G. candidum were found to inhibit conidial germination and reduce the rate of hyphal extension in different species of fungi, including P. roqueforti. Dieuleveux et al. (1998) demonstrated that G. candidum produces and excretes 2-hydroxy3-phenylpropanoic acid that has a broad-spectrum antibacterial effect. Colonization of the mold contaminant, P. caseifulvum, can occur at the surface of Blue cheese without major inhibition by any of the species investigated, thus causing color defects on the cheese (Lund et al., 1998). The occurrence of P. caseifulvum is unlikely to affect the growth and sporulation of P. roqueforti due to the different growth niches in Blue cheese.

RIPENING OF BLUE CHEESE Proteolysis and Amino Acid Catabolism Several studies have revealed extensive proteolysis in Blue cheese compared with other cheeses (González de Llano et al., 1995; Marcos et al., 1979; Zarmpoutis et al., 1997). Casein is hydrolyzed at more sites and at a considerably higher rate, and the remaining amount of intact caseins or primary breakdown products left in the ripened cheeses are limited or absent (Diezhandino et al., 2015; Fernandez-Salguero et al., 1989; Fernandez-Salguero, 2004; González de Llano et al., 1992; Hayaloglu et al., 2008; Marcos et al., 1979; Seratlic et al., 2011; Trieu-Cuot and Gripon, 1983; Zarmpoutis et al., 1997). A larger number of different peptides are also produced than in semihard cheeses (Fig. 37.3), and a high concentration of amino acids is released as a result of the peptidases, especially from mold and LAB working in concert (Coghill, 1979; Diezhandino

Blue Cheese Chapter | 37

941

FIGURE 37.3  Peptide profiles analyzed by RP-HPLC of (A) Danablu and (B) semihard yellow cheese of similar age (about 3 months). α-LA, αLactalbumin; β-LG, β-lactoglobulin; proteose-peptones, breakdown products from plasmin activity on β-casein; αs1-CN(f1-13), breakdown product from Lactococcus protease activity on the rennet derived peptide αs1-CN(f1-23).

et al., 2015; Fernandez-Salguero, 2004; Gripon et al., 1977; Ismail and Hansen, 1972; Zarmpoutis et al., 1997). The enzymes contributing to the complex proteolysis in Blue cheese originate from the milk, rennet, starter and nonstarter bacteria, molds, and yeasts, with the main contribution from the mold culture, P. roqueforti (Coghill, 1979). A significant increase in proteolysis has been observed when the mold becomes visible in the cheese, typically after 2–5 weeks of maturation depending upon the cheese variety (Diezhandino et al., 2015; Fernandez-Salguero, 2004; Seratlic et al., 2011; Trieu-Cuot and Gripon, 1983; Zarmpoutis et al., 1996). Although P. roqueforti is growing out, breakdown of the caseins is carried out mainly by rennet (Hewedi and Fox, 1984). The main activity of rennet in cheese is on αs1-casein to produce αs1-CN (f24-199) and the peptide αs1-CN (f1-23) (Table 37.6), whereas the milk protease, plasmin, hydrolyzes β-casein into γ-caseins and proteose-peptones (Fernandez-Salguero, 2004). The cell wall protease of the Lactococcus or Lactobacillus starter culture hydrolyzes the peptides produced from casein by rennet and plasmin. A limited release of amino acids carried out by the starter aminopeptidases occurs during these first weeks of ripening. After a couple of weeks, P. roqueforti dominates proteolysis, liberating both peptides and amino acids using a variety of enzymes (Table 37.6) (Diezhandino et al., 2015; Madkor et al., 1987a; Seratlic et al., 2011; Zarmpoutis et al., 1996, 1997). P. roqueforti expresses two extracellular proteases; a metalloprotease and an aspartic protease.

The activity of these enzymes is maximal in Blue cheese at the stage when P. roqueforti has grown out and begins to sporulate. Both proteases are rather stable in cheese. The metalloprotease is active at pH 4.5–8.5 with an optimum for casein hydrolysis at pH 5.5, which corresponds to the pH often found in Blue cheese during ripening. The metalloprotease has a broad specificity and hydrolyzes both αs1- and β-caseins. Hydrolysis of αs1-casein in buffer lead to eight peptides with molecular weights ranging from 7,000 to 21,000 Da (Trieu-Cuot et al., 1982b), whereas hydrolysis of β-casein in buffer gave nine peptides with molecular weights between 13,100 and 21,100 Da (TrieuCuot et al., 1982b). The peptides from αs1- and β-caseins measured in buffer can also be detected in Blue cheeses (Diezhandino et al., 2015; Le Bars and Gripon, 1981; TrieuCuot and Gripon, 1983). Of special interest is that the metalloprotease cleaves β-casein at Pro90-Glu91, which is not often hydrolyzed by proteases because of the proline residue. Like plasmin, it cleaves a bond close to Lys28-Lys29 (Le Bars and Gripon, 1981; Trieu-Cuot et al., 1982b; TrieuCuot and Gripon, 1983). The aspartic protease is stable at pH 3.5–6.0 and has two optimal pH values for hydrolysis of casein, 3.5 and 5.5, which may be explained by conformation changes in the substrate. Casein is hydrolyzed into mainly high molecular weight peptides, and it does not hydrolyze di- or tripeptides (Le Bars and Gripon, 1981; Modler et al., 1974). The first peptide released by the aspartic protease from αs1-casein in solution corresponds in isoelectric point and molecular

942 S ECTION | II  Diversity of Cheese

TABLE 37.6 Main Enzymes Involved in Proteolysis and Amino Acid Release During Ripening of Blue Cheese With Scrubbed Surfaces to Prevent Development of Slime Microflora (Ardö, 2001; Gripon, 1993) Enzyme

Specificity in Cheese

Plasmin

β-CN and αs2-CN after basic amino acids (Arg, Lys). Hydrolyzes β-CN to γ-CN and proteose-peptones; preferred cleavage sites: β-CN (28–29, 105–106, 107–108).

Rennet

Hydrolyzes αs1-CN to αs1-CN (f24-199) and αs1-CN (f1-23).

Lactococcal lactocepin

Hydrolyzes peptides produced from casein by the action of plasmin, rennet, or P. roqueforti. Produces different peptides from αs1-CN (f1-23).

Lactococcal peptidases, for example, PepN, PepC, PepX

Releases amino acids from smaller peptides. Broad aminopeptidase and dipeptidase specificity.

NSLAB, peptidases

Contribute to amino acid release.

P. roqueforti aspartic protease

Hydrolyzes β-CN preferentially to produce β-CN (98–209, 30–209, 1–29, 100–209, 1–97/99). Hydrolyzes αs1-CN.

P. roqueforti metalloprotease

Broad specificity.

P. roqueforti serine carboxypeptidase (extracellular, acid)

Releases acidic, basic and hydrophobic amino acids.

P. roqueforti metalloaminopeptidase (extracellular, alkaline)

Releases apolar amino acids (not next to Gly).

Yeast

Large variation between strains from none, to excessive proteolytic activity.

CN, Casein.

weight to αs1-CN (f24-199), indicating specificity similar to chymosin (Larsen et al., 1998; Trieu-Cuot et al., 1982a). Later, this peptide is further hydrolyzed to form four to five new peptides. The aspartic protease hydrolyzes β-casein into five peptides: initially, the peptides β-CN (f98-209), (f30-209), and (f1-29) are released, and then the peptides βCN (f100-209) and (f1-97/99) (Le Bars and Gripon, 1981; Sánchez-Rivera et al., 2014; Trieu-Cuot et al., 1982a). In Blue cheese, β-CN (f98-209) has been shown to accumulate (Diezhandino et al., 2015; Houmard and Raymond, 1979; Le Bars and Gripon, 1981; Trieu-Cuot and Gripon, 1983). Le Bars and Gripon (1981) compared commercial Blue cheeses with sterile curds inoculated with either P. roqueforti or purified aspartic protease by electrophoresis, and found that the patterns were very similar, indicating the great importance of P. roqueforti, and especially of the associated aspartic protease, for proteolysis in Blue cheese during ripening. The link between general proteolytic activity and the aspartic protease was confirmed by Fernández-Bodega et al. (2009), analyzing isolates from artisanal Spanish cheeses. They found a direct relationship between the proteolytic activity determined in agar diffusion assays and the presence and intensity of the band of aspartic protease as determined by immunodetection. A relationship was established early between the development of P. roqueforti, the activity of the aspartic protease, and the release of bitter peptides (Gripon, 1993). To break down these bitter peptides, as well as other peptides, P. roqueforti possesses several exopeptidases. An extracellular acid carboxypeptidase, with a broad specificity, releases acidic, basic, and hydrophobic amino acids and may

be important in the debittering process. It is a serine enzyme with a pH optimum for hydrolyzing an artificial substrate at pH 3.5, and it is stable at pH 5.0–5.5. P. roqueforti also produces an extracellular alkaline metalloaminopeptidase with a pH optimum of 8.0. It is specific for hydrophobic amino acids, and consequently, the debittering activity of P. roqueforti may increase with pH in Blue cheese. Several intracellular peptidases have also been detected, among them alkaline carboxy- and aminopeptidases (Gripon, 1993), but the contribution to ripening is not obvious. The proteolytic activity, as well as the level of proteases and peptidases produced by P. roqueforti, varies greatly between strains (Larsen et al., 1998; Pose et al., 2007; Seratlic et al., 2011). The growth of mold within a pierced Blue cheese leads to an increase in pH that stimulates other proteolytic activities in the cheese, such as the LAB cell wall protease (lactocepin) and the milk protease, plasmin. At this later stage of ripening, there is hardly any β-casein left in Blue cheese, and plasmin activity is limited to any remaining peptides containing specific cleavage sites. Salt inhibits the development P. roqueforti and therefore also the proteolytic activity, which explains the hard and rather tasteless zones close to the rind of Blue cheese (Godinho and Fox, 1982). Yeasts in Blue cheeses mainly belong to the genus Candida (Table 37.3). Proteolytic activity, mainly intracellular, has been detected for a few Candida strains, but this property is poorly documented for larger number of strains within the same species (Klein et al., 2002; Pereira-Dias et al., 2000). Juszczyk et al. (2005a) investigated 67 yeast isolates, representative of the species growing in Rokpol cheese, and showed extracellular activities, similar to the

Blue Cheese Chapter | 37

study by van den Tempel and Jakobsen (2000): activity on casein at 10°C was not shown by any of six tested strains of D. hansenii originating from Blue cheese, whereas five of six strains of Y. lipolytica digested all the caseins at 10°C. This shows that yeast may contribute to proteolysis, for example, a specific strain of S. cerevisiae used as an adjunct culture in Mycella was shown to enhance proteolysis and texture in the cheese (Hansen et al., 2001). Aminopeptidase activity on branched-chain amino acids was shown for all strains investigated of both D. hansenii and Y. lipolytica, and as yeasts grow to large numbers in many Blue cheeses, this activity may contribute to the release of amino acids during ripening (Klein et al., 2002). Still an excessive contribution to proteolysis may cause a detrimental effect on the cheese. NSLAB have been isolated from Blue cheese and could, as in other cheeses, be expected to take advantage of the large amount of small peptides produced by the other microorganisms present, and mainly produce similar aroma compounds from amino acids as the starter bacteria. Amino acids, which are released at high amounts in Blue cheeses (Fernandez-Salguero, 2004; Zarmpoutis et al., 1997), contribute to a background flavor, but further catabolism is needed to produce several aroma compounds characteristic of cheese (Hemme et al., 1982; Yvon and Rijnen, 2001). The specific characteristic flavors of Blue cheese originate not from amino acids, but from lipolysis and a significant production of methyl ketones. Cheese flavor compounds are produced by LAB and molds through amino acid catabolism (Hemme et al., 1982; Yvon and Rijnen, 2001). The most commonly found free amino acids in Blue cheese are glutamic acid, leucine, valine, proline, and lysine (Flórez et al., 2006b; Madkor et al., 1987a; Zarmpoutis et al., 1996, 1997). The metabolic pathways of LAB, starting with aminotransferase activity, dominate in hard and semihard cheeses, all of which have a low redox potential (Ardö et al., 2002). These activities are not very well studied in Blue cheese, but they are present. Oxidative deamination of amino acids may be carried out by P. roqueforti within the cheese because of the piercing channels, and by the microbial flora on the cheese surfaces (cf. Chapter 38, Volume 2). This activity produces ammonia in amounts that contributes to the characteristic flavor of Blue cheese varieties. Compounds resulting from different pathways of amino acid catabolism have been found in Blue cheese (as reviewed by Gripon, 1993). Glutamic acid is decarboxylated to γ-aminobutyric acid and CO2, and other amino acids are decarboxylated to amines and CO2 by P. roqueforti, as well as by adventitiously growing microorganisms in, and on Blue cheese. The concentrations of amines vary greatly between, and within cheese types (Komprda et al., 2008). Tyramine is usually observed in higher amounts than tryptamine and histamine (de Boer and Kuik, 1987; Flórez

943

et al., 2006b; Gobbetti et al., 1997; Komprda et al., 2008; Prieto et al., 2000). The levels encountered in Blue cheeses are not assumed to have a health-related significance for healthy consumers (Komprda et al., 2008). But the complex amino acid catabolism, and especially the conversion to biologically active amines in Blue cheese varieties, still needs much research.

Lipolysis Lipolysis in Blue cheeses, like proteolysis, is very intense compared with other cheeses. High amounts of free fatty acids are found during the ripening of various kinds of Blue cheeses (Table 37.7). In other cheese varieties, this extensive lipolysis would give a rancid taste; however in Blue cheeses, the free fatty acids are neutralized by the increasing pH. In general, the total level of free fatty acids increases with ripening time, especially after the mold has sporulated (Alonso et al., 1987; Contarini and Toppino, 1995; Gobbetti et al., 1997; Madkor et al., 1987b), but a decrease at the end of ripening has also been observed (Prieto et al., 2000). This decrease could be caused by conversion of the fatty acids to methyl ketones. Due to the higher NaCl concentration in the rind, which inhibits mold growth and thereby lipase production, a lower level of free fatty acids has been observed in the outer part of the cheese compared with the core (Gobbetti et al., 1997; Godinho and Fox, 1981c). This effect can be altered to some degree by selecting more NaCl-tolerant strains of P. roqueforti. A recent study showed a higher yield of FFA and methyl ketones in cheese made from homogenized pasteurized cheese milk, compared with nonhomogenized milk, when purified P. roqueforti lipase, but no other commercial lipase, was added during

TABLE 37.7 Total Concentration of Fatty Acids in Different Cheese Varieties Fatty Acids (mg kg−1)

Variety

Fatty Acids (mg kg−1)

Gamonedo3 75685

Provolone

2118

Blue (US)

35230

Gruyere

1481

Cabrales1

33153

Brie

1314

2

18905

Cheddar

1028

4562

Kopanisti6

5796-131712

Roquefort

32453

Camembert

681

Stilton2

9830-17976

Picon BejesTresviso4

58355

Parmesan

4993

Mozzarella

363

Variety

Danablu Rokpol5

Source: Adapted from Woo, A.H., Kollodge, S., Lindsay, R.C., 1984. Quantification of major free fatty acids in several cheese varieties. J. Dairy Sci. 67, 874-878, except: 1, Alonso et al. (1987); 2, Madkor et al. (1987b); 3, González de Llano et al. (1992); 4, Prieto et al. (2000); 5, Chrzanowska et al. (2003); 6, Karali et al. (2013).

944 S ECTION | II  Diversity of Cheese

the production of Blue cheese (Cao et al., 2014). Generally, the concentration of saturated and unsaturated long-chained fatty acids, C12:0–C18:3, in Blue cheese is higher than the levels of short-chained fatty acids, C4:0–C10:0 (Chrzanowska et al., 2003; Karali et al., 2013; Wolf et al., 2011) which correspond to results obtained for P. roqueforti grown in butterfat emulsions (Larsen and Jensen, 1999). Considerable differences in the levels of individual free fatty acids can be found between various types of Blue cheeses, which is not only attributable to the age of the cheeses examined, but affected as well by the microbiota present (Alonso et al., 1987; Chrzanowska et al., 2003; Karali et al., 2013; Madkor et al., 1987b; Prieto et al., 2000; Wolf et al., 2011). Degradation of lipids in Blue cheeses is mainly caused by enzymes from P. roqueforti (Coghill, 1979; Gobbetti et al., 1997; Kinsella and Hwang, 1976). The lipolytic activity of commercial strains of P. roqueforti differs significantly, resulting in different amounts of free fatty acids released (Farahat et al., 1990; Ferreira et al., 2012; Larsen and Jensen, 1999; Pose et al., 2007) and thereby different flavor profiles of the cheeses produced (Farahat et al., 1990; Gallois and Langlois, 1990). P. roqueforti produces two extracellular lipases, an acidic and an alkaline lipase (Lamberet and Menassa, 1983b; Mase et al., 1995; Menassa and Lamberet, 1982). Intracellular lipase activity has also been reported (Niki et al., 1966; Stepaniak et al., 1980), but further research on this subject has, to our knowledge, not been carried out. The acidic lipase has a pH optimum at 6.0 and a less pronounced optimum at pH 2.8, with maximum stability between pH 3.7 and 6.0 (Lamberet and Menassa, 1983b). The optimum temperature is 35–40°C, but it retains 37% of maximum activity at 5°C. The optimum pH for the alkaline lipase is 8.8–9.0 at 30°C and 9.0–10.0 at 20°C, but activity is retained between pH 4.5 and 11.0 (Lamberet and Menassa, 1983b), for example, 15 and 20% of maximum activity is retained at pH 4.5 and 6.0, respectively. The relative importance of the acidic and the alkaline lipase in cheese has not been fully determined. However, Lamberet and Menassa (1983a) investigated the lipolytic activity at pH 5.5 on tricaproin (acid lipase) and at pH 8.0 on tributyrin (alkaline lipase) in suspensions of seven French Blue cheeses. Activity at pH 5.5 dominated, and only two samples showed measurable activity at pH 8.0. Even though the pH of Blue cheeses, in general, favors activity of the acid lipase, it should be noted that the alkaline lipase has a higher activity on milk fat (Eitenmiller et al., 1970; Lamberet and Menassa, 1983a,b). P. roqueforti dominates the overall lipid degradation in Blue cheeses, but other lipolytic agents are also present. The native milk lipoprotein lipase contributes at the beginning of the ripening period, most significantly in Blue cheeses produced from homogenized milk, like Danablu and Stilton (Gripon, 1993). As lipoprotein lipase is highly inactivated by pasteurization, its activity will be most pro-

nounced in cheeses produced from raw or thermized milk. The LAB, whether they are part of the starter culture or the nonstarter microbiota, have very low lipolytic activity and are not likely to affect the lipolysis in Blue cheese (El Soda et al., 1986; Meyers et al., 1996). Yeasts probably affect lipolysis, which could be positive (Jakobsen and Narvhus, 1996), but this is very dependent upon the yeast species present. Almost all yeasts present in cheese (e.g., D. hansenii, K. lactis, S. cerevisiae, Y. lipolytica, C. catenulata, and Galactomyces geotrichum) have at least esterase activity, being able to hydrolyze short-chained fatty acids from triglycerides (Ferreira et al., 2012; Fleet and Mian, 1987; Hansen and Jakobsen, 1998; Juszczyk et al., 2005a; Roostita and Fleet, 1996a; van den Tempel and Jakobsen, 1998). Lipolysis of long-chained fatty acids has been demonstrated for Y. lipolytica, C. catenulata, and G. geotrichum, and the activity seems to be at the same level for these three yeasts (Roostita and Fleet, 1996b; van den Tempel, 2000). Strains of Y. lipolytica have strong lipolytic activity, which could be desirable in some Blue cheeses, but they also, in general, affect growth of P. roqueforti negatively; however, these interactions are strain-specific (Hansen and Jakobsen, 1997; van den Tempel and Nielsen, 2000). A positive effect of Y. lipolytica on the development of aroma components in Stilton has been described (Gkatzionis et al., 2013; Price et al., 2014). This could be used to enhance the quality of Blue cheeses made from pasteurized milk, where lipolysis and aroma formation are delayed, and often weaker unless the ripening period is prolonged.

Formation of Aroma Compounds A wide range of volatile and nonvolatile aroma compounds is produced in Blue cheese during ripening, primarily by P. roqueforti, affecting both taste and aroma of the final product. The varying proportions of these compounds determine the specific flavor profiles obtained for different Blue cheeses (Gallois and Langlois, 1990; Wolf et al., 2011). A general overview of the different groups of aroma compounds to be found, their levels and how they contribute to the overall sensory perception of the Blue cheeses will be given here. For more detailed information, excellent reviews have been published (McSweeney and Sousa, 2000; Molimard and Spinnler, 1996; Sablé and Cottenceau, 1999). The characteristic flavor and taste of Blue cheeses stem mainly from lipid degradation: the free fatty acids contribute both to the taste and aroma, but even more important are the methyl ketones produced from them, which are essential for the sensory quality of Blue cheeses (Kinsella and Hwang, 1976; Moio et al., 2000; Rothe et al., 1994). As previously mentioned, the lipolytic activity of commercial strains of P. roqueforti differs significantly, resulting in different amounts of free fatty acids produced (Larsen and Jensen, 1999; Lopéz-Díaz et al., 1996b) and thus leading

Blue Cheese Chapter | 37

945

TABLE 37.8 Total Concentration (µg kg−1 cheese) of Major Groups of Aroma Compounds in Blue Cheeses Produced With Different Strains of P. roqueforti Cheese: Strain

Roquefort: Strain PFa

Roquefort: Strain POb

Roquefort: Strain PGc

Bleu de Caussesd: Strain PGc

Bleu d’Auvergnee: Unknown Strain

Civil1: Natural Ripening

Ketones

11,095

14,350

34,940

9345

9780

3116–3221

Alcohols

4025

3305

7670

3795

8110

1634–3274

Esters

1390

2985

3835

3155

2950

518–723

Lactones

50

255

325

425

2230

Not specified

Aldehydes

5

10

15

0

250

46–172

a

Low proteolytic/low-lipolytic activity; 210 days of ripening b High proteolytic/rather high-lipolytic activity; 210 days of ripening c Medium proteolytic/medium-lipolytic activity; 210 days of ripening d 100 days of ripening e Approximately 45 days of ripening Modified from Gallois, A., Langlois, D., 1990. New results in the volatile odorous compounds of French cheeses. Lait 70, 89–106, except: 1, Cakmakci et al. (2012).

to the different flavor profiles of the cheeses (Table 37.8) (Farahat et al., 1990; Gallois and Langlois, 1990). Most volatile fatty acids (C4:0–C12:0) have rather low threshold values, pungent or rancid flavor notes, and are usually present at fairly high concentrations in Blue cheeses (Cakmakci et al., 2012; Hayaloglu et al., 2008; Karali et al., 2013; Wolf et al., 2011), but because of the high pH of Blue cheese, these acids are neutralized and hence contribute to the aroma of the cheese and not to a rancid defect (Molimard and Spinnler, 1996). Hexanoic and octanoic acids are especially important flavor compounds (Molimard and Spinnler, 1996; Rothe et al., 1994; Sablé and Cottenceau, 1999). Methyl ketones are the major aroma compounds in Blue cheeses (Table 37.8) and have been reported to constitute 50%–75% of the total volatile flavors (Cakmakci et al., 2012; de Frutos et al., 1991; Gallois and Langlois, 1990; González de Llano et al., 1990; Hansen et al., 2001; Madkor et al., 1987b; Moio et al., 2000; Ney and Wirotama, 1972; Wolf et al., 2011), and the concentration in cheese can be correlated to the intensity of a “Blue cheese” note (Rothe et al., 1982, 1986, 1994). The methyl ketones found in the highest concentrations are 2-heptanone and 2-nonanone, but 2-pentanone and 2-undecanone are also important (Cakmakci et al., 2012; Contarini and Toppino, 1995; de Frutos et al., 1991; Gallois and Langlois, 1990; González de Llano et al., 1990; Hayaloglu et al., 2008; Madkor et al., 1987b; Wolf et al., 2011). The total concentration of methyl ketones in Blue cheese depends upon manufacturing procedures, ripening times, and the strains of P. roqueforti used (Table 37.8), whereas the proportions of the individual methyl ketones in the cheese mainly depend upon the strain of P. roqueforti (Flórez et al., 2006b; Gallois and Langlois, 1990; Hayaloglu et al., 2008; Wolf et al., 2011). The odor impressions of the methyl ketones are, in general, fruity, floral, and musty, and specifically

for 2-heptanone, spicy and “Blue cheese” (Molimard and Spinnler, 1996; Sablé and Cottenceau, 1999). As increasing concentrations of free fatty acids have been shown to inhibit the growth of P. roqueforti and thereby retard lipolysis, the formation of methyl ketones from free fatty acids has been proposed to be a detoxifying mechanism (Kinsella and Hwang, 1976). Methyl ketones with one less carbon atom are produced via the β-oxidation pathway from the corresponding fatty acids. The first intermediate is a β-keto acyl-CoA, which is converted to a β-keto acid by a thiohydrolase and then decarboxylated to a methyl ketone and CO2. When at low concentrations, the acyl-CoA enters the Krebs cycle and is completely oxidized to CO2 (Molimard and Spinnler, 1996). Both conidia and mycelia are capable of producing methyl ketones (Fan et al., 1976). The majority of methyl ketones are derived directly from the fatty acid precursor, but the concentration of certain methyl ketones, for example, 2-heptanone and 2-nonanone, is generally very high compared with the concentration of the precursors, C8:0 and C10:0, and it was shown that these methyl ketones can also be derived from longer-chain fatty acids (Dartey and Kinsella, 1973a,b; Madkor et al., 1987b). Alcohols have been reported to represent between 15% and 30% of the total volatile flavor compounds (Table 37.8) (Cakmakci et al., 2012; Gallois and Langlois, 1990; Moio et al., 2000; Wolf et al., 2011), but again depending upon the cheese type. In Kuflu cheeses, ketones and alcohols were detected in similar amounts (Hayaloglu et al., 2008), whereas alcohols, of which ethanol was 80%, were the dominant group in Kopanisti cheeses, representing 60% of the total volatile compounds with ketones accounting for only 2%–3% (Karali et al., 2013). The high concentrations of ethanol also lead to high concentrations of esters. Methyl ketones can be reduced to secondary alcohols, under anaerobic conditions, and these alcohols are generally

946 S ECTION | II  Diversity of Cheese

more abundant in Blue cheese than primary alcohols. The main secondary alcohols are 2-heptanol, 2-nonanol, and 2-pentanol, depending upon the cheese type and strain of P. roqueforti used (Cakmakci et al., 2012; Gallois and Langlois, 1990; González de Llano et al., 1990; Hayaloglu et al., 2008; Wolf et al., 2011). The flavor is more or less similar to the corresponding methyl ketones, but, at higher concentrations, can give a musty or moldy impression (Kinsella and Hwang, 1976). Primary alcohols are also present, with 3-methyl-1-butanol the most abundant (Cakmakci et al., 2012; Gallois and Langlois, 1990; Hayaloglu et al., 2008; Karali et al., 2013; Moio et al., 2000; Wolf et al., 2011). Other aroma compounds with fruity and floral notes are esters and lactones; the latter are usually found at low concentrations (Table 37.8) (Gallois and Langlois, 1990; González de Llano et al., 1990; Wolf et al., 2011). The higher amount of lactones detected in Bleu d’Auvergne (Table 37.8) could be due to the use of pasteurized milk, as pasteurization of milk has been shown to increase the level of lactones. Esters have a low perception threshold, and especially ethyl esters are known for fruity notes in cheese (Karali et al., 2013). These can be formed in cheese by microbial esterification of free fatty acids with alcohols. Esters make up to 26% of the total volatile profile in Kopanisti, and in some Argentinian cheeses, ethyl esters dominate (Karali et al., 2013; Wolf et al., 2011), whereas in Kuflu and Civil cheeses, methyl esters are the most abundant which might be due to low levels of free fatty acids in these cheese types (Cakmakci et al., 2012; Hayaloglu et al., 2008). Apart from affecting the overall aroma profile of Blue cheese, the main contribution of esters is possibly to minimize the sharpness and bitterness from fatty acids and amines (Lawlor et al., 2003). Peptides and amino acids from proteolysis yield compounds which are important for the background flavor of the cheese and, furthermore, directly contribute to flavor, for example, sweet, bitter, and brothy (Nishimura and Kato, 1988; Yvon and Rijnen, 2001). Aldehydes are detected at low levels (Cakmakci et al., 2012; Gallois and Langlois, 1990; Hayaloglu et al., 2008; Karali et al., 2013; Ney and Wirotama, 1972; Wolf et al., 2011; Table 37.8), and the impact upon flavor is not well known, apart from acetaldehyde, a key component in fermented dairy products, and 3-methyl-butanal, having green and malty notes (Sablé and Cottenceau, 1999). Aldehydes are generally expected to be found at low concentrations in ripened cheese, as these can either be converted to the corresponding alcohol or oxidized to the corresponding acid (Molimard and Spinnler, 1996). The impact of the volatile and nonvolatile amines (Adda and Dumont, 1974; Ney and Wirotama, 1972) is not well known either, but it is believed that amines have an effect on the overall flavor sensation. Investigations of the impact of sulfur-containing compounds on Blue cheese flavor are limited; however, they

are supposed to make an important contribution (Gallois and Langlois, 1990; Kinsella and Hwang, 1976). Apart from P. roqueforti, yeasts also contribute to the formation of aroma compounds, either directly, especially by producing esters, or indirectly, by affecting growth and thereby enzyme production of P. roqueforti. Aroma production of D. hansenii has been investigated in cheese media models (Leclercq-Perlat et al., 2004; Sørensen et al., 2011), showing the formation of branched-chain aldehydes, aldehydes, methyl ketones, and alcohols. The use of S. cerevisiae FB7 as an adjunct culture in Mycella cheese led to an increase in the concentration of aroma compounds in the experimental cheeses compared with the reference cheeses without added yeast (Hansen et al., 2001). Positive effects on aroma production with strains of Y. lipolytica growing together with P. roqueforti have been observed in Stilton cheese (Gkatzionis et al., 2013, 2014; Price et al., 2014). Methyl ketones were found in the highest amounts in the blue veins and in the outer crust, whereas the white parts mainly contained alcohols and aldehydes (Gkatzionis et al., 2009). P. roqueforti was, however, only detected in the veins and the white parts, not in the outer crust, which was dominated by D. hansenii and to a lesser extent Y. lipolytica (Gkatzionis et al., 2014). Subsequent studies in model systems combining P. roqueforti and Y. lipolytica clearly showed a positive interaction in production of aroma compounds, confirmed by sensory analysis, where especially the “blue” note of the model system ranked higher than the real cheese (Gkatzionis et al., 2013, 2014). In addition, the effect seems to be dependent upon the inoculum level of the yeast, leaving room for altering the development of Blue cheese aroma (Price et al., 2014).

Production and Occurrence of Secondary Metabolites P. roqueforti produces a range of secondary metabolites: the alkaloids roquefortine A, B, and C and the isofumigaclavines, the marcfortines, andrastins A–D, mycophenolic acid and the mycotoxin, PR-toxin (P. roqueforti-toxin),and the precursors eremofortin A–E (Hymery et al., 2014; Nielsen et al., 2005; Scott, 1981; see Chapter 23, Volume 1, for further discussion of mycotoxins). The occurrence of these metabolites in Blue cheese is shown in Table 37.9. The sesquiterpene PR-toxin inhibits nucleic acid and protein synthesis, and is cytotoxic in human and porcine cell lines and in rat liver, in addition to being mutagenic (as cited by Scott, 1981). Many strains of P. roqueforti used commercially as starter cultures or isolated from Blue cheeses have the ability to produce PR-toxin (Boysen et al., 1996; Chang et al., 1991; Engel and Prokopek, 1979; Geisen et al., 2001; Medina et al., 1985; Orth, 1976; Wei and Liu, 1978) or one or more of the precursors, eremofortin A–E (Chang et al., 1991; Geisen et al., 2001; Moreau et al., 1980) in

Blue Cheese Chapter | 37

947

TABLE 37.9 Secondary Metabolites Produced by P. roqueforti Detected in Commercial Blue Cheeses Metabolite

No. of Cheeses Examined

No. of Positive Samples Concentration Ranges (mg kg−1)

Reference

PR-imine

60

50

0.019–0.042

2

Roquefortine

10

1

nr

3

16

16

0.05–6.8

4

12

12

0.16–0.65

5

13

13

0.2–2.29

6

30

30

0.05–1.47

9

10

10

0.8–12

10

10

9

0.1–3.6

11

Isofumigaclavine A

16

13

Traces to 4.7

4

Isofumigaclavine B

16

6

Traces

4

Mycophenolic acid

32

4

0.25–5

7

100

38

nr to 14.3

8

10

0

6

6

17.01–19.57

12

21

21

0.08–3.7

1

Andrastin A

3

nr, Not reported. 1, Nielsen et al. (2005); 2, Siemens and Zawistowski (1993); 3, Lopéz-Díaz et al. (1996a); 4, Scott and Kennedy (1976); 5, Ware et al. (1980); 6, Schoch et al. (1984); 7, Engel et al. (1982); 8, Lafont et al. (1979); 9, Finoli et al. (2001); 10, Kokkonen et al. (2005); 11, Pose et al. (2007); 12, Fernández-Bodega et al. (2009).

synthetic media. Fortunately, PR-toxin is unstable in the cheese environment and is transformed to the less toxic PRimine, which is also unstable, and PR-amide, in the presence of basic and neutral amino acids (Chang et al., 1993; Scott and Kanhere, 1979). PR-toxin has never been detected in commercial Blue cheeses or in experimental Blue cheeses made with known toxin-producing strains (Engel and Prokopek, 1979; Hymery et al., 2014; Scott and Kanhere, 1979). Roquefortine C is a typical metabolite of P. roqueforti and P. carneum (Boysen et al., 1996; Lopéz-Díaz et al., 1996a; Medina et al., 1985), and very often found in Blue cheese, where the concentration is correlated to sporulation of the fungi (Finoli et al., 2001; Pose et al., 2007; Scott and Kennedy, 1976). Isofumigaclavine A and the stereoisomer fumigaclavine A are also a characteristic secondary metabolite for P. roqueforti (Boysen et al., 1996; Geisen et al., 2001; Scott et al., 1976). Mycophenolic acid is not always produced by strains of P. roqueforti. Boysen et al. (1996) and Geisen et al. (2001) found that ca.50% of the strains investigated produced this metabolite; Engel et al. (1982) found it in 25% of the strains; and Lopéz-Díaz et al. (1996a) detected mycophenolic acid from only one strain out of nine. In contrast, Lafont et al. (1979) reported that all 16 strains of P. roqueforti investigated produced mycophenolic acid. The LD50 values determined for mycophenolic acid are high, 2500 and 700 mg/kg for mouse and rat, respectively, but subacute toxic effects have been observed for monkeys and rats (Carter et al., 1969; Scott, 1981).

Andrastins A–D was detected in various European Blue cheeses in 2005 by Nielsen et al. (2005) and later in Spanish Blue cheeses (Fernández-Bodega et al., 2009), and does not seem to be correlated to sporulation (Nielsen et al., 2005). Andrastins have anticarcinogenic properties, but the possible negative and positive effect on human health has not been clarified. Taking into account the very low levels, and the relatively low toxicity of the various mycotoxins present in the cheese, even a large consumption of Blue cheese does not pose a risk to the health of the consumer.

SELECTION OF CULTURES Blue cheese is a very complex food ecosystem with marked pH and NaCl gradients and variable, but generally low levels of O2 and CO2. This heterogeneous microenvironment creates different habitats on the surface and in the core of the cheese, which select for specific micropopulations. The technological characteristics of the LAB of the starter culture and the P. roqueforti culture have significant effects on the quality of the cheese. The primary LAB culture must be able to lower pH and survive phage attack, as acidification of the cheese milk is essential for the renneting of the milk and syneresis of the curd, and thereby the fundamental part of the cheesemaking process. The secondary culture, P. roqueforti, is often chosen for proteolytic and lipolytic activities, depending upon the type of Blue cheese, the targeted market, and the desired shelf life.

948 S ECTION | II  Diversity of Cheese

The proteolytic activity of the strain of P. roqueforti used is extremely important for texture development, whereas the lipolytic activity determines the flavor profile. Next, the culture is chosen for tolerance to NaCl, growth rate, and sporulation capacity. The right combination of these activities and characteristics is crucial for the development of a high quality product. It could, however, also be beneficial to include the interaction with LAB as a factor, and it should be considered whether to make use of the possible synergies in practice and to avoid antagonistic effects. Yeasts should also be considered as potential adjunct cultures as they are already present in the cheese and have interesting technological characteristics. There are two major objectives in using yeasts as adjunct cultures in the production of Blue cheese: (1) to secure the microenvironment by assimilating residual carbohydrates and organic acids, thereby promoting the growth of desired cultures and inhibiting the growth of spoilage and pathogenic microorganisms, and (2) to contribute directly to the desired cheese quality by the enzymatic activity and by stimulating P. roqueforti. But very careful selection is crucial to avoid undesirable antagonistic interactions between the different cultures and to avoid the production of pigments, undesirable aroma compounds, and uncontrolled enzymatic activity. A few selected yeasts will be described as potential adjunct cultures for Blue cheese. However, it is important to remember that several of the technological characteristics are strain-specific and cannot be seen as a general characteristic of the yeast species. Although D. hansenii is the yeast species most frequently isolated from Blue cheese (Tables 37.3 and 37.4), it is rarely used as an adjunct culture and only a few applications have been reported, for example, as a surface culture in the production of Roquefort (Besancon et al., 1992). D. hansenii is very weakly proteolytic and has low lipolytic activity. The strains will not enhance proteolysis, but they might alter the aroma profile slightly without changing it greatly. The potential use of D. hansenii as an adjunct culture seems to be linked with osmotolerance and good growth in Blue cheese. Furthermore, it can create a stable microenvironment which protects against undesired microbial growth by assimilation of residual carbohydrates and organic acids (van den Tempel and Jakobsen, 2000; van den Tempel and Nielsen, 2000). The potential of Y. lipolytica as a ripening culture in cheese has been evaluated by Guerzoni et al. (1998). It was demonstrated that Y. lipolytica possesses some of the essential properties for use as an adjunct culture: (1) ability to grow and compete with other naturally occurring yeasts, such as D. hansenii and S. cerevisiae, even though it assimilates only galactose and lactate; (2) compatibility with, and possible stimulation of LAB when coinoculated; and (3) remarkable lipolytic and proteolytic activities. Y. lipolytica is relatively salt tolerant, and the potential role as

an adjunct culture in Blue cheese is mainly linked with lipolysis and aroma formation, but it may also contribute to proteolysis (Gkatzionis et al., 2013; van den Tempel and Jakobsen, 2000). Y. lipolytica could be a potential adjunct culture, but should be controlled very carefully because of strong enzymatic activity, an inhibitory effect toward P. roqueforti, and the ability to discolor the cheese (Nichol and Harden, 1993; Weichhold et al., 1988). However, very positive results have been obtained for the development of aroma compounds and the typical Blue cheese note in model systems combining Y. lipolytica and P. roqueforti (Gkatzionis et al., 2013, 2014; Price et al., 2014). Strains of S. cerevisiae can stimulate the release of fatty acids by P. roqueforti, and a synergistic effect between P. roqueforti and S. cerevisiae has been demonstrated in the degradation of casein and formation of aroma compounds (Hansen et al., 2001; Hansen and Jakobsen, 2001). S. cerevisiae can assimilate residual glucose, galactose, and lactate. It has a relatively low tolerance toward NaCl and would be a suitable yeast culture only for the production of Blue cheese with low levels of NaCl, such as Gorgonzola or Mycella. The purpose of using S. cerevisiae as an adjunct culture would be to make a controlled contribution to aroma formation and proteolysis, as well as creating a stable microenvironment. For all cultures, whether they are already in use or under consideration as adjunct cultures, a thorough screening of technological characteristics and possible interactions under different environmental conditions is extremely important. Only a few selected characteristics have been investigated and described to date, but with new knowledge, especially on microbial interactions, new possibilities for applying and combining cultures in different ways have become available.

CONCLUSIONS A number of aspects of Blue cheese ripening have been discussed in the present chapter, but with the space available, not all issues can be addressed. The importance of P. roqueforti for the quality of Blue cheese is indisputable, but newly gained knowledge on the microbial interactions and the importance of adventitious microbiota points add new possibilities for improving existing Blue cheeses or developing new varieties. Adding yeast as adjunct cultures is an obvious opportunity for diversifying the product range, but there is still a need for further investigations on interactions with the other cultures present, not only concerning growth, but also on enzymatic activity. Progress has also been made in understanding the complex mechanisms of ripening, and new research on proteolysis and the impact of aspartic protease has appeared, but there remains areas to be investigated, for example, patterns of casein breakdown caused by P. roqueforti proteases in

Blue Cheese Chapter | 37

vitro and in different Blue cheeses. Furthermore, the amino acid metabolism in Blue cheeses, with all the different microorganism present, is not fully understood. This could be useful for clarifying the effect of individual cultures on proteolysis and thereby on structure development and taste. Research on texture development and the connection with the proteolytic activity of P. roqueforti, together with texture analysis of Blue cheese, is very limited. It would, however, be a valuable tool for further characterization of the cheese itself and the effect of the microbiota, especially the strains of P. roqueforti. Finally, there seems to be some potential for obtaining health benefits by consuming Blue cheeses, but at present, knowledge is scarce and built upon indications, so carefully designed clinical trials and experimental studies need to be done before any preliminary conclusions can be drawn in this interesting and new area of research.

REFERENCES Adda, J., Dumont, J.P., 1974. Les substances responsables de l’arôme des fromages à pâte molle. Lait 54, 1–21. Addis, E., Fleet, G.H., Cox, J.M., Kolak, D., Leung, T., 2001. The growth, properties and interactions of yeasts and bacteria associated with the maturation of Camembert and blue-veined cheeses. Int. J. Food Microbiol. 69, 25–36. Alegria, A., Gonzalez, R., Diaz, M., Mayo, B., 2011. Assessment of microbial populations dynamics in a blue cheese by culturing and denaturing gradient gel electrophoresis. Curr. Microbiol. 62, 888–893. Alonso, L., Juarez, J., Ramos, M., Martin-Alvarez, P.J., 1987. Overall composition, nitrogen fractions and fat characteristics of Cabrales cheese during ripening. Z. Lebensm. Unters. Forsch. 185, 481–486. Álvarez-Martín, P., Flórez, A.B., López-Díaz, T.M., Baltasar, M., 2007. Phenotypic and molecular identificatioin of yeast species associated with Spanish blue-veined Cabrales cheese. Int. Dairy J. 17, 961–967. Ardö, Y., 2001. Cheese ripening. General mechanisms and specific cheese varieties. Bulletin 369, International Dairy Federation, Brussels, pp. 7–12. Ardö, Y., Thage, B.V., Madsen, J.S., 2002. Dynamics of free amino acid composition in cheese ripening. Aust. J. Dairy Technol. 57, 109–115. Besancon, X., Smet, C., Chabalier, C., Rivemale, M., Reverbel, J.P., Ratomahenina, R., Galzy, P., 1992. Study of surface yeast flora of Roquefort cheese. Int. J. Food Microbiol. 17, 9–18. Bockelmann, W., Hoppe-Seyler, T., 2001. The surface flora of bacterial smear-ripened cheeses from cow’s and goat’s milk. Int. Dairy J. 11, 307–314. Boddy, L., Wimpenny, J.W.T., 1992. Ecological concepts in food microbiology. J. Appl. Bacteriol. 73, 23S–38S. Boysen, M.E., 1999. Molecular Identification and Quantification of the Penicillium roqueforti Group. PhD Thesis, Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden. Boysen, M., Skouboe, P., Frisvad, J., Rossen, L., 1996. Reclassification of the Penicillium roqueforti group into three species on the basis of molecular genetic and biochemical profiles. Microbiology 142, 541–549. Boysen, M.E., Jacobsson, K.G., Schnürer, J., 2000. Molecular identification of species from the Penicillium roqueforti group associated with spoiled animal feed. Appl. Environ. Microbiol. 4, 1523–1526.

949

Cakmakci, S., Gundogdu, E., Hayaloglu, A.A., Dagdemir, E., Gurses, M., Cetin, B., Tahmas-Kahyaoglu, D., 2012. Chemical and microbiological status and volatile profiles of mouldy Civil cheese, a Turkish mould-ripened variety. Int. J. Food Sci. Technol. 47, 2405–2412. Cao, M., Fonseca, L.M., Schoenfuss, T.C., Rankin, S.A., 2014. Homogenization and lipase treatment of milk and resulting methyl ketone generation in blue cheese. J. Agric. Food Chem. 62, 5726–5733. Carter, S.B., Franklin, T.J., Jones, D.F., Leonard, B.J., Mills, S.D., Turner, R.W., Turner, W.B., 1969. Mycophenolic acid: anti-cancer compound with unusual properties. Nature 233, 848–850. Cerning, J., Gripon, J.-C., Lamberet, G., Lenoir, J., 1987. Les activités biochimiques des Penicillium utilisés en fromagerie. Lait 67, 3–39. Chang, S., Wei, Y., Wei, D., Chen, Y., Jong, S., 1991. Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti. Appl. Environ. Microbiol. 57, 2581–2585. Chang, S., Lu, K., Yeh, S., 1993. Secondary metabolites resulting from degradation of PR toxin by Penicillium roqueforti. Appl. Environ. Microbiol. 59, 981–986. Chrzanowska, J., Szoltysik, M., Zarowska, B., Juszczyk, P., Wojtatowicz, M., 2003. Microbiological and physicochemical characteristics of Rokpol cheese during ripening. Milchwissenschaft 58, 505–508. Coghill, D., 1979. The ripening of blue vein cheese: a review. Aust. J. Dairy Technol. 34, 72–75. Contarini, G., Toppino, P.M., 1995. Lipolysis in Gorgonzola cheese during ripening. Int. Dairy J. 5, 141–155. Dartey, C.K., Kinsella, J.E., 1973a. Oxidation of sodium (U-14C) palmitate into carbonyl compounds by Penicillium roqueforti spores. J. Agric. Food Chem. 21, 721–726. Dartey, C.K., Kinsella, J.E., 1973b. Metabolism of (U-14C) lauric acid to methyl ketones by the spores of Penicillium roqueforti. J. Agric. Food Chem. 21, 933–936. de Boer, E., Kuik, D., 1987. A survey of the microbiological quality of blue veined cheeses. Neth. Milk Dairy J. 41, 227–237. de Frutos, M., Sanz, J., Martínez-Castro, I., 1991. Characterization of artisanal cheeses by GC and GC/MS analysis of their medium volatility (SDE) fraction. J. Agric. Food Chem. 39, 524–530. Devoyod, J.J., Desmazeaud, M., 1971. Les associations microbiennes dans le fromage de Roquefort. III. Action des entérocoques et des levures fermentant le lactose vis-à-vis des lactobacilles. Lait 51, 399. Devoyod, J.J., Sponem, D., 1970. La flore microbienne du fromage de Roquefort. VI. Les levures. Lait 50, 524–542. Devoyod, J.J., Bret, G., Auclair, J.E., 1968. La flore microbienne du fromage de Roquefort. I. Son évolution au cours de la fabrication et de l’affinage du fromage. Lait 48, 479–480. Dieuleveux, V., Lemarinier, S., Guéguen, M., 1998. Antimicrobial spectrum and target site of D-3-phenyllactic acid. Int. J. Food Microbiol. 40, 177–183. Diezhandino, I., Fernándes, D., González, L., McSweeney, P.L.H., Fresno, J.M., 2015. Microbiological, physico-chemical and proteolytic changes in a Spanish blue cheese during ripening (Valdeón cheese). Food Chem. 168, 134–141. Dixon, N.M., Kell, D.B., 1989. The inhibition by CO2 of the growth and metabolism of micro-organisms. J. Appl. Bacteriol. 67, 109–136. Dörge, T., Carstensen, J.M., Frisvad, J.C., 2000. Direct identification of pure Penicillium species using image analysis. J. Microbiol. Meth. 41, 121–133. Eitenmiller, R.R., Vakil, J.R., Shahani, K.M., 1970. Production and properties of Penicillium roqueforti lipase. J. Food Sci. 35, 130–133.

950 S ECTION | II  Diversity of Cheese

El Soda, M., Korayem, M., Ezzat, N., 1986. The esterolytic and lipolytic activities of lactobacilli. III. Detection and characterization of the lipase system. Milchwissenschaft 41, 353–355. Eliskases-Lechner, F., Ginzinger, W., 1995. The yeast flora of surfaceripened cheeses. Milchwissenschaft 50, 458–462. Engel, G., 1986. Hefen in silagen und rohmilch. Milchwissenschaft 41, 633–637. Engel, G., Prokopek, D., 1979. Kein nachweis von Penicillium roquefortitoxin in Käse. Milchwissenschaft 34, 272–274. Engel, G., von Milczewski, K.E., Prokopek, D., Teuber, M., 1982. Strainspecific synthesis of mycophenolic acid by Penicillium roqueforti in blue-veined cheese. Appl. Environ. Microbiol. 43, 1034–1040. Ercolini, D., Hill, P.H., Dodd, C.E.R., 2003. Bacterial community structure and location in Stilton cheese. Appl. Environ. Microbiol. 69, 3540–3548. Fan, T.Y., Hwang, D.H., Kinsella, J.E., 1976. Methyl ketone formation during germination of Penicillium roqueforti. J. Agric. Food Chem. 24, 443–448. Farahat, S.M., Rabie, A.M., Faraq, A.A., 1990. Evaluation of the proteolytic and lipolytic activity of different Penicillium roqueforti strains. Food Chem. 36, 169–180. Fernández-Bodega, M.A., Mauriz, E., Gómez, A., Martín, J.F., 2009. Proteolytic activity, mycotoxins and andrastin A in Penicillium roqueforti strains isolated from Cabrales, Valdeón and Bejes-Tresviso local varieties of blue-veined cheeses. Int. J. Food Microbiol. 136, 18–25. Fernandez-Salguero, J., 2004. Internal mould-ripened cheeses: characteristics, composition and proteolysis of the main European blue vein varieties. Ital. J. Food Sci. 16, 437–445. Fernandez-Salguero, J., Marcos, A., Alcala, M., Esteban, M.A., 1989. Proteolysis of Cabrales cheese and other European blue vein cheese varieties. J. Dairy Res. 56, 141–145. Ferreira, C.O., Alvarez-Martin, P., Flórez, A.B., Diaz, M., Mayo, B., 2012. Biochemical and technological properties of Penicillium roqueforti and Geotrichum candidum strains isolated from Cabrales, a Spanish traditional, blue-veined, starter-free cheese. Ital. J. Food Sci. 24, 115–124. Filtenborg, O., Frisvad, J.C., Thrane, U., 1996. Moulds in food spoilage. Int. J. Food Microbiol. 33, 109–113. Finoli, C., Vecchio, A., Galli, A., Dragoni, I., 2001. Roquefortine C occurrence in blue cheese. J. Food Prot. 64, 246–251. Fleet, G., 1990. Yeast in dairy products—a review. J. Appl. Bacteriol. 68, 199–211. Fleet, G., 1992. Spoilage yeasts. Crit. Rev. Biotechnol. 12, 1–44. Fleet, G., Mian, M.A., 1987. The occurrence and growth of yeasts in dairy products. Int. J. Food Microbiol. 4, 145–155. Flórez, A.B., Mayo, B., 2006a. PCR-DDGE as a tool for characterizing dominant microbial populations in the Spanish blue-veined Cabrales cheese. Int. Dairy J. 16, 1205–1210. Flórez, A.B., Mayo, B., 2006b. Microbial diversity and succession during the manufacture and ripening of traditional, Spanish, blue-veined Cabrales cheese, as determined by PCR-DGGE. Int. J. Food Microbiol. 110, 165–171. Flórez, A.B., López-Díaz, T.M., Álvarez-Martín, P., Baltasar, M., 2006a. Microbial characterization of the traditional Spanish blue-veined Cabrales cheese: identification of dominant lactic acid bacteria. Eur. Food Res. Technol. 223, 503–508. Flórez, A.B., Ruas-Madiedo, P., Alonso, L., Baltasar, M., 2006b. Microbial, chemical and sensorial variables of the Spanish traditional blueveined Cabrales cheese, as affected by inoculation with commercial Penicillium roqueforti spores. Eur. Food Res. Technol. 222, 250–257.

Flórez, A.B., Álvarez-Martín, P., López-Díaz, T.M., Baltasar, M., 2007. Morphotypic and molecular identification of filamentous fungi from Spanish blue-veined Cabrales cheese, and typing of Penicillium roqueforti and Geotrichum candidum isolates. Int. Dairy J. 17, 350–357. Flórez, A.B., Belloch, C., Alvarez-Martin, P., Querol, A., Mayo, B., 2010. Candida cabralensis sp. nov., a yeast species isolate from traditional Spanish blue-veined Cabrales cheese. Int. J. Syst. Evol. Microbiol. 60, 2671–2674. Fox, P.F., Law, J., 1991. Enzymology of cheese ripening. Food Biotechnol. 5, 239–262. Fox, P.F., Wallace, J.M., Morgan, S., Lynch, C.M., Niland, E.J., Tobin, J., 1996. Acceleration of cheese ripening. Antonie van Leeuwenhoek 70, 271–297. Freitas, A.C., Pintado, A.E., Pintado, M.E., Malcata, F.X., 1999. Role of dominant microflora of Picante cheese on proteolysis and lipolysis. Int. Dairy J. 9, 593–603. Frisvad, J.C., 1982. Metoder til karakterisering af levnedsmiddelbårne toxinogene skimmelsvampe (Penicillium og Aspergillus). Licentiatafhandling. Laboratoriet for Levnedsmiddelindustri, DTH, København. Gallois, A., Langlois, D., 1990. New results in the volatile odorous compounds of French cheeses. Lait 70, 89–106. Galzin, M., Galzy, P., Bret, G., 1970. Etude de la flore de levure dans le fromage de Roquefort. Lait 50, 1–37. Geisen, R., Larsen, M.D., Hansen, T.K., Holzapfel, W.H., Jakobsen, M., 2001. Characterization of Penicillium roqueforti strains used as cheese starter cultures by RAPD typing. Int. J. Food Microbiol. 65, 183–191. Gkatzionis, K., Linforth, R.S.T., Dood, C.E.R., 2009. Volatile profile of Stilton cheeses: differences between zones within a cheese and dairies. Food Chem. 113, 506–512. Gkatzionis, K., Hewson, L., Hollowood, T., Hort, J., Dood, C.E.R., Linforth, R.S.T., 2013. Effect of Yarrowia lipolytica on blue cheese odour development: flash profile sensory evaluation of microbiological models and cheeses. Int. Dairy J. 30, 8–13. Gkatzionis, K., Yunita, D., Linforth, R.S.T., Dickinson, M., Dodd, C.E.R., 2014. Diversity and activities of yeasts from different parts of a Stilton cheese. Int. J. Food Microbiol. 177, 109–116. Glas, N., Donaldson, G., 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microbiol. 61, 1323–1330. Gobbetti, M., Burzigotti, R., Smacchi, E., Corsetti, A., De Angelis, M., 1997. Microbiology and biochemistry of Gorgonzola cheese during ripening. Int. Dairy J. 7, 519–529. Godinho, M., Fox, P.F., 1981a. Effect of NaCl on the germination and growth of Penicillium roqueforti. Milchwissenschaft 36, 205–208. Godinho, M., Fox, P.F., 1981b. Ripening of Blue cheese. Salt diffusion rate and mould growth. Milchwissenschaft 36, 329–333. Godinho, M., Fox, P.F., 1981c. Ripening of Blue cheese. Influence of salting rate on lipolysis and carbonyl formation. Milchwissenschaft 36, 476–478. Godinho, M., Fox, P.F., 1982. Ripening of Blue cheese. Influence of salting rate on proteolysis. Milchwissenschaft 37, 72–75. González de Llano, D., Ramos, M., Rodriguez, A., Montilla, A., Juarez, M., 1992. Microbiological and physicochemical characteristics of Gamonedo blue cheese during ripening. Int. Dairy J. 2, 121–135. González de Llano, D., Ramos, M., Polo, C., Sanz, J., Martinez-Castro, I., 1990. Evolution of the volatile components of an artisanal blue cheese during ripening. J. Dairy Sci. 73, 1676–1683.

Blue Cheese Chapter | 37

González de Llano, D., Polo, C.M., Ramos, M., 1995. Study of proteolysis in artisanal cheeses: high performance liquid chromatography of peptides. J. Dairy Sci. 78, 1018–1024. Gourama, H., 1997. Inhibition of growth and mycotoxin production of Penicillium by Lactobacillus species. Food Sci. Technol. 30, 279–283. Gourama, H., Bullerman, L.B., 1995. Antimycotic and antiaflatoxigenic effect of lactic acid bacteria: a review. J. Food Prot. 57, 1275–1280. Gripon, J.C., 1993. Mould-ripened cheeses. In: Fox, P.F. (Ed.), Cheese: Chemistry, Physics and Microbiology. Vol. 2. Major Cheese Groups. second ed. Chapman & Hall, London, pp. 111–136. Gripon, J.C., Desmazeaud, M.J., le Bars, D., Bergere, J.L., 1977. Role of proteolytic enzymes of Streptococcus lactis, Penicillium roqueforti and Penicillium caseicolum during cheese ripening. J. Dairy Sci. 60, 1532–1538. Guerzoni, M.E., Gobbetti, M., Lanciotii, R., Vannini, L., Chaves Lòpez, C., 1998. Yarrowia lipolytica as potential ripening agent in milk products. In: Proceedings of IDF Symposium: Yeasts in the Dairy Industry: Positive and Negative Aspects (Copenhagen, 1996), pp. 23–33. Hansen, T.K., 2001. Microbial Interactions in Blue Veined Cheeses. PhD Thesis, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark. Hansen, T.K., Jakobsen, M., 1997. Possible role of microbial interactions for growth and sporulation of Penicillium roqueforti in Danablu. Lait 77, 479–488. Hansen, T.K., Jakobsen, M., 1998. Interactions between Penicillium roqueforti and yeast of importance in the production of blue cheese. In: Proceedings of IDF Symposium: Yeasts in the Dairy Industry: Positive and Negative Aspects (Copenhagen, 1996), pp. 50–58. Hansen, T.K., Jakobsen, M., 2001. Taxonomical and technological characteristics of Saccharomyces spp. associated with blue veined cheese. Int. J. Food Microbiol. 69, 59–68. Hansen, T.K., Cantor, M.D., van den Tempel, T., Jakobsen, M., 2001. Saccharomyces cerevisiae as a starter culture in Mycella. Int. J. Food Microbiol. 69, 101–111. Hayaloglu, A.A., Kirbag, S., 2007. Microbial quality and presence of moulds in Kuflu cheese. Int. J. Food Microbiol. 115, 376–380. Hayaloglu, A.A., Brechany, E.Y., Deegan, K.C., McSweeney, P.L.H., 2008. Characterization of the chemistry, biochemistry and volatile profile of Kuflu cheese, a mould-ripened variety. LWT-Food Sci. Technol. 41, 1323–1334. Hemme, D., Bouillanne, C., Métro, F., Desmazeaud, M.J., 1982. Microbial catabolism of amino acids during cheese ripening. Sci. Alim. 2, 113–123. Hewedi, M.M., Fox, P.F., 1984. Ripening of Blue cheese: characterization of proteolysis. Milchwissenschaft 39, 198–201. Houmard, J., Raymond, M.N., 1979. Further characterization of the Penicillium roqueforti acid protease. Biochimie 61, 979–982. Hymery, N., Vasseur, V., Coton, M., Mounier, J., Jany, J., Barbier, G., Coton, E., 2014. Filamentous fungi and mycotoxins in cheese: a review. Comp. Rev. Food Sci. Food Safety 13, 437–456. Ismail, A.A., Hansen, K., 1972. Accumulation of free amino acids during cheese ripening of some types of Danish cheese. Milchwissenschaft 27, 556–559. Ison, R.W., Gutteridge, C.S., 1987. Determination of the carbonation tolerance of yeasts. Lett. Appl. Microbiol. 5, 11–13. Jakobsen, M., Narvhus, J., 1996. Yeasts and their possible beneficial and negative effects on the quality of dairy products. Int. Dairy J. 6, 755–768.

951

Juszczyk, P., Wojtatowicz, M., Zarowska, B., Chrzanowska, J., Malicki, A., 2005a. Diversity of physiological and biochemical properties within yeast species occurring in Rokpol cheese. Pol. J. Food Nutr. Sci. 14, 257–261. Juszczyk, P., Wojtatowicz, M., Zarowska, B., Chrzanowska, J., Malicki, A., 2005b. Growth and sporulation of Penicillium roqueforti strains in the presence of yeasts selected as co-starters for cheese production. Electr. J. Pol. Agric. Univ. 8 (1), Article no. 10. Kaminarides, S.E., 1986. Quality of Kopanisti cheese. Bull. Greek Nat. Dairy Committee I, 7–13. Kaminarides, S.E., Anifantakis, E.M., 1989. Evolution of the microflora of Kopanisti cheese during ripening. Study of the yeast flora. Lait 69, 537–546. Kaminarides, S.E., Laskos, N.S., 1992. Yeast’s in factory brine of Feta cheese. Aust. J. Dairy Technol. 47, 68–71. Kaminarides, S.E., Anifantakis, E.M., Balis, C., 1992. Changes in Kopanisti cheese during ripening using selected pure microbial cultures. J. Soc. Dairy Technol. 45, 56–60. Karali, F., Georgala, A., Massouras, T., Kaminarides, S., 2013. Volatile compounds and lipolysis levels of Kopanisti, a traditional Greek rawmilk cheese. J. Sci. Food Agric. 93, 1845–1851. Kinsella, J.E., Hwang, D., 1976. Enzymes of Penicillium roqueforti involved in the biosynthesis of cheese flavor. CRC Crit. Rev. Food Sci. Nutr. 8, 191–228. Klein, N., Zourari, A., Lortal, S., 2002. Peptidase activity of four yeast species frequently encountered in dairy products—comparison with several dairy bacteria. Int. Dairy J. 12, 853–861. Kloster, H., 1980. Mælkestrømmens historie og kvalitet. Special-Trykkeriet Viborg A/S. Kokkonen, M., Jestoi, M., Rizzo, A., 2005. Determination of selected mycotoxins in mold cheeses with liquid chromatography coupled to tandem with mass spectrometry. Food Addi. Contam. 22, 449–456. Komprda, T., Dohnal, V., Závodníková, R., 2008. Contents of some biologically active amines in a Czech blue-vein cheese. Czech J. Food Sci. 26, 428–440. Kulmyrzaev, A., Bertrand, D., Dufour, E., 2008. Characterization of different blue cheeses using a custom-design multispectral imager. Dairy Sci. Technol. 88, 537–548. Lafont, P., Siriwardana, M.G., Combemale, I., Lafont, J., 1979. Mycophenolic acid in marketed cheeses. Food Cosmet. Toxicol. 17, 147–149. Lamberet, G., Menassa, A., 1983a. Détermination et niveau des activités lipolytiques dans les fromages à pâte persillée. Lait 63, 333–344. Lamberet, G., Menassa, A., 1983b. Purification and properties of an acid lipase from Penicillium roqueforti. J. Dairy Res. 50, 459–468. Larsen, T.O., Frisvad, J.C., 1995a. Characterization of volatile metabolites from 47 Penicillium taxa. Mycol. Res. 99, 1153–1166. Larsen, T.O., Frisvad, J.C., 1995b. Chemosystematics of Penicillium based on profiles of volatile metabolites. Mycol. Res. 99, 1167–1174. Larsen, M.D., Jensen, K., 1999. The effects of environmental conditions on the lipolytic activity of strains of Penicillium roqueforti. Int. J. Food Microbiol. 46, 159–166. Larsen, M.D., Kristiansen, K.R., Hansen, T.K., 1998. Characterization of the proteolytic activity of starter cultures of Penicillium roqueforti for production of blue veined cheeses. Int. J. Food Microbiol. 43, 215–221. Lawlor, J.B., Delahunty, C.M., Sheehan, J., Wilkinson, M.G., 2003. Relationships between sensory attributes and the volatile compounds, nonvolatile and gross compositional constituents of six blue-type cheeses. Int. Dairy J. 13, 481–494.

952 S ECTION | II  Diversity of Cheese

Le Bars, D., Gripon, J.C., 1981. Role of Penicillium roqueforti proteinases during blue cheese ripening. J. Dairy Res. 48, 479–487. Le Dréan, G., Mounier, J., Vasseur, V., Arzur, D., Habrylo, O., Barbier, G., 2010. Quantification of Penicillium camemberti and P. roqueforti mycelium by real-time PCR to assess their growth dynamics during ripening cheese. Int. J. Food Microbiol. 138, 100–107. Leclercq-Perlat, M.-N., Corrieu, G., Spinnler, H.-E., 2004. Comparision of volatile compounds produced in model cheese medium deacidification by Debaryomyces hansenii or Kluyveromyces marxianus. Am. Dairy Sci. Assoc. 87, 1545–1550. Lecocq, J., Gueguen, M., 1994. Effects of pH and sodium chloride on the interactions between Geotrichum candidum and Brevibacterium linens. J. Dairy Sci. 77, 2890–2899. Lenoir, J., 1984. The surface flora and its role in the ripening of cheese. Bulletin 171, International Dairy Federation, Brussels, pp. 3–20. López-Díaz, T.M., Alonso, C., Roman, C., García-López, M.L., Moreno, B., 2000. Lactic acid bacteria isolated from a hand-made blue cheese. Food Microbiol. 17, 23–32. Lopéz-Díaz, T.M., Santos, J., Prieto, M., García-López, M.L., Otero, A., 1995. Mycoflora of a traditional Spanish blue cheese. Neth. Milk Dairy J. 49, 191–199. Lopéz-Díaz, T.M., Román-Blanco, C., García-Arias, M.T., GarcíaFernández, M.C., García-López, M.L., 1996a. Mycotoxins in two Spanish cheese varieties. Int. J. Food Microbiol. 30, 391–395. Lopéz-Díaz, T.M., Santos, J., Otero, A., García, M.L., Moreno, B., 1996b. Some technological properties of Penicillium roqueforti strains isolated from a home-made blue cheese. Lett. Appl. Microbiol. 23, 5–8. Lumsden, W.B., Duffus, J.H., Slaughter, J.C., 1986. Effects of CO2 on budding and fission yeasts. J. Gen. Microbiol. 133, 877–881. Lund, F., Frisvad, J.C., Filtenborg, O., 1995. Associated mycoflora of cheese. Food Microbiol. 12, 173–180. Lund, F., Filtenborg, O., Frisvad, J.C., 1998. Penicillium caseifulvum, a new species found on fermented Blue cheese. J. Food Mycol. 1, 95–101. Madkor, S., Fox, P.F., Shalabi, S.I., Metwalli, N.H., 1987a. Studies on the ripening of Stilton cheese: proteolysis. Food Chem. 25, 13–29. Madkor, S., Fox, P.F., Shalabi, S.I., Metwalli, N.H., 1987b. Studies on the ripening of Stilton cheese: lipolysis. Food Chem. 25, 93–109. Marcos, A., 1993. Water activity in cheese in relation to composition, stability and safety. In: Fox, P.F. (Ed.), Cheese: Chemistry, Physics and Microbiology. Vol. 1. General Aspects. second ed. Chapman & Hall, London, pp. 439–470. Marcos, A., Esteban, M.A., León, F., Fernández-Salguero, J., 1979. Electrophoretic patterns of European cheeses: comparison and quantitation. J. Dairy Sci. 62, 892–900. Marcos, A., Millan, R., Esteban, M.A., Alcala, M., Fernández-Salguero, J., 1983. Chemical composition and water activity of Spanish cheeses. J. Dairy Sci. 66, 2488–2493. Martley, F.G., Crow, V.L., 1993. Interactions between non-starter microorganisms during cheese manufacture and ripening. Int. Dairy J. 3, 461–483. Mase, T., Matsumiya, Y., Matsuura, A., 1995. Purification and characterization of Penicillium roqueforti IAM 7268 lipase. Biosci. Biotechnol. Biochem. 59, 329–330. Masui, K., Yamada, T., 1996. In: Moore, A. (Ed.), French cheeses. Dorling Kindersley, London. McSweeney, P.L.H., Sousa, M.J., 2000. Biochemical pathways for the production of flavour compounds in cheeses during ripening: a review. Lait 80, 293–324.

Medina, M., Gaya, P., Nuñez, M., 1985. Production of PR toxin and roquefortine by Penicillium roqueforti isolates from Cabrales blue cheese. J. Food Prot. 48, 118–121. Menassa, A., Lamberet, G., 1982. Contribution à l’étude du système lipolytique de Penicillium roqueforti. Caractères comparés de deux activités exocellulaires. Lait 62, 32–43. Meyers, S.A., Cuppett, S.L., Hutkins, R.W., 1996. Lipase production by lactic acid bacteria and activity on butter oil. Food Microbiol. 13, 383–389. Modler, H.W., Brunner, J.R., Stine, C.M., 1974. Extracellular protease of Penicillium roquefort. II. Characterization of a purified enzyme preparation. J. Dairy Sci. 57, 528–534. Moio, L., Piombino, P., Addeo, F., 2000. Odour-impact compounds of Gorgonzola cheese. J. Dairy Res. 67, 273–285. Molimard, P., Spinnler, H.E., 1996. Review: compounds involved in the flavor of surface mold-ripened cheeses: origins and properties. J. Dairy Sci. 79, 169–184. Moreau, S., Lablache-Combier, A., Biguet, J., 1980. Production of eremofortins A, B and C relative to formation of PR toxin by Penicillium roqueforti. Appl. Environ. Microbiol. 39, 770–776. Mounier, J., Monnet, C., Vallaeys, T., Arditi, R., Sarthhou, A.-S., Hélias, A., Irlinger, F., 2008. Microbial Interactions within a Cheese Microbial Community. Appl. Environ. Microbiol. 74, 172–181. Muir, D.D., Hunter, E.A., Watson, M., 1995. Aroma of cheese. 1. Sensory characterisation. Milchwissenschaft 50, 499–503. Ney, K.H., Wirotama, I.P.G., 1972. Untersuchung von Edelpilzkäse-aroma. Z. Lebensm. Unters. Forsch. 149, 275–279. Nichol, A.W., Harden, T.J., 1993. Enzymatic browning in mould-ripened cheeses. Aust. J. Dairy Technol. 48, 71–73. Nielsen, M.S., Frisvad, J.C., Nielsen, P.V., 1998. Colony interaction and secondary metabolite production of cheese-related fungi in dual culture. J. Food Prot. 61, 1023–1029. Nielsen, K.F., Dalsgaard, P.W., Smedsgaard, J., Larsen, T.O., Andrastins, A.-D., 2005. Penicillium roqueforti metabolites consistently produced in blue-mold-ripened cheese. J. Agric. Food Chem. 53, 2908–2913. Niki, T., Yoshioka, Y., Ahiko, K., 1966. Proteolytic and lipolytic activities of Penicillium roqueforti isolated from Blue cheese. In: Proc. XVII Int. Dairy Congr., Munich, vol. D:2, pp. 531–537. Nishimura, T., Kato, H., 1988. Taste of free amino acids and peptides. Food Rev. Int. 4, 175–194. Nuñez, M., 1978. Microflora of Cabrales cheese: changes during maturation. J. Dairy Res. 45, 501–508. Nuñez, M., Medina, M., 1979. La flore lactique du fromage bleu de Cabrales. Lait 59, 497–513. Nuñez, M., Medina, M., Gaya, P., Dias-Amado, C., 1981. Les levures et les moisissures dans le fromage Bleu de Cabrales. Lait 61, 62–79. Ordonez, J.A., Masso, J.A., Marmol, M.P., Ramos, M., 1980. Contribution à l’étude du fromage “Roncal”. Lait 60, 283–294. Orth, R., 1976. PR-toxinbildung bei Penicillium roqueforti-Stämmen. Z. Lebensm. Unters. Forsch. 160, 131–136. Ottogalli, G., Bianchi-Salvadori, B., Galli, A., Cecchi, L., Resmini, P., Volonterio, G., 1974. Etudes chimiques et microbiologiques sur la maturation de fromages persillés. Influence de la flore microbienne selon différentes technologies. Lait 54, 656–674. Pereira-Dias, S., Potes, M.E., Marinho, A., Malfeito-Ferreira, M., Loureiro, V., 2000. Characterisation of yeast flora isolated from artisanal Portuguese ewe’s cheese. Int. J. Food Microbiol. 60, 55–63. Petyaev, I.M., Bashmakov, Y.K., 2012. Could cheese be the missing piece in the French paradox puzzle? Med. Hypotheses 79, 746–749.

Blue Cheese Chapter | 37

Philip, S., 1985. Special cultures—significance and application in cheese factories. Dtsch. Molkerei Zeitung 106, 1706–1710. Pitt, J.I., 1979. The Genus Penicillium and Its Teleomorphic States Eupencillium and Talaromyces. Academic Press, Inc, New York, pp. 634. Pitt, J.I., Hocking, A.D., 1997. Fungi and Food Spoilage, second ed. Blackie Academic and Professional, London. Pose, G., Ludemann, V., Gomez, A., Segura, J., 2007. Comparison of growth characteristics and roquefortin C production of Penicillium roqueforti from blue-veined cheese. Mycotoxin Res. 23, 122–126. Price, J.E., Linforth, R.S.T., Dodd, C.E.R., Phillips, C.A., Hewson, L., Hort, J., Gkatzionis, K., 2014. Study of the influence of yeast inoculum concentration (Yarrowia lipolytica and Kluyveromyces lactis) on blue cheese aroma development using microbiological models. Food Chem. 145, 464–472. Prieto, B., Urdiales, R., Franco, I., Tornadijo, M.E., Fresno, J.M., Carballo, J., 1999. Biochemical changes in Picón Bejes-Tresviso cheese, a Spanish blue-veined variety, during ripening. Food Chem. 67, 415–421. Prieto, B., Franco, I., Fresno, J.M., Bernardo, A., Carballo, J., 2000. Picón Bejes-Tresviso blue cheese: an overall biochemical survey throughout the ripening process. Int. Dairy J. 10, 159–167. Roostita, R., Fleet, G.H., 1996a. The occurrence and growth of yeasts in Camembert and blue-veined cheeses. Int. J. Food Microbiol. 28, 393–404. Roostita, R., Fleet, G.H., 1996b. Growth of yeast’s in milk and associated changes to milk composition. Int. J. Food Microbiol. 31, 205–219. Rothe, M., Engst, W., Erhardt, V., 1982. Studies on characterization of Blue cheese flavour. Die Nahrung 26, 591–602. Rothe, M., Ruttloff, H., Herrmann, H., Engst, W., 1986. Problems of technical production and characterization of Blue cheese aroma concentrate. Die Nahrung 30, 791–797. Rothe, M., Kornelson, C., Schröder, R., 1994. Key components of food flavour: a sensory study on Blue cheese flavor. In: Maarse, H., van der Heij, D.G. (Eds.), Trends in Flavour Research. Elsevier Science B.V., London, pp. 221–232. Roy, U., Batish, V.K., Grover, S., Neelakantan, S., 1996. Production of antifungal substance by Lactococcus lactis subsp. lactis CHD-28.3. Int. J. Food Microbiol. 32, 27–34. Sablé, S., Cottenceau, G., 1999. Current knowledge of soft cheeses flavor and related compounds. J. Agric. Food Chem. 47, 4825–4836. Salvadori, P., Bianche-Savadori, B., Gower, G., 1974. Caratterstiche fisiologiche e culturali di associazoni microbiche costitute lattici termofili “Penicillium roqueforti”. Industria del Latte 10, 23–37. Samson, R.A., Eckardt, C., Orth, R., 1977. The taxonomy of Penicillum species from fermented cheeses. Antonie Van Leeuwenhoek 43, 341–350. Samson, R.A., Hoekstra, E.S., Frisvad, J.C., Filtenborg, O., 1995. Introduction to Food-Borne Fungi, fourth ed. Centraalbureau voor Schimmelcultures, Baarn, The Netherlands. Sánchez-Rivera, L., Diezhandino, I., Gomez-Ruiz, J.A., Fresno, J.A., Miralles, B., Recio, I., 2014. Peptidomic study of Spanish blue cheese (Valdeon) and changes after simulated gastrointestinal digestion. Electrophoresis 35, 1627–1636. Schmidt, J.L., Lenoir, J., 1980a. Contribution à l’etude de la flore levure du fromage de Camembert. Lait 60, 272–282. Schmidt, J.L., Lenoir, J., 1980b. Contribution à l’étude des apitudes biochimiques de levures isolées du fromage de Camembert. II. Essais et interprétations complémentaires. Lait 60, 343–350. Schoch, U., Lüthy, J., Schlatter, C., 1984. Mykotoxine von P. roqueforti und P. camemberti in Käse. I. Vorkommen chemisch identifizierter Mykotoxine. Milchwissenschaft 39, 76–80.

953

Scott, P.M., 1981. Toxins of Penicillium species used in cheese manufacture. J. Food Prot. 44, 702–710. Scott, P.M., Kanhere, S.R., 1979. Instability of PR toxin in blue cheese. J. Assoc. Off. Anal. Chem. 62, 141–147. Scott, P.M., Kennedy, B.P.C., 1976. Analysis of blue cheese for roquefortine and other alkaloids from Penicillium roqueforti. J. Agric. Food Chem. 24, 865–868. Scott, P.M., Merrien, M.A., Polonsky, J., 1976. Roquefortine and isofumigaclavine A, metabolites from Penicillium roqueforti. Experientia 32, 140–142. Seiler, H., Busse, M., 1990. The yeasts of cheese brines. Int. J. Food Microbiol. 11, 289–304. Sensidoni, A., Rondinini, G., Peressini, D., Maifreni, M., Bortolomeazzi, R., 1994. Presence of an off-flavour associated with the use of sorbates in cheese and margarine. Italian J. Food Sci. 6, 237–242. Seratlic, S.V., Miloradovic, Z.N., Radulovic, Z., Macej, O.D., 2011. The effect of two types of mould inoculants on the microbiological composition, physicochemical properties and protein hydrolysis in two Gorgonzola-type cheese varieties during ripening. Int. J. Dairy Technol. 64, 408–416. Siemens, K., Zawistowski, J., 1993. Occurrence of PR imine, a metabolite of Penicillium roqueforti in blue cheese. J. Food Prot. 56, 317–319. Sørensen, L.M., Gori, K., Petersen, M.A., Jespersen, L., Arneborg, N., 2011. Flavor compound production by Yarrowia lipolytica, Saccharomyces cerevisiae and Debaryomyces hansenii in a cheese-surface model. Int. Dairy J. 21, 970–978. Stepaniak, L., Kornacki, K., Grabska, J., Rymaszewski, J., Cichosz, G., 1980. Lipolytic and proteolytic activity of Penicillium roqueforti, Penicillium candidum and Penicillium camemberti strains. Acta Alim. Polon. 6, 155–164. Stolk, A.C., Samson, R.A., Frisvad, J.C., Filtenborg, O., 1990. The systematics of the terverticillate penicilla. In: Samson, R.A., Pitt, J.I. (Eds.), Modern Concepts in Penicillium and Aspergillus Classification. Plenum Press, New York, pp. 121–137. Suzuki, I., Nomura, M., Morichi, T., 1991. Isolation of lactic acid bacteria which suppress mold growth and show antifungal action. Milchwissenshaft 46, 635–639. Taniwaki, M.H., 1995. Growth and Mycotoxin Production by Fungi under Modified Atmospheres. PhD Thesis, University of New South Wales, Kensington, NSW. Taniwaki, M.H., Hocking, A.D., Pitt, J.I., Fleet, G.H., 2001. Growth of fungi and mycotoxin production on cheese under modified atmospheres. Int. J. Food Microbiol. 68, 125–133. Tariq, V.N., Campell, M., 1991. Influence of volatile metabolites from Geotrichum candidum on other fungi. Mycol. Res. 95, 891–893. Thom, C., Currie, J., 1913. The dominance of Roquefort mould in cheese. J. Biol. Chem. 15, 249–258. Trieu-Cuot, P., Gripon, J.C., 1983. Etude électrophorétique de la protéolyse au cours de l’affinage des fromages à pâte persillée du type bleu d’Auvergne. Lait 63, 116–128. Trieu-Cuot, P., Archieri-Haze, M.J., Gripon, J.C., 1982a. Effect of aspartyl proteinases of Penicillium caseicolum and Penicillium roqueforti on caseins. J. Dairy Res. 49, 487–500. Trieu-Cuot, P., Archieri-Haze, M.-J., Gripon, J.-C., 1982b. Etude comparative de l’action des métalloprotéases de Penicillium caseicolum et Penicillium roqueforti sur les caséines alpha s1 et bêta. Lait 62, 234–249. Trihaas, J. (2005). Implementation of Electronic Nose Technology in Quality Control of Mould Ripened Danish Cheese. PhD Thesis, BioCentrum-DTU, Technical University of Denmark.

954 S ECTION | II  Diversity of Cheese

Tudor, D.A., Board, R.G., 1993. Food spoilage yeast. In: Rose, A.H., Harrison, J.S. (Eds.), The Yeasts. Vol. 5. Yeasts Technology. second ed. Academic Press, pp. 436–516. Tzanetakis, N., Hatzikamari, M., Litopoulou-Tzanetaki, E., 1998. Yeasts of the surface microflora of Feta cheese. In: Proceedings of IDF Symposium: Yeasts in the Dairy Industry: Positive and Negative Aspects (Copenhagen 1996), pp. 34–43. Valerio, F., Favilla, M., De Bellis, P., Sisto, A., De Candia, S., Lavermicocca, P., 2009. Antifungal activity of strains of lactic acid bacteria isolated from a semolina ecosystem against Penicillium roqueforti, Aspergillus niger and Endomyces fibuliger contaminating bakery products. Syst. Appl. Microbiol. 32, 438–448. Valik, L., Baranyi, J., Görner, F., 1998. Predicting fungal growth: the effect of water activity on Penicillium roqueforti. Int. J. Food Microbiol. 47, 141–146. van den Tempel, T., 2000. The Role of Yeast in the Ripening of Blue Mould Cheese. PhD Thesis, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark. van den Tempel, T., Jakobsen, M., 1998. Yeasts associated with Danablu. Int. Dairy J. 8, 25–31. van den Tempel, T., Jakobsen, M., 2000. The technological characteristics of Debaryomyces hansenii and Yarrowia lipolytica and their potential as starter cultures for production of Danablu. Int. Dairy J. 10, 263–270. van den Tempel, T., Nielsen, M.S., 2000. Effects of atmospheric conditions, NaCl and pH on growth and interactions between moulds and yeasts related to blue cheese production. Int. J. Food Microbiol. 57, 193–199. van den Tempel, T., Gundersen, J.K., Nielsen, M.S., 2002. The microdistribution of oxygen in Danablu cheese measured by a microsensor during ripening. Int. J. Food Microbiol. 75, 157–161. Vasdinyei, R., Deak, T., 2003. Characterization of yeast isolates originating from Hungarian dairy products using traditional and molecular identification techniques. Int. J. Food Microbiol. 86, 123–130. Viljoen, B.C., Greyling, T., 1995. Yeast’s Associated with Cheddar and Gouda Making. Int. J. Food Microbiol. 28, 79–88.

Viljoen, B.C., Knox, A.M., De Jager, P.H., Laurens-Hattingh, A., 2003. Development of Yeast Populations during Processing and Ripening of Blue Veined Cheese. Food Technol. Biotechnol. 41, 291–297. Visagie, C.M., Houbraken, J., Frisvad, J.C., Hong, S.-B., Klaassen, C.H.W., Perrone, G., Seifert, K.A., Varga, J., Yaguchi, T., Samson, R.A., 2014. Identification and nomenclature of the genus Penicillium. Stud. Mycol. 78, 343–371. Vivier, D., Rivemale, M., Reverbel, J.P., Ratomahenina, R., Galzy, P., 1992. Some observations on the physiology of Penicillium roqueforti Thom and Penicillium cyclopium Westling. Lait 72, 277–283. Ware, G.M., Thorpe, C.W., Pohland, A.E., 1980. Determination of roquefortine in blue cheese and blue cheese dressing by high pressure liquid chromatography with ultraviolet and electrochemical detectors. J. Assoc. Off. Anal. Chem. 63, 637–641. Wei, R.D., Liu, G.X., 1978. PR toxin production in different Penicillium roqueforti strains. Appl. Environ. Microbiol. 35, 797–799. Weichhold, U., Seiler, H., Busse, M., Klostermeyer, H., 1988. Rotverfärbungen bei Käse und deren Ursachen. Deutsche Milchwirkschaft 46, 1671–1765. Williams, A.G., Withers, S.E., 2007. Tyrosine metabolism in pigmentforming Yarrowia lipolytica strains isolated from English and European speciality mould-ripened cheese exhibiting a brown discolouration defect. Int. J. Dairy Technol. 60, 165–174. Wolf, I.V., Perotti, M.C., Zalazar, C.A., 2011. Composition and volatile profiles of commercial Argentinean blue cheeses. J. Sci. Food. Agric. 91, 385–393. Yvon, M., Rijnen, L., 2001. Cheese flavour formation by amino acid catabolism. Int. Dairy J. 11, 185–201. Zarmpoutis, I.V., McSweeney, P.L.H., Beechinor, J., Fox, P.F., 1996. Proteolysis in the Irish farmhouse Blue cheese, Chetwynd. Irish J. Agric. Food Res. 35, 25–36. Zarmpoutis, I.V., McSweeney, P.L.H., Fox, P.F., 1997. Proteolysis in blue-veined cheeses: an intervarietal study. Irish J. Agric. Food Res. 36, 219–229.