Yeasts and Molds: Penicillium camemberti

SA Jackson and ADW Dobson, University College Cork, Cork, Ireland

Ó 2016 Elsevier Inc. All rights reserved.

Introduction 1
Growth Characteristics of Penicillium camemberti 1
Enzymes Produced by Penicillium camemberti 2
Penicillium camemberti as a Biocontrol Agent in Cheese 2
Secondary Metabolism in Penicillium camemberti 2
Penicillium camemberti and Cheese Flavor 2
Types of Cheese Involving Penicillium camemberti 3
Camembert Cheese 3
Brie Cheese 3
Penicillium camemberti in Other Food-Related Applications 4
Advanced Methods for the Identification of Penicillium camemberti 4
Genetics 4
Further Reading 4
Change History 4

Introduction

Penicillium camemberti was first described by Thom and is thought to be a domesticated form of Penicillium commune. A number of
synonyms exist for the species including Penicillium rogeri, Penicillium candidum, Penicillium album, and Penicillium caseicolum. The
fungus is mainly (almost exclusively) found either on cheese or in the cheese factory environment and is rarely found away
from this environment. Penicillium camemberti is used in the production of Camembert and Brie cheeses, on which colonies of
the fungus form a white crust. It is also used as a starter culture for fermented meat products and is often found as a spontaneous
colonizer of fermented sausages originating from the mycobiota of the production facility. There have been reports on the wider
exploitation of P. camemberti especially in the decontamination of softwood bleach effluents, which contain high levels of ecolog-
ically undesirable phenolic and chlorinated phenolic compounds. Therefore, P. camemberti, which to date has been predominantly
used in the dairy industry, may also find future utility in other nondairy-related areas.

Growth Characteristics of Penicillium camemberti

Colony diameter on Czapek yeast extract agar (CYA) is typically 19–27 mm at room temperature (24–26
C) after 10 days. Colonies
on CYA appear yellow/orange or green to fawn to pale brown/blue in color. The reverse sides of the colonies on CYA typically
appear either yellow/orange or green brown in color. Colony diameter on malt extract agar (MEA) is typically 12–27 mm. Colonies
on MEA also appear yellow/orange or green in color, with the reverse sides of the colonies being red, olive, green, or brown in color
depending on the growth medium. The optimum growth temperature range is 20–25
C, with growth being recorded at 5
C but
not at 37
C. With respect to pH, growth can take place in the pH range of 3.5–6.5. Penicillium camemberti has similar water activity
(aw) limits for growth as Penicillium roqueforti with an optimum aw value of 0.998 for growth at 25
C and an ability to grow in the aw
range from 0.91 to 0.94. Thus, from a pH standpoint, P. camemberti is ideal as a starter culture given that the pH of Camembert and
related types of cheese reaches about 4.6 during the first 24 h and eventually following maturation increases to around 5.5 in the
center of the cheese and around 7.0 in the outer part of the cheese. Similarly, it is suitable as a starter culture from an aw standpoint
given that the aw of the surface and center of Camembert cheese has been recorded as 0.93 and 0.97, respectively. The salt tolerance
of the fungus coupled with its ability to grow at an aw of 0.93 results in it growing on the surface of Camembert cheese during the
cheese maturation process. However, it is only after 1 week of ripening that P. camemberti is observed, and within 2–3 weeks it covers
the entire surface of the cheese. During this process, the fungus also metabolizes lactate to CO2 and H2O at the surface of the cheese
to establish a pH gradient, which is a key factor in the maturation process, and results in a higher pH. This effect is pronounced on
the surface of the cheese, with a pH gradient becoming established toward the center of the cheese, resulting in lactate migrating
toward the surface, where it is assimilated as a carbon source by the fungus. This depletion of lactate in the center of the cheese
results in casein being degraded primarily by enzymes from the rennet, the plasmin from the milk, and by enzymes from the lactic
acid starter cultures. Ammonia is formed at the surface of the cheese from amino acids, the consequence of which is a further
increase in pH. The proteinases from P. camemberti are activated by the increasing pH and they migrate only slowly into the cheese.
During ripening, the dynamics of sporulation of P. camemberti is affected by the concentration of CO2 in the atmosphere. For
example, the number of P. camemberti spores present in the rind is fairly constant at around 104 CFU g1 during the first 6 days
of ripening at 6% CO2. However, at 2% CO2, the fungus is known to sporulate at a faster rate and spore counts can reach levels

Reference Module in Food Sciences http://dx.doi.org/10.1016/B978-0-08-100596-5.01091-X 1

2 Yeasts and Molds: Penicillium camemberti

as high as 106 CFU g1 on the sixth day of growth. After day 11 and until day 40, sporulation remains stationary, close to
106 CFU g1.
Regardless of CO2 concentration, the mycelium of P. camemberti begins to grow from day 4 onward with both mycelium and
aerial mycelia being visible. Between days 5 and 12, the mycelia grow and uniformly cover the entire cheese surface. From day
10 to 16, if the cheese is wrapped, the rind color remains white and is around 3 mm thick. Increases in CO2 concentration above
2% negatively affect the growth of P. camemberti on cheese. Because in Camembert-type cheese, P. camemberti is generally inoculated
in a mixed culture with Geotrichum candidum, CO2 is known to alter the equilibrium between these two strains, with higher CO2
concentrations favoring G. candidum and resulting in poorer development of P. camemberti mycelium.
The degree of water permeability in cheese wrapping films has been reported to have a marked effect on dynamics of cheese
ripening in Camembert cheeses. The degree of water permeability within the wrapped cheeses appears to be a key factor in controlling
the cheese ripening progress, particularly with respect to increases in the pH of the cheese core and in thickness of the underrind. Water
losses from around 3 to 6% over a 23-day ripening period appear optimal with respect to good ripening dynamics and cheese quality.

Enzymes Produced by Penicillium camemberti

Penicillium camemberti produces a variety of different proteinases including two extracellular endopeptidases. One of the two extra-
cellular endopeptidases is a metalloprotease and is the principal proteolytic enzyme active at close to neutral pH values (pH 6.5). It
is similar to the metalloprotease produced by P. roqueforti. At acidic pH (pH 4.0), P. camemberti produces an acid protease. Other
proteolytic enzymes produced by P. camemberti include an aminopeptidase and a carboxypeptidase. These proteolytic enzymes play
an important role in cheese ripening. There are some differences between strains with respect to the production of different types of
proteinases. There is, however, greater variation between P. camemberti strains in their ability to produce extracellular lipolytic
enzymes. The lipase system is active within broad pH (5.5–9.5) and temperature (1–35
C) ranges.

Penicillium camemberti as a Biocontrol Agent in Cheese

Starter cultures are known to contribute to the inhibition of the undesirable growth of fungal contaminants and mycotoxin produc-
tion in fermented foods. When P. camemberti is used as a secondary starter culture, it exerts a powerful inhibitory effect on many
common cheese contaminants such as Cladosporium herbarum, P. roqueforti, Penicillium caseifulvum, and P. commune. The antagonistic
power of P. camemberti is strain dependent in that the growth inhibition of C. herbarum is not affected by the choice of the strain of
P. camemberti, whereas the Penicillium contaminants are very sensitive to the choice of strain. The antagonistic activity is much
stronger when P. camemberti is used as a pure culture, with the inhibitory activity being reduced considerably if the fungus is
used in a mixed culture, for example with G. candidum.

Secondary Metabolism in Penicillium camemberti

A number of Penicillium toxins have been identified in contaminated cheese, including roquefortin C, isofumigaclavine A, cyclopiazonic
acid (CPA), mycophenolic acid, and, much less frequently, ochratoxin A and PR toxin. A few strains of P. camemberti are known to
produce a number of secondary metabolites such as cyclopaldic acid, rugulovasine A, and rugulovasine B as well as palitantin. However,
CPA, which is a neurotoxic and immunosuppressive compound, remains the most significant toxin produced by P. camemberti, partic-
ularly at higher storage temperatures. The toxicity of CPA results in a large part from its specific inhibition of calcium-dependent ATPase
in the sarcoplasmic reticulum, leading to altered cellular (Ca2þ) levels and resulting in increased muscle contractions.
CPA is almost exclusively found in the rind but not in the core of the cheese. This is due to the inability of P. camemberti to grow
in the cheese core. CPA does not appear to constitute a major threat to the consumer with the highest levels in cheese being reported
to date at >2 ppm, which would constitute <4 mg CPA in a portion of the most highly contaminated cheeses. It has also been re-
ported that CPA can be produced by P. camemberti in submerged culture, at levels <4 ppm following a 96 h fermentation. Produc-
tion of CPA is known to be strain specific and appears to be unrelated to spore yield.
No noticeable mutagenic activity was detected when crude extracts of several strains of P. camemberti containing a pool of metab-
olites were assessed, suggesting that undesired long-term effects from the consumption of P. camemberti-ripened cheese are unlikely.
This may explain the fact that despite many P. camemberti strains possessing the ability to produce CPA, no acute toxicity associated
with the consumption of food produced by the fungus has been reported to date. The possibility exists that this may also be due to
the fact that many other metabolites are likely to be produced at the same time as CPA and that these metabolites may in some way
have an antagonistic effect that could neutralize or negate the toxicity of CPA. However, no clear scientific evidence exists to support
this hypothesis and further work is required to substantiate this theory.

Penicillium camemberti and Cheese Flavor

The production of numerous flavor compounds in Camembert cheese can be directly attributed to the enzymatic activity of
P. camemberti. While the presence of the fungus at the surface of the cheese gives the cheeses their characteristic appearance, it
 Yeasts and Molds: Penicillium camemberti 3

is known that low molecular weight compounds produced by the fungus contribute significantly to taste. Volatile
compounds are an important component of these low molecular weight molecules, with volatile fatty acids being the
most abundant compounds within the volatile fraction. In fact, lipolysis is particularly important in soft cheeses such as
Camembert where free fatty acids can reach up to 10% of the total fatty acids present. As previously mentioned,
P. camemberti produces a lipase, which is similar to the alkaline lipase produced by P. roqueforti, and proteases. Lipase
and protease activities are involved in the degradation of short-chain fatty acids and peptides in the cheese. The resulting
products are subsequently transformed into important taste and aroma compounds, such as ammonia, methyl ketones,
primary and secondary alcohols, esters, aldehydes, lactones, and sulfur compounds. The methyl ketones are by far the
most abundant neutral compounds in the volatile fraction of mold-ripened cheeses. Such methyl ketones include
2-nonanone, 2-undecanone, 2-heptanone, and 2-pentanone, and their corresponding secondary alcohols, and contribute
to the musty flavor of the cheese. Due to their typical odors and their low odor thresholds as well as their concentration
in cheese, ketones and methyl ketones play a key role in the overall flavor of surface mold-ripened cheeses. The secondary
alcohol 1-octen-3-ol, in particular, is responsible for the characteristic mushroom flavor of Camembert-type cheese. Most
esters have floral and fruity notes and are believed to contribute to the overall aroma by minimizing the sharpness and
bitterness imparted by fatty acids and amines, respectively.
In work by the Corrieu group interactions between temperature and relative humidity (RH) have been shown to play an
important role in P. camemberti sporulation, carbon substrate consumption rates, and thickening of the cheese underrind. At
98% RH, the specific growth rate of P. camemberti spores was up to between 2 (8
C) and 106 (16
C) times higher. While the
best ripening conditions to achieve an optimum between growth and biochemical kinetics were shown to be 13
C and
94% RH. In more recent work by this group where the effect of processing parameters on the appearance, odor and rind
color, creamy underrind thickness and consistency, and core hardness was assessed they reported that the best ripening
conditions to achieve an optimum balance between these cheese sensory qualities and marketability were 16  1
C and
94 þ 1% RH.

Types of Cheese Involving Penicillium camemberti

Camembert Cheese
In the traditional manufacture of the surface mold-ripened Camembert cheese, whole pasteurized milk is warmed to 29–33
C and
ripened using a lactic acid bacteria starter culture. The high acidity of the milk assists in whey drainage and suppresses the growth of
undesirable organisms. Coagulating enzyme is added to allow the formation of a firm curd within a 1–2 h period. The resulting curd
is then dipped into small, perforated forms and allowed to drain for 1–2 days, with frequent turning. The cheese is then removed
and salted, and is typically inoculated with a culture containing both mold and bacteria. The curing of Camembert cheese is quite
a complex process and involves not only the uniform and progressive development of certain ripening agents, but also the gradual
drying out of the curd. To help achieve this, the curing rooms are usually maintained at temperatures of around 13
C and at a RH of
90%. The creamy, semiliquid interior consistency characteristics of Camembert are largely due to the activity of P. camemberti. The
mold can be mixed with the milk, sprinkled on the curd, or rubbed on the cheese along with salt. After 2 weeks, the primary surface
of mold growth forms a thin, gray-white, feltlike rind but does not penetrate the cheese. The cheese is then wrapped in parchment
and foil, and boxed. The cheese is regarded as being in prime condition after a 4- to 5-week-period at which time it should be
consumed.
Ammonia, which has a low odor threshold (5 mmol kg1), is associated with a ripened aroma when its concentration is
within the accepted limit. However, overripened Camembert cheeses can develop a strong ammonia odor as a result of the intense
deamination activity of P. camemberti. Thus, a pronounced aroma of ammonia is indicative of overripening of Camembert. Flavor
defects characterized by a typical celluloid flavor originating from the production of styrene by the mold sometimes appear
during ripening or storage of mold-ripened cheese. It has recently been shown that P. camemberti can produce styrene from
phenylalanine by phenylalanine ammonia lyase activity followed by a decarboxylation reaction catalyzed by a cinnamic acid
decarboxylase.

Brie Cheese
Brie, a cheese that is surface ripened by mold, is very similar to Camembert. Differences exist, however, in the internal ripening
and in the characteristic flavor and aroma of the cheeses. The traditional manufacture of brie cheese involved initially warming
the milk to 32
C and then adding coagulating enzyme to initiate curd development within 2–3 h. The curd is then dipped into
small forms and hoops and allowed to drain for about 24 h. The hoops are removed, and the cheese is turned and dry-salted.
Initial ripening for about 8 days occurs in a well-ventilated drying room maintained at 13–16
C. During this time, the curd
softens rapidly and becomes slightly yellow and translucent in color, and a feltlike layer of white mold appears on the surface.
The cheese is then moved to a dark, moist room or cellar that is maintained at 11
C and at a RH of 85% for 2–4 weeks. The
initial white mold layer formed by P. camemberti eventually changes to a yellow color and is subsequently overgrown with
gram-positive organisms similar to those found on smear-ripened cheese appearing red in color. The cheese becomes less acidic
and the curd is yellow and creamy. The surface growth of both P. camemberti and smear organisms during ripening is respon-
sible for the characteristic flavor of brie. Like Camembert, brie ripens rapidly, is perishable, and must be consumed soon after
ripening.
4 Yeasts and Molds: Penicillium camemberti

Penicillium camemberti in Other Food-Related Applications

Penicillium camemberti has successfully been used to improve the quality of sausage meat, through the superficial inoculation and
growth of an atoxigenic strain of P. camemberti on the surface of the sausage. This has been reported to result in strong proteolysis
and lipolysis, which produce an intense increase in the diglyceride, monoglyceride, phospholipid, and free fatty acid concentrations,
and in volatile compounds, and a corresponding decrease in triglyceride levels. Compounds such as branched aldehydes and the
corresponding alcohols, acids, and esters, derived from the catabolism of amino acids, are responsible for the ripened flavor. The
development of the fungal mycelia on the surface of the sausages not only protects lipids from oxidation, resulting in lower
2-thiobarbituric acid values and lipid oxidation-derived compounds, such as aliphatic aldehydes and alcohols, but also completely
eliminates the growth of undesirable naturally occurring mold contaminants. Thus, the use of P. camemberti results not only in the
protection of the sausage from fungal contamination but also in an improvement in the odor and flavor of the sausage.

Advanced Methods for the Identification of Penicillium camemberti

Numerous industries are currently employing electronic nose (e-nose)-based detection systems to monitor food quality control,
storage, and spoilage by both bacteria and fungi. E-nose involves an analysis of the chemicals contained in an extract using gas chro-
matography coupled to mass spectrometry (GC–MS) and liquid chromatography coupled to mass spectrometry (LC–MS). A fungal
taxon can be identified by the metabolites it produces. Thus, profiling the pool of volatile and nonvolatile metabolites produced by
Penicillium species, in defined conditions, can be used as a chemotaxonomic tool to differentiate efficiently even closely related
species and to distinguish between cheese-related fungi. Karlshøj and colleagues analyzed the pool of volatile metabolites produced
by several fungi species, including P. camemberti, using the e-nose approach and showed an increasing difference between fungal
species throughout time. These authors indicated that P. camemberti can be unambiguously identified after 3 days of growth on yeast
extract glucose medium, with no CPA being detected. Thus, they clearly demonstrated the ability of e-nose to correctly identify
closely related fungi, grown on given conditions, to a species level. Because these species have also been shown to differ in myco-
toxin production, it also demonstrates the potential use of e-nose as a powerful tool for the identification of mycotoxigenic fungi in
food and feedstuffs.

Genetics

Modern molecular based approaches have recently been applied to monitor P. camemberti mycelium growth kinetics using real-time
PCR and the BETcam1F and BETcam1R primers targeting the beta-tubulin gene. This work indicates that lactose is the main energy
source for growth of the fungal mycelium at the beginning of ripening and that lactose depletion triggers stress, resulting in spor-
ulation, particularly in cheeses ripened at 16
C and 98% RH. A real-time quantitative PCR method has also been developed to
evaluate the growth dynamics of a cheese ripening culture containing P. camemberti on the surface of soft-cheese model curds.
Twenty three microsatellite markers have been developed for both P. camemberti and P. roqueforti with a number of markers being
identified such as the microsatellite locus PC4 that may be useful candidates for barcoding of these fungal strains. Molecular-based
approaches have also been employed to monitor the metatranscriptome of P. camemberti over a 77-day ripening period and have
identified sets of genes involved in functions ranging from metabolic processes, cell growth, and stress responses which appear to be
particularly active in the first 2 weeks of the ripening process. It is clear that approaches such as this will lead to an increased knowl-
edge of the important role that particular sets of genes play in the sensory process with ripening process in Camembert cheese.

Further Reading

Brunaa, J.M., Hierroa, E.M., de la Hoza, L., Mottramb, D.S., Fernandeza, M., Ordonez, J.A., 2003. Changes in selected biochemical and sensory parameters as affected by the
superficial inoculation of Penicillium camemberti on dry fermented sausages. Int. J. Food Microbiol. 85, 111–125.
Karlshøj, K., Nielsen, P.V., Larsen, T.O., 2007. Differentiation of closely related fungi by electronic nose analysis. J. Food Sci. 72, 187–192.
Le Bars, J., 1979. Cyclopiazonic acid production by Penicillium camemberti Thom and natural occurrence of this mycotoxin in cheese. Appl. Environ. Microbiol. 38, 1052–1055.
Nielsen, M.S., Frisvad, J.C., Nielsen, P.V., 1998. Protection by fungal starters against growth and secondary metabolite production of fungal spoilers of cheese. Int. J. Food
Microbiol. 42, 91–99.
Lessard, M.-H., Viel, C., Boyle, B., St-Gelais, D., Labrie, S., 2014. Metatranscriptome analysis of fungal strains Penicillium camemberti and Geotrichum candidum reveal cheese
matrix breakdown and potential development of sensory properties of ripened Camembert-type cheese. BMC Genomics 15, 235.

Change History

Update of Dobson and Abbas. Yeasts and Molds: Penicillium camemberti. Encyclopedia of Dairy Sciences, 2011, Pages 776–779.
July 2015. Dobson and Jackson updated text and references.