Cheese Production Using Penicillium Species

 Cheese is a dairy product that made the dietary benefits of milk into a transportable, longer lasting, and more readily digestible form. The production of cheese was used to make protein rich food available year around. The first evidence of cheese making was seen in the sixth millennium BC from specific animal proteins found in pottery with holes, that could have been used as a cheese sieve (Salque et. al., 2013). Cheese is a complex microbial environment that can host multiple species of microbes at once, such as bacteria from starter cultures to lower the pH of the dairy, from bacterial smears on the outer surface of the cheese block, or from fungal mycelial inhabitation of the cheese block. The basics of cheese making is the result of coagulation of large proteins such as casein through the addition of the enzyme rennin; which is separated from the whey, the liquid portion that contains a majority of lactose (Milk Facts). After the separation of curd from whey, many options are available for ripening, or letting microorganisms effect the flavor, texture, and appearance of the solid cheese mass over time. This paper explores a specific subsection of cheese production; how the genus Penicillium is used in the ripening stage of cheese production to make specific types of well-known cheese, the phylogenic relationship between species and Penicillium as a course of contamination.

 The genus Penicillium is within the phylum Ascomycota, and in a cheese environment is known to reproduce asexually through conidia. The two main Penicillium species that are used in cheese production is P. camemberti, which is used to make Camembert and Brie cheese, and P. roqueforti, which is used to make blue-veined cheeses (Lund Filtenborg, and Frisvad, 1995). Camembert and Brie cheeses are characterized by thick, white mycelial growth on the outside of the cheese that produces a soft interior. Blue-veined cheeses are characterized by blue to green colored mycelial growth through the interior of the cheese, that produces a specific flavor and odor profile. The majority of cheese production until the early 1900’s was imported from Europe, until research led to an understanding of how to improve the process of cheese making on a mass scale (History of cheese). The key to mass production of certain types of cheese, such as blue-veined, was the use of homogenized milk; which lead to a more characteristic flavor to the mature cheese, reduction of yellow coloring from milk when cows were out on pasture, and a more uniform inoculation of ripening microflora (Babel, 1953).

 In cheese production before the rennin is added a lactic acid bacterium (LAB) starter is added that metabolize lactose, which lowers the pH of the curd (McSweeney, 2004). The low pH of the curd is important because it decreases possible sources of contamination as well as changing the chemistry of the dairy product by making casein easier to break down later during the ripening stage of production (McSweeney, 2004). After the starter culture is added it sets for a prescribed amount of time, which depends upon the cheese type, and then the enzyme rennin is added to coagulate the curds. Once the curds have coagulated into a matt, they are cut and strained from the whey, where salt is added (Babel, 1953). In blue-veined cheeses when salt is added P. roqueforti powder is also added which inoculates the curd, but in Camembert cheeses, the cheese is inoculated via natural inoculation by being placed in the ripening room (Babel, 1953). After the cheese curds are removed from the whey, they are placed in shaping hoops that have holes, which are turned at regular interval to insure an even drainage of residual whey, and for uniform inoculation in Camembert cheeses (Babel, 1953). Unlike Camembert cheeses, in blue-veined cheeses the entire curd is not only inoculated, but during the ripening stage the solid block of cheese are skewered to allow the escape of carbon dioxide and a limited amount of oxygen flow to promote the growth of P. roqueforti only (Babel, 1953). The skewering process is an essential step in the production of blue-veined cheeses, or without it the characteristic coloring on the interior of the cheese as well as the crumbly texture would not be noticeable. The Penicillium molds used in cheese production use the cheese as a carbon source through the breakdown of casein, the main protein in cheese, which leads to as softer texture of the cheese. The bi-products of mycelial growth and reproduction lead to characteristic flavor profiles and odors. Research has been conducted to determine exactly what chemicals formed by molds such as P. roqueforti that produce blue-veined cheeses characteristic rancid flavor and a crumbly interior due to the mycelial growth. Penicillium breaks down proteins through a series of chemical pathways that eventually lead to methyl ketone and free fatty acids, which are responsible for the characteristic rancid flavor and odor (Stokoe, 1927). P. camemberti is a surface mold exclusively due to its large oxygen requirements and produces a proteolytic enzyme that makes its way into the interior of the cheese to produce a characteristic creamy flavor and water consistency (Babel, 1953). Camembert and brie cheese have to be under a specific thickness in order for the entire cheese to ripen uniformly, otherwise parts of the cheese may become inedible before the entire block is ripe.

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 Fungi are only once species of microorganism that is used in a specific stage during the production of cheese, the ripening stage, which can last few days to months, possibly even years. Blue-veined cheese and Camembert cheeses are aged and ready for market in a relatively short time span of a few weeks (Babel, 1953). A part of cheese production that is always an area of concern is food safety, and in the case of cheese, possibilities of undesirable or detrimental contaminations of the cheese block. Undesirable contamination is that of an isolate of P. roqueforti in blue-veined cheeses that produces a orange to red color instead of the characteristic blue to green color. Incorrect or off coloring, undesirable or off flavoring or odor of the cheese, or unusual texture could all be considered a side effect of undesirable contamination and would lower or stop commercial sale of the cheese product. A detrimental contamination is the infestation of a cheese block of species of microorganism that is a human pathogen and can cause illness if consumed. The initial source for inhibiting bacterial growth in the cheese blocks is in the lactic acid bacterial (LAB) starter cultures used before rennin coagulation, which lowers the pH of the dairy and limits the organisms that can survive in such an environment (Irlinger and Mounier, 2009). Another way to combat contamination is inoculation of the cheese block by the desired fungus. Fungi grow rapidly and can be resistant to certain environmental factors such as salt content or pH viability (Babel, 1953). This rapid growth and ability to tolerate certain environmental factors leads to a decrease in other possible contaminates inhabiting the cheese block through competition. To optimize mycelial growth and to further inhibit contaminates the ripening rooms are kept at specific temperature and humidity, which can either be completely man-made in a facility or in places such as caves that have been used for centuries (Salque et. al., 2013).

 In order to optimize mycelial growth a 25C temperature ripening room was established as preferred for a majority of fungal species and has been reiterated in several research studies such as G. Le Dréan et. al. (2010). In the study conducted a relationship between mycelial growth and DNA extraction was found in order to determine the biomass of fungi in cheese, which could be used to further optimize growth (G. Le Dréan et. al., 2010). In order to determine this correlation in both P. roqueforti and in P. camemberti the growth of both species was observed. It was found that “P. roqueforti grew in two phases of rapid growth and then remained stable” and “P. camemberti growth rate was high till day 11 and then a stationary phase was reached” (G. Le Dréan et. al., 2010). The importance of this research is that mycelial growth can be monitored, and an understanding of what should be considered “normal” growth patterns have been quantified. An understanding of mycelial growth patterns also provides information on the critical times during the ripening phase where a contaminate could infect the cheese block.

 There is a major limitation to cheese molds, and it is that until recently they only reproduced asexually. This limits the genomic diversity of the fungi, which could lead to adaptions of contaminates that the fungi could not compensate for or to the evolution of a parasite of the fungus itself. Recently however, under lab conditions, P. roqueforti has been observed with an induced sexual reproduction phase (Ropars et. al., 2014). The implications of this being that under lab conditions through sexual recombination desirable phenotypes can be expressed in higher percentages and undesirable mutations can be removed from the genomic sequence. This could lead to specialized strains, or isolates, within each species that produce specific results and could possibly affect the cheese market with new and desirable cheese flavors and aromas. The ability to manipulate the genome through sexual reproduction could also lead to greater diversity, an increased resistance to other microorganism as parasites and increase in competitive capabilities on cheese.

 This research could change the cheese market globally, yet the ability to identify fungi or effect the genome has only been accomplished in the last two decades. When sequencing was first used, it was determined that identification of specific species could be found near regions that were unlikely to vary, such as those that transcribed for 18S rRNA (Cappa and Cocconcelli, 2001), but in recent years that has been further revolutionized. Today a barcoding sequence is used to identify species, even possibly isolates, using an ITS region of fungal DNA (Schoch et. al., 2012). This barcoding region is a sequence that is highly variable between regions of the DNA that are almost constant, and thus can be used to identify species. This ITS region is now a standard today within the mycology field, even though there are some limitations in specific lineages, and other regions of DNA in conjunction with ITS should be used to resolve differences (Schoch et. el., 2012).

 A study was conducted in order to determine the majority of fungal species from dairy environments in order to understand the fungal species possible adaptions to a limited environment on cheese (Ropars et. al., 2012). It was found that the majority of species within the dairy environment were in the genus Penicillium, and only a few of the samples were from Roquefort or Camembert cheeses, which suggests that the genera Penicillium is a source of major contamination in undesired cheese environments (Ropars et. al., 2012). This is possibly due to species within the Penicillium genus being able to inhabit a wide range despite a wide range of inhibitory environmental factors. It was also found that in the phylogenic tree that “cheese fungi are interspersed among other fungi, suggesting that they might be inadvertent products of domestication that could have occurred several times” (Ropars et. al., 2012). The importance of understanding the phylogenic relationship between species of fungi that inhabit cheeses is that it is of economic importance to determine the likelihood of reversals that could be detrimental to the industry or harmful to consumers. It was found that the fungi found in both the cheese environment in a natural environment as control covered wide range of orders such as Onygenales, Eurotiales, Hypocreales, Microascales, and others that could not be determined down to specific orders only to class (Ropars et. al., 2012). From this research other studies found evidence of a horizontal gene transfer, which at the time was extremely uncommon in the kingdom Eukarya (Cheeseman et. al., 2014).

 In the fungi from cheese environments, specifically Penicillium a certain section of DNA can be used to correctly identify down to the species, called Wallaby, and it was found and used as evidence of a horizontal gene transfer by Cheeseman et. al. (2014). Wallaby was determined to be the result of horizontal gene transfer due to the exact same sequence being found in several Penicillium species found on cheese but differing in locations on the genome (Cheeseman et. al., 2014). It is suspected that the Wallaby region codes for regulation in spore production or in “antimicrobial activities”, which could lead to a competitive edge in the cheese environment (Cheeseman et. al., 2014). Wallaby has yet to be found in a fungal species that did not inhabit cheese, and it is possible that the gene transfer could have been influenced by human activity (Cheeseman et. al., 2014). In general fungi are unable to compete with bacteria in term of colonization of the cheese block unless specific environmental factors are present, which can be adjusted according to mycelial growth.

 Fungi have the upper hand on cheese blocks over bacteria as the cheese ages due to the lower water availability, which fungi are able to overcome unlike extremely sensitive bacteria (Haasum and Neilson, 1998). It was found that fungi are extremely inhibited by temperature either increase or reduction, slight inhibition with higher carbon dioxide, and in certain species lower inhibition in lower oxygen concentrations (Haasum and Neilson, 1998). Certain fungi that are sensitive to oxygen concentration are P. camemberti, which is why it only grows on the rind of the cheese block rather than through the cheese block. The fungi that were slightly inhibited by a decrease in carbon dioxide concentration, were able to overcome this limitation, but the stunt in mycelial growth originally could lead to undesirable bacterial contamination of the cheese block (Haasum and Neilson, 1998). This study showed that certain fungi have a preferred temperature range, not only for mycelial growth but for conidia formation as well, such as in relation to one another P. roqueforti requires a higher ambient temperature than P. camemberti (Haasum and Neilson, 1998). These are important factors in cheese production in order to achieve high mycelial growth, inhibit undesirable colonization of the cheese block, and to have the desired characteristics the of cheese type be evident as a by-product of mycelial inhabitation.

 Since fungi are able to inhabit a wide range of environments and there is concern over contamination of cheese blocks. P. roqueforti and P. camemberti are used to make a specific type of cheese, but on any other variety of cheese they are seen as a contaminate and are undesirable. P. camemberti unlike P. roqueforti is unable to survive in other environment other than cheese blocks, this is due to the domestication and of the fungi centuries ago (Cheeseman et. al., 2014). Since P. roqueforti can survive only other surfaces or media than cheese, it is a possible source of contamination in many cheese environments other than blue-veined cheese. The genus Penicillium also contains a multitude of other species that can inhabit a cheese block as a contaminate with P. commune being the most common (Lund, Filtenborg, and Frisvad, 1995). Before genomic analysis was available P. commune was seen as the ancestral species of the genus Penicillium and gave rise to almost all of the cheese associated mycoflora (Lund, Filtenborg, and Frisvad, 1995). 

 There is a major aspect of using fungi to ripen cheese which has yet to be discussed, which is the fact that fungi are known for producing mycotoxins that can be harmful to humans. In general mycotoxins are a secondary metabolite, have a low-molecular weight, and can be resistant to industrial processing (Hymery et. al., 2014). The low molecular weight of mycotoxins does mean however, that if the toxin is heated to a certain degree, like other proteins, it will denature and should no longer be harmful for consumption. The being said, some cheeses would be inedible if heated to the point of denaturing all of the possible mycotoxins it could be infected with.
 The origin of the fungus on a cheese block can influence the type and concentration of a mycotoxin (Hymery, 2014). P. roqueforti is known to produce the mycotoxins Roquefortine C, Mycophenolic acid, and PR toxin (Hymery, 2014). PR toxin is chemically unstable in cheese and is converted into a less toxic molecule, thus it is not a possible source of toxicity during consumption (Hymery, 2014). Roquefortine C is a known neurotoxin that is produced by all P. roqueforti and has been linked to several cases of animal toxicity, yet no known human cases are known (Hymery, 2014). Mycophenolic acid “is presently used as an immunosuppressant in kidney, heart, and liver transplant patients to avoid organ rejection” (Hymery, 2014). All three possible mycotoxins that are produced by P. roqueforti are either converted into a non-toxic form, have had no known cases of toxicity after centuries of consumption, or is used as a treatment for some patients. P. roqueforti is seen as an edible and safe to consume fungi, however it does still produce a mycotoxin and has to be monitored for mutations. A simple mutation in one of the mycotoxins could lead to a detrimental outcome from consumption.

 P. camemberti produces only one mycotoxin, which is cyclopiazonic acid (Hymery, 2014). This toxin targets the kidneys and hind gut and could possibly be associated with aflatoxicosis cases (Hymery, 2014). There are also no known cases of toxicity in humans from consumption of P. camemberti (Hymery, 2014). These two species of fungi are used to produce cheese for human consumption, but they still produce mycotoxins. Just because there are no known cases where the mycotoxins have caused an illness does not mean that the mycotoxins could potentially cause an illness in humans. Mycotoxin toxicity and concentration have to monitored regularly, and isolates that produce higher concentrations of the mycotoxins in the cheese environment must be removed.

 The genus Penicillium contains species that are used to make cheeses that have great economic value worldwide. The production of these cheese varieties has become both a science and an art form, with further research leading to desirable reproducible results on a large scale. Research on the specific species used to make blue-veined cheese or Camembert cheeses has led to optimization of mycelial growth and a reduction in contamination from other sources. The phylogenetic relationship between Penicillium species has also been resolved to a certain degree, which can indicate how the species evolved to its present-day form and how humans can further manipulate the genome for desirable products. Evidence of horizontal gene transfer indicate a recent evolutionary step that could possibly have implications in cheese production, if the Wallaby sequence does infer a competitive advantage over other possible sources of contamination, such as bacteria. The main challenge that is still seen today in chees production of fungi ripened cheeses is maximizing mycelial growth that imparts specific characteristic to the cheese block, while limiting contaminates and possible mycotoxins that could be produced. The cheese environment is a complex system where microbes are either working in a mutualistic relationship that produces desired dairy products, or microbes are trying to outgrow other microbial competitors that are undesirable. These challenges are something that cheese producers have to constantly monitor, and there is always room for improvement through further research and understanding of the relationship between fungi and the cheese environment.

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