Survey of Bacteria Used in Cheese Production

yes, i wrote another paper about cheese. 

some music while you read:

Microbes are commonly thought of as dangerous pathogens, especially in the food service industry, however many microbes contribute to the preparation or flavor of popular consumables.  One of these products is cheese.  In 2012, americans consumed more than 33 pounds of cheese per capita (“USDA Economic Research Service - Dairy Data,” n.d.) and its general enjoyment is due to the variety of bacteria that are added in the cheese making process.  Cheese has been produced for hundreds of years in many countries around the world and while modern techniques are being employed in its current production, the key to unique and flavorful cheeses continues to be in the selection and addition of bacteria.  Studying microbiology is important to continued production of excellent cheeses as it helps makers (and consumers) understand the science behind traditional cheese making and how modern biotechnology influences future production and food safety.
The simplest explanation of cheese production is milk solidified by acid.  Milk is an emulsion of proteins, mainly casein, and lipids with a smaller fraction of minerals and other particles.  When acid is added lipid combines with casein to create curds.  Water and lactose leave the solid and is called whey.  The curd is collected and pressed together to form a solid block, which is known as cheese.
            Building upon this process, different types of cheese arise from the different moisture content and flavor, characteristics determined by the method of curdling and ingredients added.  Coagulation techniques used today, not so different from techniques invented decades ago, are in one of three categories- acid coagulated, acid-heat coagulated, and rennet coagulated.  In all three techniques, lactic acid bacteria (LAB) can be added as the acid source.  LAB use lactose in milk for energy and produce lactic acid.  Modulating the temperature, pH and moisture can change the amount of lactic acid produced.  Acid can be added directly to milk, such as in ricotta cheese, but a wider variety of flavors can be achieved by adding bacteria instead; each bacteria has its own flavor profile and a gentle acidification.  Purified lactic acid can also be added to milk to simulate bacterial metabolism but is not cost efficient; it is cheaper to just add bacteria instead of going through the process of purifying lactic acid. 
Once acidified, rennet can be added to improve the texture of the curd.  Rennet causes the casein to aggregate in chains and trap LAB, lipids, and some whey into a matrix.  This results in a more gelatinous texture like in parmesan (uses rennet) compared to crumbly paneer (doesn’t use rennet).  Rennet is a group of enzymes that traditionally come from the stomach of mammals (i.e. cows) and contains protease and lipases.  Rennet can also be of plant or fungal origin.  Called “vegetable rennet”, a popular alternative for animal rennet is produced naturally by fungus Mucor miehei.  With the advent of molecular techniques, a genetically modified fungus, such as the ubiquitous Aspergillus niger is made to ferment components of rennet.  The fermented products will participate in milk coagulation but the organism that produced it will die.  This has led to the FDA approval of this substitute because the actual genetically modified organism is not alive in the final product.  The enzymatically active components of rennet are collectively called chymosin and makes up about 80% of rennet.  Although in cooking chymosin and rennet are often used interchangeably, the wider use of vegetable rennet and fermentation produced chymosin (FPC) is more often called “chymosin”, and only animal produced enzymes are called rennet, because the additional proteins found in animal rennet are not present in plant based enzymes.  Comparing the products, FPC is the most similar to rennet because the transformed organisms had genes originally isolated from mammals.(Salgado et al., 2013)  Both types of chymosin are as popular as rennet because it is easy to obtain in large quantities for a low price and have an appeal to vegan or vegetarian consumers.
Historically, LAB were the accidental addition to unrefrigerated milk that incidentally created cheese.  Over time, recipes were perfected by the isolation of specific LAB to create signature blends and more consistent products.  Regional cheeses take on the flavors of the bacteria of the locale and specific maker and are perpetuated by the back slopping process.  Back slopping is the use of a starter from the last prepared batch or a particularly desirable batch for the next batch.  Although it is not a controlled process, desirable bacteria are more likely to out-compete unwanted bacteria in the next batch.  A more precise and hygienic way to add live cultures to food is to completely characterize bacteria and use specific amounts in the production stages.
Traditionally, bacteria from food samples can be identified and characterized by biochemical tests and use of selective and differential media.  A modern and efficient way alternative to this is 16s ribosomal RNA sequencing.  The 16s ribosomal subunit is found in prokaryotes and is highly conserved.  PCR amplification is used in combination with sequencing to identify bacteria.  The sequence produced from PCR with 16s [universal] primers are compared to sequences in databases such as NCBI BLAST to identify the genus and species. 
Golija cheese is a regional cheese of Serbia (Amarela Terzic-Vidojevic, 2014) that is made in small without starters or back slopping.  Rennet is added to milk and the resulting curd is cut into blocks and stored in brine in sealed containers at ~16ºC (above refrigeration but below room temperature) for several months.  A 16s analysis revealed a variety of LAB from the milk source and curd preparation.  A total of 188 gram positive bacteria were characterized by this molecular method and compared with reference strains and traditional biochemical results to show antimicrobial properties and enzymatic activity.  This research has shown a variable amount of bacteria is present in the cheese depending on the stage of preparation.  The specimen with the largest variety of microbes was a 20-day brined cheese.  The most commonly isolated organism is a well-known dairy industry LAB, Leuconostoc mesenteroides, and was found in most young cheeses.  The bacteria responsible for Golija’s signature texture and flavor are Enterococcus faecium and Enterococcus durans.  Along with Enterococcus faecalis, these organisms are also the most commonly recovered bacteria in European cheeses and dairy products. (Cosentino et al., 2004) Interestingly, E. faecium is also an opportunistic pathogen known to cause infantile meningitis but their antibacterial properties prevent more dangerous pathogens such as Listeria monocytogenes from growing in cheeses.  However, it is still advisable to refrain from eating unpasteurized dairy products to avoid acquiring health problems.  This research is an example of how 16s is an effective way to identify and characterize bacteria and may lend itself to large-scale production using the same types of bacteria. 
Microbes for flavor and texture more prominently recognized in the cheese ripening process.  Ripening, also called maturing, is when cheese acquires its signature pungent flavors.  This process can take weeks to years, depending on the type.  After the curds are pressed, they are transferred to frames called hoops and more of the whey is removed by air and gravity.  The resulting form is then brined with salt and bacteria or fungi are added.  Yeasts use the lactic acid produced by the starter.  This lowers the pH and adds different enzymes to the cheese, allowing secondary bacteria to participate in the ripening process.  (Viljoen et al., 2003)  Cheeses with a “crust” or coating, such as brie are only coated with molds on the outside.  Mold can added to the brining solution or after brining, may be sprayed with yeast.  The yeast begin to ferment the cheese starting at the surface and begins penetrating the main body of the cheese from the outside.  In younger cheeses, the crust may be thin and mild.  In older soft cheeses, the crust may be thicker and more pungent as the yeast is allowed to grow progressively toward the body.  In some cases, the crust yeast makes the cheese body softer instead of more firm like the crust itself.  Cheeses that are harder take longer for the yeast to penetrate so the crust is very thin like in asiago or Parmesan cheese. 
The yeast or bacteria can alternately be added directly to the cheese milk instead of just as crust.  For example, Streptococcus thermophilus, a LAB commonly used in fermented dairy products like yogurt, is incorporated in hard cheeses to improve texture due to its secreted polysaccharides.  Low-fat cheeses can be produced with low fat milk when S. thermophilus is added but will still maintain its traditional texture from bacterial produced exopolysaccharides.  Another famous variety, swiss cheese like Emmenthaler, incorporates the bacterium Propionibacterium freudenreichii subsp. shermanii.  The holes, or “eyes” of the cheese is from the CO2 gas produced in citrate metabolism of the organism.  (Mukdsi et al., 2014)  Eyes in Emmenthaler are incidental, even though their presence is a well known cheese characteristic.  P. freuenrichii is added to cheese mainly for its lipolytic action.  The enzymes produced from lipolysis create pungent and fruity flavors.  The lipolysis takes place early in the cheese making process.  Even in the coagulation process, lipolysis is taking place.  In the ripening process, lipolysis takes place while the bacteria are still alive; since this is an early stage in the ripening, the cheese is still soft enough to be stretched into large gas bubbles.  The bubbles get trapped inside the body instead of released from the cheese because of the tough crust.  pH and temperature can also be used to control bubble size.
Another distinct variety of cheese is blue cheese.  The name is descriptive of the colored veins of Penecillium sp. mold growing throughout the cheese.  Different species of Penecillium are used, depending on the region of Europe the cheese is found in (due to cultural regulations).  The regional bacteria give the cheese distinct flavors.  To achieve the deep penetration of mold, the organism is actually injected into the cheese. (Fernández-salguero, 2004) blue cheese, particularly Roquefort, is an old variety that got contaminated by microorganisms from the environment, a cave.  Today, the process is much more controlled so a consistent flavor can be achieved.  In one characterization study, cheese that was not pierced did not maintain full penetration by the surface mold.  When the Penecillium sp. cannot reach the cheese body, less proteolysis occurs and there is a drastic difference in flavor and scent. 
Incidental molds and bacteria enter the cheese in the ripening process over time.  after brining, many cheeses are left exposed to ambient air in storage or aging rooms.  In modern cheese making facilities, the rooms can be temperature and humidity controlled.  Microbes can even be aerosolized and applied to the cheese.   The environmental conditions influence the metabolic process of the organisms in the cheese.  During this time, other microbes from the environment or from the handler can enter the cheese.  In some cases this is a beneficial addition, such as in the E. faecalis addition to Golija previously mentioned.  In other cases, this is an opportunity for pathogens to infiltrate the cheese.  Staphylococcus aureus is a ubiquitous microbe commonly found in skin.  It is an opportunistic pathogen but is a danger when certain serotypes cause toxins.  Cheese is potentially a healthy environment for S. aureus to grow because of high salt factor, storage temperature, and protein content.  Bacteria in crusts and in the cheese body prevent contamination because other bacteria out-compete the growth of S.aureus.  Cheese is also a good traveling food, compared to milk, because it is preserved and will last longer without spoilage.  Pasteurizing the milk in the initial cheese making process removes raw milk pathogens and the proper brining and ripening methods will prevent pathogenic bacteria from reentering the mixture, creating a safe and delicious food product. 
Cheese is a widely consumed food product whose origins reach back many years.  While the original production of cheeses may have been accidental, modern cheese making is a specific science.  The process uses specific bacteria species to control the outcome of the products.  This combination of new and old techniques yields a great variety of cheeses and perfection to the popular food.  The current direction of microbiology will enhance this process and create even more cheeses than the large variety already available to choose from.  Microbiology is essential to the preservation of the art of cheese making.



Cosentino, S., Pisano, M.B., Corda, A., Fadda, M.E., Piras, C., 2004. Genotypic and technological characterization of enterococci isolated from artisanal Fiore Sardo cheese. J. Dairy Res. 71, 444–450. doi:10.1017/S002202990400041X

Fernández-salguero, J., 2004. Internal Mould - Ripened Cheeses: Characteristics, Composition and Proteolysis of the Main European Blue Vein Varieties. FORMAGGI Matur. CON MUFFE INTERNECARATTERISTICHE Compos. E PROTEOLISI DELLE PIÙ IMPORTANTI Var. Eur. VENATURA BLU 16, 437–445.

Kindstedt, P.S., 2013. The Making of Great Cheeses. Microbe 361–367.

Kindstedt, P.S., 2013. Making Great Cheeses, Part 2. Microbe 401–407.

Mukdsi, M.C.A., Falentin, H., Maillard, M.-B., Chuat, V., Medina, R.B., Parayre, S., Thierry, A., 2014. The Secreted Esterase of Propionibacterium freudenreichii Has a Major Role in Cheese Lipolysis. Appl. Environ. Microbiol. 80, 751–756. doi:10.1128/AEM.03640-13

Pearson, H., 2003. Sequencing reveals secrets of Stilton. Nature News. doi:10.1038/news030609-18

Salgado, J.A.G., Kangwa, M., Fernandez-Lahore, M., 2013. Cloning and expression of an active aspartic proteinase from Mucor circinelloides in Pichia pastoris. BMC Microbiol. 13, 250. doi:10.1186/1471-2180-13-250

USDA Economic Research Service - Dairy Data [WWW Document], n.d. URL (accessed 12.9.13).

Viljoen, B.C., Khoury, A.R., Hattingh, A., 2003. Seasonal diversity of yeasts associated with white-surface mould-ripened cheeses. Food Res. Int. 36, 275–283. doi:10.1016/S0963-9969(02)00169-2

1 comment:

  1. possible evidence of cheese making at least 7000 years ago:

    it never occurred to me that in addition to preserving milk and making it more storable/transportable, cheese may have also helped mitigate lactose intolerance.





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