Cultivation of a Cheesemonger

A Blog of Cheese Culture and Cultures


Leave a comment

Lysozyme

AlexanderFleming_microscopeB2Lysozyme is an enzyme discovered and named by Alexander Fleming- the same guy who discovered Penicillin. The discovery was completely accidental when “nasal drippings were inadvertently introduced to a petri dish.”

Lysozyme is an antibacterial found in abundance in the human body – in tears and saliva; and is also present in the spleen, lung, kidney, white blood cells, plasma, and breast milk. Lysozyme has antibacterial activity against a number of bacterial species. 

Lysozyme Use in Food

Just as lysozyme is an antibacterial in humans, lysozyme from hens’ egg albumen has been used in cheese making to prevent a production defect called butyric late blowing due to the Clostridium bacteria.

ClassicCaseofLateBlowingcausedbyClostridia_Hoiz

An example of a cheese with the Butryic Late Blowing defect.

Research for using Lysozyme from hens’ egg albumen began in the late 1960s and 1970s. This research was followed by trials on cheese carried out in Europe in the early 1980s. These trials demonstrated the efficacy of lysoyzyme to prevent the late blowing defect in  different types of cheese. Since then, hens’ egg albumen lysozyme has been recognized as by the FDA as “The most   ‘generally recognized as safe'” effective organic anti-bacterial for use in food (Rulis). Because lysozyome in cheese is from egg, some cheese ingredients list may mention that the product contains egg by FDA allergen regulations.

The Role of Chlostridia in Butyric Late Blowing in Cheese

Many species of the genus Clostridium, also known as butyric acid bacteria or BAB for short, are endospore-forming obligately anaerobic bacteria able to ferment carbohydrates. Clostridia species have been identified as a significant milk contaminant by having been frequently detected in cheese samples that show the presence of Butyric late blowing. Clostridium tyrobutyricum, Clostridium butyricum, and Clostridium sporogenes are the most commonly observed species. Together, these species are called “butyric acid spores.”  Among the three species, C. tyrobutyricum has been the most extensively studied. It is acid-tolerant, characterized by the ability to ferment lactate (lactic acid sans a hydrogen) to butyric acid, carbon dioxide, and hydrogen. It is these carbon dioxide and hydrogen gasses that cause larger than normal eyes in the cheese and large cracks and bursts within the paste. Silage, a forage conservation technique, is frequently pointed as the principal source of butyric acid spores of ruminant feed.

Silage

ManinSorghumSilage

Man in a silo of harvested sorghum

Silage is harvested crops, mainly grass and corn, and stored in silos to ferment. Compared to hay, silage can provide a richer source of available provitamins A and other carotenoids and tocopherols. Because silage is full of vitamins and important nutrients that grazing animals need, silage has markedly exceeded hay production in many countries and has been the prevailing type of preserved forage.  Some farming systems are based on year-round silage feeding to cattle.

However, Silage can also be a pool of the undesirable bacteria. Bacteria such as Bacillus cereus, Clostridium tyrobutyricum and Listeria monocytogenes can carry over some components affecting sensory properties from silage to cow’s milk creating a concern to those in the dairy industry.

However, in some parts of the world, the off flavors and late blowing in cheese is so unwanted, the use of silage has been prohibited. P.F. Fox wrote in the second volume of Cheese: Chemistry, Physics and Microbiology text that “Since lactate decomposition by Clostridium butyricum and Cl. tyrobutyricum into butyric acid, acetic acid, carbon dioxide and hydrogen causes the cheese loaf to blow. Even in small amounts, butyric acid is unfavourable to flavour development. Therefore, in Switzerland, silage feeding of cows is prohibited for cheese production.”

The crops that are harvested for silage are placed in a silo and within 48 hours the stack will start and finish an aerobic fermentation phase in which trapped oxygen is consumed and is then followed by anaerobic fermentation that will convert sugars to acids. Complete fermentation takes approximately two weeks.

In the past the fermentation was conducted by indigenous microorganisms, but today the chemical reactions that occur in the stack are regulated. Some bulk silage is inoculated with specific microorganisms to speed fermentation or improve the resulting silage. Silage inoculants contain one or more strains of lactic acid bacteria, and the most common is Lactobacillus plantarum. Other bacteria used in inoculants include Lactobacillus buchneri, Enterococcus faecium andPediococcus species.

Chlostridia

Clostridia are bacteria found in the soil and on dirty plant material. Pollution by earth and dead plant elements further increase the risk of butyric fermentation by increasing the introduction of Clostridia into the silaged crop.

Clostridia in silage impair milk quality due to the fact that clostridial spores can survive the passage through the alimentary tract of a dairy cow. Clostridial spores present in silage are transferred to milk via faeces and faecal contamination of the udder.

HarvestingCornSilage_withadditions

Clostridia are endospore-forming anaerobic bacteria. Many Clostridia ferment carbohydrates as well as proteins. In addition to carbohydrate fermentation, it is C. tyrobutyricum that can degrade lactic acid to butyric acid and carbon dioxide and hydrogen gasses according to the following overall reaction:

Sugar or 2 Lactic –> Butyric + 2 CO2 + 2H

How does Lysozyme Work as an Anti- Chlostridium Bacterial?

Lysozyme is such a strong component of the immune system because it is able to degrade peptidoglycan present in bacterial cell walls. It destroys the Chlostridium cell wall by cleaving the Beta (1-4) N-acetyl-D-glucosamine polymer located in the peptidoglycan cell wall of the bacteria. Some Clostridia can cause serious health problems. One extremely toxic species is Clostridium botulinum.

ChlostridiumBotulinumfullImage

Lysozyme Application in Cheesemaking

We now know that butyric acid, which makes cheese unpalatable, and an accumulation of carbon dioxide and hydrogen gas, that cause defects in the cheese, both result when Chlostridia ferments sugars or lactic acid during ensilage.  Hen egg albumen lysozome has been approved by the FDA as an anti-bacterial to destroy the cell walls of any present Chlostridia to prevent these unfavorable results in the cheese making process.

During cheese making, lysozyme is added to cheese vats in either a liquid or spray dried and granulated form. At the pH of the milk, lysozyme is positively charged, and as the curd forms, lysozyme is electrostatically attracted and adsorbed to the negatively charged casein molecules that form the curd. After separation of the curd from the whey, greater than 90% of the lysozyme activity resides with the curd.

Lysozyme will not begin to destroy Clostridia spores until partway through the aging process of the cheese.  As the center of the wheel develops anaerobic conditions, the contaminating spores of Clostridium tyrobutyricum slowly begin to germinate.  Lysozyme that was introduced to the milk and has remained on the curd and will break down the Chlostridium cell wall and continue to do so for the duration of the aging process of the cheese. (A.S. Naidu)

From a consumer’s point of view, lysozyme is completely harmless.

Bibliography

Batt, Carl A., Totorello, Mary Lou ed. Encyclopedia of Food Microbiology 2 ed. Elsevier 2014

Drouin, Pascal  and Lafrenière, Carole . Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health. Chapter 18: Clostridial Spores in Animal Feeds and Milk. Published: September 26, 2012 under CC BY 3.0 license  http://cdn.intechopen.com/pdfs-wm/39470.pdf

Fox, P.F. ed., Cheese: Chemistry, Physics and Microbiology: Volume 2 Major Cheese Groups. Aspen Publishers, 1999

Goodling, A. et. al Hen Egg-White (HEW) Lysozyme. Protopedia.  http://proteopedia.org/wiki/index.php/Lysozyme

Muck, R.E. Butyric Acid In Silage: Why It Happens. Powerpoint. U.S. Dairy Forage Research Center, USDA. http://www.ars.usda.gov/sp2UserFiles/Place/36553000/dairyexpo/2006/WDE2006_butyric_acid.pdf

Naidu A.S., ed. Natural Food Antimicrobial Systems. CRC Press, Jun 21, 2000

Pavel Kalac. The effects of silage feeding on some sensory and health attributes of cow’s milk: A review. Food Chemistry 125 (2011) 307–317 http://ssu.ac.ir/cms/fileadmin/user_upload/Mtahghighat/tfood/ARTICLES/milk/The_effects_of_silage_feeding_on_some_sensory_and_health_attributes.pdf

Rulis, Alan M. Ph.D. Agency Response Letter GRAS Notice No. GRN 000064. Letter written April 2, 2001. http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory/ucm153975.htm Page Last Updated: 10/15/2014

Stefanie J.W.H. Oude Elferink, Frank Driehuis, Jan C. Gottschal, and Sierk F. Spoelstra. Paper 2.0: Silage Fermentation Processes and their Manipulation. Agriculture and Consumer Protection Dept. Food and Agriculture Organization of the United Nations. http://www.fao.org/docrep/005/x8486e/x8486e09.htm

LYZ lysozyme [ Homo sapiens (human)]The National Center for Biotechnology Information updated on 7-Feb-2016    http://www.ncbi.nlm.nih.gov/gene/4069

Clostridia – the underestimated danger in dairy farms. Addcon Green Chemistry.

http://www.addcon.com/en/feed/silage-additives/ensiling-tips/clostridia/

Images

Alexander Fleming Laboratory Museum. Fleming-at-Microscope.  Flickr. <https://www.flickr.com/photos/47516639@N04/4356123114/in/photostream/&gt;

Lysolac: Safely & Naturally Protecting Your Cheese. “Image of Cheese deformity caused by Butyric Late Blowing” Chestnut Hill Sciences LLC. http://www.seedtoday.com/articles/corn_silage_hybrids_and_seeding_rates-112712.html

Kesmodel, David. U.S. Farmers’ Latest Hot Crop: Sorghum. Wall Street Journal. Jan. 21, 2015

Seed Today. Corn Silage Hybrids and Seeding Rates. August 15, 2011 http://www.seedtoday.com/articles/corn_silage_hybrids_and_seeding_rates-112712.html

DB Science 3. Bacteria. https://dbscience3.wikispaces.com/Gabby