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Tag words: lactic acid bacteria, LAB, homolactic, heterolactic, Lactococcus, Lactobacillus, acidophilus, Streptococcus thermophilus, Leuconostoc, lactic acid, cheese, curds, yogurt, butter, buttermilk, sour cream, cheesemaking, bacteriocins, nicin, lantibiotics, vaccine delivery, lactococcus genome, probiotics.





Kenneth Todar currently teaches Microbiology 100 at the University of Wisconsin-Madison.  His main teaching interest include general microbiology, bacterial diversity, microbial ecology and pathogenic bacteriology.

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Lactic Acid Bacteria (page 5)

(This chapter has 5 pages)

© Kenneth Todar, PhD



The following section that focuses on Lactococcus lactis as the prototypical lactic acid bacterium. Lactococcus lactis exhibits virtually all of the foregoing characteristics and applications of a lactic acid bacterium. This bacterium is chosen because of its preeminence in cheese making and its status as the natural nominee as the state microbe of the dairy state of Wisconsin.


 
Lactococcus lactis. UW Department of Bacteriology strain LcL325UW. Magnification 20000X. Scanning electron micrograph by Joseph A. Heintz, University of Wisconsin-Madison.

Lactococcus lactis is a microbe classified informally as a Lactic Acid Bacterium because it ferments milk sugar (lactose) to lactic acid. Lactococci are typically spherical or ovoid cells, about 1.2µm by 1.5µm, occurring in pairs and short chains. They are Gram-positive, non motile, and do not form spores. Lactococci are found associated with plant material, mainly grasses, from which they are easily inoculated into milk. Hence, they are found normally in milk and may be a natural cause of souring. Lactococcus lactis has two subspecies, lactis and cremoris, both of which are essential in manufacture of many varieties of cheese and other fermented milk products.

Lactococcus lactis is related to other lactic acid bacteria such as Lactobacillus acidophilus in our intestinal tract and Streptococcus salivarius in the mouth. However, Lactococcus does not normally colonize human tissues and differs from many other lactic acid bacteria in its pH, salt, and temperature tolerances for growth, which are important characteristics relevant to its use as a starter culture in the cheesemaking industry.

Lactococcus lactis is vital for manufacturing cheeses such as Cheddar, Colby, cottage cheese, cream cheese, Camembert, Roquefort and Brie, as well as other dairy products like cultured butter, buttermilk, sour cream and kefir. It may also be used for vegetable fermentations such as cucumber pickles and sauerkraut. The bacterium can be used in single strain starter cultures, or in mixed strain cultures with other lactic acid bacteria such as Lactobacillus and Streptococcus species.

When Lactococcus lactis is added to milk, the bacterium uses enzymes to produce energy (ATP) from lactose. The byproduct of ATP production is lactic acid. The lactic acid curdles the milk that then separates to form curds, which are used to produce cheese and whey. Curdling the milk is not the bacterium's only role in cheese production. The lactic acid produced by the bacterium lowers the pH of the product and preserves it from the growth by unwanted bacteria and molds while other metabolic products and enzymes produced by Lactococcus lactis contribute to the more subtle aromas and flavors that distinguish different cheeses.


Fermented dairy products wherein Lactococcus lactis is the primary organism involved in manufacture.

Product

Principal acid producers

Secondary microflora

Cheese

Colby, Cheddar, cottage, cream

Lactococcus lactis ssp. cremoris

None

Lactococcus lactis ssp. lactis

 

Blue

Lactococcus lactis ssp. cremoris

Citrate+ Lactococcus lactis ssp. lactis
Penicillium roqueforti

Lactococcus lactis  ssp. lactis

 

Fermented milk

Buttermilk

Lactococcus lactis ssp. cremoris

Leuconostoc spp.
Citrate+Lactococcus lactis ssp. lactis

Lactococcus lactis ssp. lactis

 

Sour cream

Lactococcus lactis ssp. cremoris

None

Lactococcus lactis ssp. lactis





Lactococcus lactis. Magnification 1500X. Phase micrograph courtesy of T.D. Brock, University of Wisconsin-Madison.


Cheese

Cheese making is essentially a dehydration process in which milk casein, fat and minerals are concentrated 6 to 12-fold, depending on the variety. The basic steps common to most varieties are acidification, coagulation, dehydration, and salting. Acid production is the major function of the starter bacteria. Lactic acid is responsible for the fresh acidic flavor of unripened cheese and is important in coagulation of milk casein, which is accomplished by the combined action of rennet (an enzyme) and lactic acid produced by the microbes. During the ripening process the bacteria play other essential roles by producing volatile flavor compounds (e.g. diacetyl, aldehydes), by releasing proteolytic and lipolytic enzymes involved in cheese ripening, and by producing natural antibiotic substances that suppress growth of pathogens and other spoilage microorganisms. For Cheddar and Colby cheese production, starter cultures include strains of Lactococcus lactis ssp. cremoris and/or lactis. Likewise, blue cheeses  require Lactococcus lactis ssp. cremoris or lactis, but the mold Penicillium roqueforti is also added as a secondary culture for flavor and blue appearance.

 
Wisconsin's unique cheese curds, Colby, and dozens of varieties of Cheddar are made exclusively with strains of Lactococcus lactis. Images courtesy of Wisconsin Cheese Mart, Milwaukee Wisconsin.


Cultured Butter, Buttermilk and Sour Cream

Sour cream is made from cream to which a starter culture of Lactococcus lactis has been added to coagulate the cream and to enhance its flavor. Buttermilk is also made with Lactococcus lactis in order to acidify, preserve and flavor the milk. Diacetyl, made from citrate by Lactococcus, gives buttermilk its distinct taste and enhances its storage properties. Lactococcus lactis or mixed cultures that contain Lactococcus lactis, plus a Leuconostoc species are used. In the making of cultured butter, fat (cream) is separated from skim milk by centrifugation of milk. The cream is pasteurized and inoculated with selected starter cultures. The ripened cream is then churned. The cream separates again into cream butter and its byproduct, sour buttermilk.


Nisin

Nisin is an antibiotic-like substance, called a bacteriocin, produced by the "food grade" starter strain, Lactococcus lactis ssp. lactis. It is a natural antimicrobial agent with activity against a wide variety of Gram-positive bacteria, including food-borne pathogens such as Listeria, Staphylococcus and Clostridium. The primary target of nisin is believed to be the cell membrane. Unlike some other antimicrobial peptides, nisin does not need a receptor for its interaction with the cell membrane; however, the presence of a membrane potential is required. Nisin is a natural preservative present in cheese made with Lactococcus lactis ssp. lactis, but it is also used as a preservative in heat processed and low pH foods. Since nisin cannot be synthesized chemically, the nisin-producing Lactococcus lactis strains are used for its industrial synthesis.

The first established use of nisin was as a preservative in processed cheese products, but numerous other applications in preservation of foods and beverages have been identified. It is currently recognized as a safe food preservative in approximately 50 countries. Nisin has been used as a preservative in various pasteurized dairy products and canned vegetables, baked, high-moisture flour products, and pasteurized liquid eggs. There is interest in the use of nisin in natural cheese production. Considerable research has been carried out on the anti-listerial properties of nisin in foods and a number of applications have been proposed. Uses of nisin to control spoilage lactic acid bacteria have been identified in beer, wine, alcohol production, and high acid foods such as salad dressings. Production of highly purified nisin preparations has led to interest in the use of nisin for human ulcer therapy and mastitis control in cattle.



Lactococcus lactis and the molecule, Nisin. Modified Scanning EM from  profoodinternational.com with permission.


Starter Cultures

Starter cultures have crucial roles to play during all phases of the cheese making and maturation process. As the culture grows in the milk, it converts lactose to lactic acid. This ensures the correct pH for coagulation and influences the final moisture content of the product. The rate of acid production is critical in the manufacture of certain products, e.g. Cheddar cheese. In mechanized operations, starters are often required to produce acid at a consistently fast rate through the manufacturing period each and every day. During ripening, culture, lipolytic and proteolytic enzymes are released from the bacteria that add a balanced aroma, taste, texture, and surface appearance to the product. The negative redox potential created by starter growth in cheese also aids in preservation and the development of flavor in Cheddar and similar cheeses. Additionally, antibiotic-like substances produced by starters (e.g. nisin) may also have a role in preservation.


A commercially-available starter culture. The description reads "Direct set Mesophilic Culture- Lactococcus lactis and Lactococcus cremoris. For hard and fresh cheeses - Cheddar, Colby, Feta, Chevre and others. Use 1/4 tsp. per 2-3 gallons of milk for hard cheeses and 1/4 tsp. per 3-5 gallons of milk for soft cheeses. This package contains enough Direct set Mesophilic culture to set 16 gallons of milk. Store refrigerated (40-45 Deg F). This culture does not contain rennet." http://www.thegrape.net


Lactococcus and Vaccine Delivery

A recently discovered application of Lactococcus lactis is in the development of vaccine delivery systems. The bacterium can be genetically engineered to produce proteins from pathogenic species on their cell surfaces. Intra nasal inoculation of an animal with the modified strain will elicit an immune response to the cloned protein and provide immunity to the pathogen. For example, if one wished to provide immunity to Streptococcus pyogenes, the causative agent of strep throat, Lactococcus could be engineered to present the conserved portion of the streptococcal M protein required for streptococcal adherence and colonization to the nasopharyngeal mucosa. The resulting local immune response could protect the individual from strep throat caused by the streptococcus that exhibits that form of the M protein. This approach theoretically can be adapted to any pathogen that colonizes and or/enters via a mucosal surface in humans or animals. This includes human pathogens such as Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Bordetella pertussis and Neisseria meningitidis, among others.

More than 4 million deaths per year are due to respiratory diseases. Economical and effective vaccines against respiratory pathogens are needed for implementation in poorer countries where the disease burden is highest. Following respiratory tract infection, some pathogens may also invade the epithelial tissue, achieving systemic circulation and spread to other organs. Nasal administration of different antigen formulations using Lactococcus lactis as a delivery vehicle has shown promising results in the induction of immune responses that defeat of the pathogens at the site of infection.

Lactococcus lactis has been shown to deliver antigens that stimulate mucosal immunity to nonrespiratory pathogens, as well, including HIV, Human papilloma virus and the malarial parasite.

Some of the research papers that have employed Lactococcus lactis as a vector for vaccine delivery are cited below.

Lee, M.H., et al. 2001. Expression of Helicobacter pylori urease subunit B gene in Lactococcus lactis MG1363 and its use as a vaccine delivery system against H. pylori infection in mice. Vaccine 2001. 19:3927-3931.

Ribeiro, L.A., et al. 2002. Production and Targeting of the Brucella abortus Antigen L7/L12 in Lactococcus lactis: a First Step towards Food-Grade Live Vaccines against Brucellosis. Applied and Environmental Microbiology 2002. 68:910-916.

Xin, K.Q., et al. 2003 Immunogenicity and protective efficacy of orally administered recombinant  Lactococcus lactis expressing surface-bound HIV Env. Blood 2003. 10:223-228.

Robinson, K., et al. 2004. Mucosal and cellular immune responses elicited by recombinant strains of Lactococcus lactis expressing tetanus toxin fragment C. Infection and Immunity 2004. 72: 2753–2756.

Bermudez-Humaran, L.G., et al. 2005. A Novel Mucosal Vaccine Based on Live Lactococci Expressing E7 Antigen and IL-12 Induces Systemic and Mucosal Immune Responses and Protects Mice against Human Papillomavirus Type 16-Induced Tumors. The Journal of immunology 2005. 175:7297-7302.

Buccato, S., et al. 2006. Use of Lactococcus lactis Expressing Pili from Group B Streptococcus as a Broad-Coverage Vaccine against Streptococcal Disease. The Journal of Infectious Diseases 2006. 194:331-340.

Ramasamay, R. et al. 2006. Immunogenicity of a malaria parasite antigen displayed by Lactococcus lactis in oral immunisations. Vaccine 2006. 24:3900-3908.

Hanniffy, S.B., et al. 2007. Mucosal Delivery of a Pneumococcal Vaccine Using Lactococcus lactis Affords Protection against Respiratory Infection. The Journal of Infectious Diseases 2007. 195:185–93.


The Lactococcus Genome

Partly due to their industrial relevance, both Lactococcus lactis subspecies (lactis and cremoris) are widely used as generic LAB models for research. L. lactis ssp. cremoris, used in the production of hard cheeses, is represented by the laboratory strains LM0230 and MG1363. Similarly, L. lactis ssp. lactis is employed in soft cheese fermentations, with the workhorse strain IL1403, ubiquitous in LAB research laboratories. In 2001, the genome of strain IL1403 was sequenced leading to increased understanding of LAB genomics and related applications. Currently, there are two L. lactis ssp. cremoris that have been sequenced for public release.

A French group sequenced the genome of Lactococcus lactis ssp. lactis. The genome sequence reveals 12 enzymes called aminotransferases, some of which are used to break down complex, branched, ring-shaped, and sulfur-containing amino acids. The molecules produced when the amino acids are degraded are very important for cheese flavor. Understanding which amino acids are broken down by which enzymes could give cheese makers greater control over flavor and fragrance of their fromage.


Genome atlas of the chromosome of L. lactis MG1363. ifr.ac.uk

The sequence also led to the identification of 29 genes that are required to build the mesh-like peptidoglycan component of the bacterium's cell wall. Inducing some of these enzymes can accelerate the slow, expensive process of cheese ripening, during which the cheese ages and develops its characteristic flavor. Learning how to selectively activate some of these enzymes could revolutionize the cheese manufacturing process.

The Lactococcus lactis ssp. lactis genome has 2,365,589 units (bp) of DNA, which contain 2,310 predicted genes. About 64 percent of the genes have assigned roles in the cell, while 20 percent match other hypothetical genes with unknown function. Almost 16 percent of the genes bear no resemblance to genes from other species and are considered to be unique to this bacterium.

The L. lactis genome contains six prophages (carrying nearly 300 genes or about 14 percent of the total coding capacity) and 43 insertion elements. Sequence data also revealed new possibilities for fermentation pathways and confirmed the total lack of genes and enzymes involved in the TCA cycle although, unexpectedly, certain genes necessary for aerobic respiration were found encoded in the genome.

It is anticipated that understanding the physiology and genetic make-up of this bacterium will prove invaluable for food manufacturers as well as the pharmaceutical industry, which is exploring the capacity of L. lactis to serve as a vehicle for delivering drugs and vaccines.

For example, genomic analysis of Lactococcus lactis subsp lactis revealed the presence of several genes that encode enzymes involved in the reduction of pyruvate to various end-product other than homolactic acid, including ethanol, acetic acid, formic acid, diacetyl, acetoin and butanediol. Manipulation and understanding of the regulation of these genes could defeat or enhance the synthesis of these end products leading to strain improvement in starter cultures for cheeses, buttermilk, and other dairy products.



The pyruvate metabolism of Lactococcus lactis. LDH: lactate dehydrogenase; PDH: pyruvate dehydrogenase complex; PFL: pyruvate formate-lyase; ADHE: acetaldehyde dehydrogenase; ADHA: alcohol dehydrogenase; PTA: phosphotransacetylase; ACKA: acetate kinase, ALS/ILVB: catabolic and anabolic 2-acetolactate synthase; ALDB: acetolactate decarboxylase; BUTA: diacetyl reductase; BUTB: acetoin reductase; NOX: NADH oxidase. Oliveira et al. BMC Microbiology. 5:39 (2005).




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