Lactococcus lactis Wisconsin State Microbe

Bacteriology at UW- Madison

The Microbial World

University of Wisconsin - Madison

Lactococcus lactis Wisconsin's State Microbe

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 molecules, called 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. But 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
Colby, Cheddar, cottage, cream	Lactococcus lactis ssp. lactis
Blue	Lactococcus lactis ssp. cremoris	Citrate⁺ Lactococcus lactis ssp. lactis Penicillium roqueforti
Blue	Lactococcus lactis ssp. lactis
Fermented milk
Buttermilk	Lactococcus lactis ssp. cremoris	Leuconostoc spp. Citrate⁺ Lactococcus lactis ssp. lactis
Buttermilk	Lactococcus lactis ssp. lactis
Sour cream	Lactococcus lactis ssp. cremoris	None
Sour cream	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 egg. 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."

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

Due to their industrial importance, both Lactococcus lactis subspecies are widely used as models in lactic acid bacteria research. L. lactis ssp. cremoris is represented by the laboratory strains LM0230 and MG1363. Lactococcus lactis ssp. lactis is represented in research laboratories by the "workhorse strain", IL1403. Beginning in 2001, the genomes of these three strains have been sequenced, which is leading to an ever increased understanding of these bacteria especially related to their applications.

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

Comparative genomics will provide information about how the various strains of Lactococcus have adapted to their environment, and how they use available nutrients. Analysis of the genome has also revealed several surprising features, including genes suggesting that the bacterium can perform aerobic respiration and can undergo horizontal gene transmission by the process of transformation. This research marks a critical step towards understanding and manipulating Lactococcus lactis, in particular for improving the flavor, texture, and preservation of 10 million tons of cheese produced annually. Knowledge of the genome sequence will also facilitate current and future work that aims to exploit Lactococcus lactis for a variety of medical and health maintenance applications.