"This work will impact the dairy fermentation industry by providing more avenues to improve cultures. Improved cultures should increase revenue for the industry and provide better tasting and safer food for the consumer."-- Daniel O'Sullivan

The use of lactic cultures for the production of fermented food products such as cheese, yogurt and fermented milks is a multi-billion dollar industry. The success of the industry depends on the consistent performance of live cultures in the fermentations. One problem that can occur is if the culture is attacked by phage (viruses that attack bacteria). Phage are particularly troublesome for dairy fermentations and if left unchecked they could quickly wipe out an entire culture, resulting in a failed fermentation. Our approach in this research project (MIN-18-055) is to study natural defense systems against phage that have naturally evolved, with the goal of incorporating them in cultures used for specific fermentations. The more defense systems that are built into cultures the more resistant they will be to phage attack. Using just a single defense is a short term fix as the phage quickly evolve and overcome the defense. Multiple defenses that attack different parts of the phage developmental cycle greatly limit the ability of phage to evolve resistance.

In this project, we have characterized two novel phage defenses that were isolated from a strain of Lactococcus lactis, a common cheese starter culture. One of these defenses is a restriction modification (R/M) system, which means it degrades the phage DNA when it enters the culture. This R/M system functions differently than the other R/M systems that have been characterized, suggesting it should be a useful addition to the arsenal of phage defenses available for cultures. Through its natural evolution, it also acquired an IS element, which is a piece of DNA that can help move the R/M genes, possibly onto the phage. This has been shown to occur in the past when a new phage took out a number of commercial cheese fermentations by acquiring the R/M resistant gene via a nearby IS element. We therefore precisely removed it, resulting in an improved R/M defense for lactic cultures.

The other phage defense system we have characterized is termed an abortive infection system (Abi). This means the reproduction of the phage is impeded. Specifically, in this case the Abi system prevents the phage from replicating its DNA. The combination of an R/M and Abi system is like a one-two punch, where the phage reproduction is hit at two different steps. An analogous situation is the development of new drugs to combat HIV infection, whereby the 'cocktail' of drugs that patients now take hit the virus at different steps in its reproduction cycle.

Another problem that can occur with foods is allowing the growth of spoilage or pathogenic microorganisms. Lactic cultures can combat them by producing bacteriocins, which are small proteins that can kill other bacteria. The fact that they are proteins and readily digested by humans differentiates them from antibiotics. Prehaps the best known bacteriocin is nisin, which is produced by certain strains of Lactococcus lactis. This bacteriocin is very effective at controlling the growth of many spoilage and pathogenic bacteria. Purified nisin would be considered an ingredient and would therefore require specific approval by the FDA. Currently, it is only approved in the United States as an ingredient for processed cheese. However, the use of culture fermentates from nisin-producing lactococci is a very effective way of extending the shelf life and safety of foods. This is permitted by the FDA as lactic cultures have GRAS (generally regarded as safe) status and therefore do not require specific approval. To ensure consistent production of nisin by the culture, it is necessary to understand all the nuances involved in its production. We have uncovered novel ways in which the culture switches on and off production. Understanding these mechanisms will provide the information to fine tune the fermentation to maximize production.

As only a select few strains of Lactococcus lactic can produce nisin, this limits the use of this antimicrobial compound. Engineering other cultures that are used in different foods to produce nisin would expand the uses for this effective compound. We are currently working with a dairy Enterococcus culture that contains the genes for nisin production, but doesn't produce it. After analyzing nisin gene expression in this bacterium, we found that the genes involved in immunity to nisin were expressed, but the genes involved in nisin production were not. As expression of the nisin genes requires an induction system that is activated by nisin molecules outside the bacterial cell, we added some nisin to the culture medium. This had the effect of switching on the nisin genes and enabling some nisin to be produced. This was the first time that a bacterium other than L. lactis produced nisin. Furthering the understanding of how the nisin gene systems function will enable us to improve production and expand the avenues of use for this versatile protein.

This work will impact the dairy fermentation industry by providing more avenues to improve cultures. Improved cultures should increase revenue for the industry and provide better tasting and safer food for the consumer. Currently, slowed fermentations are costly to manufacturers in time lost and also can result in a lower-quality product, which sells for a lower price.

Better and safer foods should improve the quality of life for many in the future.

Minnesota County
All Counties

Publication for Further Information
Chandrapati, S., and D. J. O'Sullivan. (2002). 'Characterization of the promoter regions involved in galactose and nisin mediated induction of the nisA gene in Lactococcus lactis ATCC 11454'. Mol. Micorbiol. 46:467-477.

Web Site for Further Information

Primary Researcher
Daniel O'Sullivan
dosulliv@umn.edu

Participating Individuals
Larry McKay
4 Undergraduate Students
6 Graduate Students
4 Post Docs

Participating Institutions
USDA Minnesota South Dakota Dairy Foods Research Center Dairy Management Inc

Department
Food Science and Nutrition

Government Funding Type
External
Federal
State

Additional Funding Information
Average annual funding is $200,000.00