Strategy for Manipulation of Cheese Flora Using Combinations of Lacticin 3147-Producing and -Resistant Cultures

Abstract

The aim of the present study was to develop adjunct strains which can grow in the presence of bacteriocin produced by lacticin 3147-producing starters in fermented products such as cheese. A Lactobacillus paracasei subsp. paracasei strain (DPC5336) was isolated from a well-flavored, commercial cheddar cheese and exposed to increasing concentrations (up to 4,100 arbitrary units [AU]/ml) of lantibiotic lacticin 3147. This approach generated a stable, more-resistant variant of the isolate (DPC5337), which was 32 times less sensitive to lacticin 3147 than DPC5336. The performance of DPC5336 was compared to that of DPC5337 as adjunct cultures in two separate trials using either Lactococcus lactis DPC3147 (a natural producer) or L. lactis DPC4275 (a lacticin 3147-producing transconjugant) as the starter. These lacticin 3147-producing starters were previously shown to control adventitious nonstarter lactic acid bacteria in cheddar cheese. Lacticin 3147 was produced and remained stable during ripening, with levels of either 1,280 or 640 AU/g detected after 6 months of ripening. The more-resistant adjunct culture survived and grew in the presence of the bacteriocin in each trial, reaching levels of 10 7 CFU/g during ripening, in contrast to the sensitive strain, which was present at levels 100-to 1,000-fold lower. Furthermore, randomly amplified polymorphic DNA-PCR was employed to demonstrate that the resistant adjunct strain comprised the dominant microflora in the test cheeses during ripening.Members of the lactic acid bacteria play an essential role in a wide variety of food fermentations, with Lactococcus lactis representing the primary starter cultures exploited for cheddar cheese manufacture. In addition, a population of adventitious microflora, otherwise known as nonstarter lactic acid bacteria (NSLAB), can proliferate during ripening and often form the dominant flora in the cheese. The precise role of NSLAB strains in flavor development remains unclear; however, whether their effect is positive or negative, they certainly contribute to the unpredictability associated with cheddar cheese quality (7). Importantly, NSLAB have also been associated with defects in cheese such as the formation of calcium lactate crystals (27) and slit formation.Given increasing consumer demand, the challenge now facing the cheesemaker is the development of a safe, well-flavored, consistent cheddar. One approach to improving cheese flavor has been the deliberate addition of selected adjunct lactobacilli during manufacture which would impart beneficial qualities (7). Although some studies reported cheese with improved flavor, in other studies adjunct cultures were responsible for flavor defects (13,14,20,23). In addition, comparisons with control vats were difficult to interpret due to contamination with the adjunct strain or with adventitious NSLAB, which eventually reach levels similar to those in the test vat (1, 28). Hence, in order to study the relevance of NSLAB, as well as examine the role of adjunct cultures in cheddar flavor, th...

Preservation and fermentation: past, present and future

“…Since this microflora is often associated with quality defects in the product, such as crystal formation, flavour defects and slit defects, use of these bacteriocin-producing cultures should offer manufacturers greater predictability in terms of product quality. In an extension of this concept, Lactobacillus strains exhibiting greater resistance to the bacteriocin have now been isolated which can be used as adjuncts to lacticin-producing starters (Ryan et al, 2001). Consequently, it should be possible to isolate lacticin-resistant derivatives of either flavour adjunct or probiotic strains to be used with lacticin-producing starters.…”

Use of lacticin 481 to facilitate delivery of the bacteriophage resistance plasmid, pCBG104 to cheese starters

“…In some cases components of the starter system are derived from the nonstarter flora because of their positive contribution to cheese flavour. A method for rendering these strains resistant to bacteriocin has previously been developed in this laboratory (Ryan et al 2001) whereby a Lactobacillus paracasei strain which had been isolated from the nonstarter Cheddar cheese flora was made tolerant to bacteriocin. This approach readily permits the controlled use of nonstarter strains which have a beneficial effect on cheese flavour.…”

Insertional Mutagenesis To Generate Lantibiotic Resistance in Lactococcus lactis

“…Despite being one of the most extensively characterized lantibiotics, relatively few studies have investigated the development of resistance to this antimicrobial. Studies so far have established that low-level resistance can occur at low frequencies after a single exposure to low concentrations of lacticin 3147 (7,17,26) and that mutants can become moderately more resistant following repeated exposure to increasing lantibiotic concentrations (17,27). To date, the identity of the genes that contribute to lacticin 3147 resistance is unknown.…”

“…This could potentially give control over the biota in food fermentations with the addition of lantibiotics for food safety purposes. This principle was previously investigated using a spontaneously arising lacticin 3147-resistant variant of Lactobacillus paracasei in conjunction with a lacticin 3147-producing starter and resulted in increased control of the nonstarter lactic acid bacteria in cheese manufacture (27). However, as the stability of a spontaneously generated mutant can be variable, the creation of stable lantibiotic-resistant starters and adjuncts could be greatly beneficial.…”