A Multibacteriocin Cheese Starter System, Comprising Nisin and Lacticin 3147 in Lactococcus lactis, in Combination with Plantaricin from Lactobacillus plantarum

Abstract

Functional starter cultures demonstrating superior technological and food safety properties are advantageous to the food fermentation industry. We evaluated the efficacies of single- and double-bacteriocin-producing starters of Lactococcus lactis capable of producing the class I bacteriocins nisin A and/or lacticin 3147 in terms of starter performance. Single producers were generated by mobilizing the conjugative bacteriophage resistance plasmid pMRC01, carrying lacticin genetic determinants, or the conjugative transposon Tn5276, carrying nisin genetic determinants, to the commercial starter L. lactis CSK2775. The effect of bacteriocin coproduction was examined by superimposing pMRC01 into the newly constructed nisin transconjugant. Transconjugants were improved with regard to antimicrobial activity and bacteriophage insensitivity compared to the recipient strain, and the double producer was immune to both bacteriocins. Bacteriocin production in the starter was stable, although the recipient strain proved to be a more efficient acidifier than transconjugant derivatives. Overall, combinations of class I bacteriocins (the double producer or a combination of single producers) proved to be as effective as individual bacteriocins for controlling Listeria innocua growth in laboratory-scale cheeses. However, using the double producer in combination with the class II bacteriocin producer Lactobacillus plantarum or using the lacticin producer with the class II producer proved to be most effective for reducing bacterial load. As emergence of bacteriocin tolerance was reduced 10-fold in the presence of nisin and lacticin, we suggest that the double producer in conjunction with the class II producer could serve as a protective culture providing a food-grade, multihurdle approach to control pathogenic growth in a variety of industrial applications.IMPORTANCE We generated a suite of single- and double-bacteriocin-producing starter cultures capable of generating the class I bacteriocin lacticin 3147 or nisin or both bacteriocins simultaneously via conjugation. The transconjugants exhibited improved bacteriophage resistance and antimicrobial activity. The single producers proved to be as effective as the double-bacteriocin producer at reducing Listeria numbers in laboratory-scale cheese. However, combining the double producer or the lacticin-producing starter with a class II bacteriocin producer, Lactobacillus plantarum LMG P-26358, proved to be most effective at reducing Listeria numbers and was significantly better than a combination of the three bacteriocin-producing strains, as the double producer is not inhibited by either of the class I bacteriocins. Since the simultaneous use of lacticin and nisin should reduce the emergence of bacteriocin-tolerant derivatives, this study suggests that a protective starter system produced by bacteriocin stacking is a worthwhile multihurdle approach for food safety applications.

Keywords: bacteriocins; food safety; protective culture.

FIG 1

PCR amplification using pMRC01-specific primers (orf27, orf49, orf51, and orf52) to detect the presence of pMRC01 in L. lactis CSK3594 and CSK3533 and using primers designed to regions of the nisin operon (nisA and nisFEG) to confirm the presence of nisin genetic determinants in L. lactis CSK3281 and L. lactis CSK3533. Lane M, 100-bp DNA ladder (New England BioLabs).

FIG 2

Colony mass spectrometry analysis of L. lactis CSK2775, L. lactis CSK3281 (nisin transconjugant, Nis⁺), L. lactis CSK3594 (lacticin transconjugant, Ltn⁺), and L. lactis CSK3533 (nisin and lacticin double producer, Nis⁺, Ltn⁺). Masses corresponding to the bacteriocins are indicated. Inset photos show inhibition zones produced by each strain against the indicator strain L. lactis HP.

FIG 3

Acidification profiles of L. lactis CSK2775 (□), L. lactis CSK3594 (lacticin) (■), L. lactis CSK3281 (nisin) (○), and L. lactis CSK3533 (nisin and lacticin) (●) grown in 10% reconstituted skim milk.

FIG 4

Counts of viable L. innocua cells in laboratory-scale cheeses. Bacteriocin-containing vats were compared to vat 1 (no bacteriocin) at each week. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

MALDI-TOF MS analysis of vat 7 (lacticin, nisin, and plantaricin) (A), vat 4 (lacticin and nisin) (B), vat 5 (nisin and plantaricin) (C), and vat 6 (lacticin and plantaricin) (D). Masses corresponding to the bacteriocins are indicated. Inset photos show inhibition zones produced by correct-mass-containing fractions against the indicator strain L. lactis HP (nisin and lacticin) or L. innocua (plantaricin). F, fraction.

FIG 5

MALDI-TOF MS analysis of vat 7 (lacticin, nisin, and plantaricin) (A), vat 4 (lacticin and nisin) (B), vat 5 (nisin and plantaricin) (C), and vat 6 (lacticin and plantaricin) (D). Masses corresponding to the bacteriocins are indicated. Inset photos show inhibition zones produced by correct-mass-containing fractions against the indicator strain L. lactis HP (nisin and lacticin) or L. innocua (plantaricin). F, fraction.

FIG 5

MALDI-TOF MS analysis of vat 7 (lacticin, nisin, and plantaricin) (A), vat 4 (lacticin and nisin) (B), vat 5 (nisin and plantaricin) (C), and vat 6 (lacticin and plantaricin) (D). Masses corresponding to the bacteriocins are indicated. Inset photos show inhibition zones produced by correct-mass-containing fractions against the indicator strain L. lactis HP (nisin and lacticin) or L. innocua (plantaricin). F, fraction.

FIG 5

MALDI-TOF MS analysis of vat 7 (lacticin, nisin, and plantaricin) (A), vat 4 (lacticin and nisin) (B), vat 5 (nisin and plantaricin) (C), and vat 6 (lacticin and plantaricin) (D). Masses corresponding to the bacteriocins are indicated. Inset photos show inhibition zones produced by correct-mass-containing fractions against the indicator strain L. lactis HP (nisin and lacticin) or L. innocua (plantaricin). F, fraction.