Production and Antimicrobial Activity of Nisin Under Enological Conditions

Abstract

Lactic acid bacteria (LAB) are responsible for the malolactic fermentation of wines, and, therefore, controlling the growth of these bacteria is a key factor for elaborating premium wines. Sulfur dioxide has been traditionally used as an efficient antimicrobial and antioxidant agent, however, nowadays consumers' demand tends toward a reduction of sulfur dioxide levels in wine and other fermented foods. A previous study of our research group had demonstrated the effectiveness of the bacteriocin nisin to inhibit the growth of enological LAB, and its activity had been tested in culture broths. The aim of this study was to investigate the possibility of controlling the growth of bacteria in wine by the use of nisin in combination with sulfur dioxide, and to study nisin production by the natural producer Lactococcus lactis LM29 under enological conditions. Our results showed that L. lactis LM29 produced nisin in the presence of 2 and 4% ethanol (v/v), while higher concentrations of ethanol fully inhibited the production of nisin. We obtained a nisin enriched active extract (NAE) from the cell-free supernatant of a culture of L. lactis LM29 in MRS broth containing 60% (v/v) sterile grape juice, and the extract was fully active in inhibiting the growth of the enological LAB tested by the microtiter method. Moreover, the nisin concentration of the obtained NAE could actually prevent the formation of an undesirable biofilm of LAB strains. Finally, our results of wine ageing under winery conditions showed that the use of 50 mg/L nisin decreased fourfold the concentration of sulfur dioxide required to prevent LAB growth in the wines.

Keywords: Lactococcus lactis; bacteriocin; lactic acid bacteria; nisin; sulfur dioxide; wine; winemaking.

FIGURE 1

LAB populations in wines submitted to aging with addition of different combinations of nisin and potassium metabisulphite. Bacterial populations (log₁₀CFU/mL) were measured after 3 months of aging. Potassium metabisulphite concentrations: 18 mg/L; 25 mg/L; 100 mg/L nisin concentrations: A = 0 mg/L, B = 0.78 mg/L, C = 6.25 mg/L, and D = 50 mg/L. Different letters (a, b, c, d, e) indicate significant differences (p < 0.05; variable parameter: nisin concentration) among data obtained for the wines treated with the same concentration of potassium metabisulphite.

FIGURE 2

Biofilm formation and bacterial growth in presence of nisin and ethanol in the culture broth of planktonic cells, (A) biofilm was quantified after 48 h incubation without nisin (solid color bars) or with 6 μg/mL nisin (diagonal striped bars) in the culture broth and with (0–8%) ethanol: dashed line (1 a.u.): above which it is considered positive biofilm formation, (B) bacterial growth was quantified after 48 h incubation without nisin (solid color bars) or with 6 μg/mL nisin (diagonal striped bars) in the culture broth and with (0–8%) ethanol; dashed line: initial bacterial population above which it is positive the bacterial growth of L. mesenteroides J32 (black bars) and O. oeni IS151 (gray bars).

FIGURE 3

Addition of fresh culture broth containing binary combinations of nisin and ethanol on a preformed bacterial biofilm. The preformed biofilm of L. mesenteroides J32 in absence of antimicrobials, was quantified after 24 h incubation with fresh culture broth containing: 0 μg/ml nisin (black bars), 6 μ/mL nisin (diagonal striped bars), 200 μg/ml (horizontal striped bars) in binary combinations with ethanol (0–8%); dashed line: initial preformed biofilm.

FIGURE 4

Growth curve and bacteriocin activity of L. lactis LM29. Bacterial population (log₁₀CFU/mL) (gray squares) was measured over a period of 48 h incubation in MRS broth, Bacteriocin activity (AU) (black bars) was measured by the microtiter assay against the indicator strain P. pentosaceus FBB63.

FIGURE 5

Fluorescence microscopy images of L. lactis LM29 after 24 h incubation either in presence or in absence of ethanol in the culture broth. (A,B) Cells incubated in MRS broth. (C,D) Cells incubated in MRS broth with 12% ethanol. (A,C) Non-viable bacterial cells, with damaged cytoplasmic membrane, stained with propidium iodide that fluoresce red. (B,D) Viable cells, with intact cytoplasmic membrane, stained with Syto 9 that fluoresce green.

FIGURE 6

Growth curves and bacteriocin activity of L. lactis LM29 in presence of different ethanol concentrations. Bacterial population (log₁₀CFU/mL) (continuous line) was measured over a period of 48 h incubation. Bacteriocin activity (AU) (dashed line) was measured by the microtiter assay against the indicator strain P. pentosaceus FBB63. Ethanol concentrations: 0, 2, 4, 8, 12%.