Impact of Nisin and Nisin-Producing Lactococcus lactis ssp. lactis on Clostridium tyrobutyricum and Bacterial Ecosystem of Cheese Matrices

Abstract

Clostridium tyrobutyricum spores survive milk pasteurization and cause late blowing of cheeses and significant economic loss. The effectiveness of nisin-producing Lactococcus lactis ssp. lactis 32 as a protective strain for control the C. tyrobutyricum growth in Cheddar cheese slurry was compared to that of encapsulated nisin-A. The encapsulated nisin was more effective, with 1.0 log₁₀ reductions of viable spores after one week at 30 °C and 4 °C. Spores were not detected for three weeks at 4 °C in cheese slurry made with 1.3% salt, or during week 2 with 2% salt. Gas production was observed after one week at 30 °C only in the control slurry made with 1.3% salt. In slurry made with the protective strain, the reduction in C. tyrobutyricum count was 0.6 log₁₀ in the second week at 4 °C with both salt concentration. At 4 °C, nisin production started in week 2 and reached 97 µg/g after four weeks. Metabarcoding analysis targeting the sequencing of 16S rRNA revealed that the genus Lactococcus dominated for four weeks at 4 °C. In cheese slurry made with 2% salt, the relative abundance of the genus Clostridium decreased significantly in the presence of nisin or the protective strain. The results indicated that both strategies are able to control the growth of Clostridium development in Cheddar cheese slurries.

Keywords: Cheddar cheese slurry; Clostridium tyrobutyricum; Lactococcus lactis; Nisin; antimicrobial peptides; dairy products; protective starter.

Figure 1

Total lactic acid bacteria count (starter culture) in Cheddar cheese slurries made with (A) 1.3% NaCl and (B) 2% NaCl, stored for four weeks at 4 °C, pH 5.3 (n = 3), different letters indicate significant difference (p < 0.05).

Figure 1

Total lactic acid bacteria count (starter culture) in Cheddar cheese slurries made with (A) 1.3% NaCl and (B) 2% NaCl, stored for four weeks at 4 °C, pH 5.3 (n = 3), different letters indicate significant difference (p < 0.05).

Figure 2

Total lactic acid bacteria count in Cheddar cheese slurries made with (A) 1.3% NaCl and (B) 2% NaCl stored for two weeks at 30 °C, (n = 3), Different letters indicate significant difference (p < 0.05).

Figure 2

Total lactic acid bacteria count in Cheddar cheese slurries made with (A) 1.3% NaCl and (B) 2% NaCl stored for two weeks at 30 °C, (n = 3), Different letters indicate significant difference (p < 0.05).

Figure 3

Clostridium tyrobutyricum count in Cheddar cheese slurries adjusted to pH 5.3 and stored for four weeks at 4 °C, (A) 1.3% NaCl and (B) 2% NaCl (n = 3), Different letters indicate significant difference (p < 0.05).

Figure 3

Clostridium tyrobutyricum count in Cheddar cheese slurries adjusted to pH 5.3 and stored for four weeks at 4 °C, (A) 1.3% NaCl and (B) 2% NaCl (n = 3), Different letters indicate significant difference (p < 0.05).

Figure 4

Gas swelling of Cheddar cheese slurry (made with 1.3% NaCl) by Clostridium tyrobutyricum after one week at 30 °C, left: control; right: containing encapsulated nisin-A.

Figure 5

Nisin production in Cheddar cheese slurry containing bacteriocin-producing Lactococcus lactis ssp. lactis 32 (protective starter), during 4 weeks at 4 °C (n = 3), Different letters indicate significant difference (p < 0.05).

Figure 6

Nisin released in Cheddar cheese slurry containing the encapsulated bacteriocin, during 4 weeks at 4 °C (n = 3), W = weeks, Different letters indicate significant difference (p < 0.05).

Figure 7

Nisin production in Cheddar cheese slurry containing bacteriocin-producing Lactococcus lactis ssp. lactis 32 (protective starter), during two weeks at 30 °C (n = 3), Different letters indicate significant difference (p < 0.05).

Figure 8

Nisin released in Cheddar cheese slurry containing the encapsulated bacteriocin, during two weeks at 30 °C (n = 3), Different letters indicate significant difference (p < 0.05).

Figure 9

Relative abundance of bacterial genera, based on 16S rRNA, in Cheddar cheese slurries containing C. tyrobutricum made with 1.3% NaCl. CLOTY) control, EN) Encapsulated nisin and LACLA32) protective strain treated group, stored for 4 weeks at 4 °C or for two weeks at 30 °C (D1 = time zero).

Figure 10

Relative abundance of bacterial genera, based on 16S rRNA, in Cheddar cheese slurries containing C. tyrobutricum made with 2% NaCl. CLOTY) control, EN) Encapsulated nisin and LACLA32) protective strain treated group, stored for four weeks at 4 °C or for two weeks at 30 °C (D1 = time zero).