Inactivation Effect of Germination Combined with Cold Plasma Treatment on Bacillus licheniformis Spores

Abstract

Food spoilage, primarily caused by spore-forming bacteria, has become a critical concern since it results in substantial economic losses within the food industry. Past investigations have successfully identified Bacillus licheniformis as the main bacterium responsible for spoilage in roast chicken. In this study, we screened a new sterilization combination from 16 germinants and 4 cold plasma conditions, respectively. Among them, the combination of "A"GFNa-1 (composed of 60 mmol/L L-alanine, 10 mmol/L D-glucose, 10 mmol/L D-fructose, and 1 g/L NaCl) with cold plasma treatment (packed with 100% argon at 70 kV) proved effective in deactivating B. licheniformis spores, resulting in a reduction of approximately 2.1 log CFU/mL. Furthermore, we exposed the spores to different conditions: CK (no germination, no cold plasma), MF (germination only), CP (no germination, 100% argon packed, 70 kV cold plasma treatment for 3 min), and MF + CP (germination for 5 h, 100% argon packed, 70 kV cold plasma treatment for 3 min). The results of heat inactivation and dipicolinic acid (DPA) release rate demonstrated that cold plasma treatment effectively inactivated both spores and vegetative cells without inducing germination. Additionally, the reduced survival under hyperosmotic conditions and the presence of distinct red fluorescence patterns observed through confocal laser scanning microscopy (CLSM) collectively suggest that cold plasma treatment disrupts the inner membrane structure and leads to the inactivation of B. licheniformis. Overall, our findings indicate a spore clearance rate of 99.2% and suggest that the combination of efficient germinants and cold plasma treatment holds promise as a viable approach to mitigate spore contamination in the food industry.

Keywords: Bacillus licheniformis; cold plasma; germination; inactivation; spores.

Figure 1

Preliminary screening of the germination effect of germinants. (A–I) Growth curves of spores under the influence of different concentrations (30 mmol/L, 40 mmol/L, 50 mmol/L, 60 mmol/L, 70 mmol/L) of D-glucose (G), D-fructose (F), D-galactose (Gal), L-Alanine (Ala), L-Asparagine (Asp), L-Valine (Val), L-Proline (Pro), L-Lysine (Lys), and L-Histidine (His). (J,K) Growth curves of spores under the influence of inosine at concentrations of 4 mmol/L, 6 mmol/L, and 8 mmol/L, and DPA at concentrations of 4 mmol/L, 6 mmol/L, and 8 mmol/L. (L) Growth curve of spores under the influence of AGFK (10 mmol/L Asp, 10 mmol/L G, 10 mmol/L F, 50 mmol/L KCl). (M–P) Growth curves of spores under the influence of Ala (60 mmol/L), G (10 mmol/L), and F (10 mmol/L) in the presence of KCl/NaCl/MgCl₂/CaCl₂ at concentrations of 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, and 5 g/L. For detailed information on the treatment groups, please refer to Table 1.

Figure 2

Further screening of the germination effect of germinants. (A) Thermal inactivation and germination rate of spores under the influence of eight different germinants: Ala (60 mmol/L), AGFK (10 mmol/L Asp, 10 mmol/L G, 10 mmol/L F, 50 mmol/L KCl), “A”GFNa-1 (60 mmol/L Ala, 10 mmol/L G, 10 mmol/L F, 1 g/L NaCl), “A”GFNa-5 (60 mmol/L Ala, 10 mmol/L G, 10 mmol/L F, 5 g/L NaCl), “A”GFMg-0.05 (60 mmol/L Ala, 10 mmol/L G, 10 mmol/L F, 0.05 g/L MgCl₂), “A”GFCa-1 (60 mmol/L Ala, 10 mmol/L G, 10 mmol/L F, 1 g/L CaCl₂), and “A”GFCa-5 (60 mmol/L Ala, 10 mmol/L G, 10 mmol/L F, 5 g/L CaCl₂). (B) Fluorescence excitation and emission spectra of the TbCl₃·6H₂O-DPA complex. (C) Standard curve depicting the relationship between DPA concentration and fluorescence intensity. (D) Effects of the eight different germination factors on DPA release for B. licheniformis spores. Detailed information regarding the treatment groups can be found in Table 1. Error bars represent the standard deviations of the mean (n = 3). Means with different letters were significantly different (p < 0.05).

Figure 3

(A) Thermal inactivation of B. licheniformis under different conditions: air (100% air, no cold plasma), Ar (100% argon, no cold plasma), C-50 (100% argon packed, 50 kV cold plasma), C-60 (100% argon packed, 60 kV cold plasma), and C-70 (100% argon packed, 70 kV cold plasma). (B) Thermal inactivation of B. licheniformis under different treatments: CK (no germination, no cold plasma), MF (germination only), CP (no germination, 100% argon packed, 70 kV cold plasma for 3 min), and MF + CP (germination for 5 h, 100% argon packed, 70 kV cold plasma for 3 min). (C) Determination of B. licheniformis growth curves under different treatments: CK, MF, CP, and CP + MF. (D) DPA release rate of B. licheniformis spores under different treatments: CK, MF, CP, and CP + MF. (E) Survival status of B. licheniformis spores in hypertonic medium with different conditions: NA, NA + 1 mol/L NaCl, and NA + 1 mol/L NaCl + 50 mmol/L glucose. (F) Analysis of B. licheniformis spores using confocal laser scanning microscope (CLSM) under different treatments: MF, CP, and MF + CP. Error bars represent the standard deviations of the mean (n = 3). Upper case letters indicate significant differences between groups (p < 0.05) and lowercase letters indicate significant differences within groups (p < 0.05).