Inhibition and Mechanism of Citral on Bacillus cereus Vegetative Cells, Spores, and Biofilms.

Bacillus cereus causes food poisoning by producing toxins that cause diarrhea and vomiting and, in severe cases, endocarditis, meningitis, and other diseases. It also tends to form biofilms and spores that lead to contamination of the food production environment. Citral is a potent natural antibacterial agent, but its antibacterial activity against B. cereus has not been extensively studied. In this study, we first determined the minimum inhibitory concentrations and minimum bactericidal concentrations, growth curves, killing effect in different media, membrane potential, intracellular adenosine triphosphate (ATP), reactive oxygen species levels, and morphology of vegetative cells, followed by germination rate, morphology, germination state of spores, and finally biofilm clearance effect. The results showed that the minimum inhibitory concentrations and minimum bactericidal concentrations of citral against bacteria ranged from 100 to 800 µg/mL. The lag phase of bacteria was effectively prolonged by citral, and the growth rate of bacteria was slowed down. Bacteria in Luria-Bertani broth were reduced to below the detection limit by citral at 800 µg/mL within 0.5 h. Bacteria in rice were reduced to 3 log CFU/g by citral at 4000 µg/mL within 0.5 h. After treatment with citral, intracellular ATP concentration was reduced, membrane potential was altered, intracellular reactive oxygen species concentration was increased, and normal cell morphology was altered. After treatment with citral at 400 µg/mL, spore germination rate was reduced to 16.71%, spore morphology was affected, and spore germination state was altered. It also had a good effect on biofilm removal. The present study showed that citral had good bacteriostatic activity against B. cereus vegetative cells and its spores and also had a good clearance effect on its biofilm. Citral has the potential to be used as a bacteriostatic substance for the control of B. cereus in food industry production.

Antimicrobial Activity of Eugenol Against Bacillus cereus and Its Application in Skim Milk.

Bacillus cereus is a foodborne pathogen widely distributed in the large-scale catering industry and produces spores. The study explored the antibacterial activity, potential mechanism of eugenol against B. cereus, and spores with germination rate. The minimum inhibitory concentration (MIC; 0.6 mg/mL) of eugenol to six B. cereus strains was compared with the control; B. cereus treated with eugenol had a longer lag phase. Eugenol at a concentration of more than 1/2MIC decreased viable B. cereus (∼5.7 log colony-forming unit [CFU]/mL) counts below detectable limits within 2 h, and eugenol of 3MIC reduced B. cereus (∼5.9 log CFU/mL) in skim milk below detectable limits within 30 min. The pH values of skim milk were unaffected by the addition of eugenol. The ΔE values below 2 show that the color variations of skim milk were not visible to the human eye. For sensory evaluation, eugenol did not significantly affect the color or structural integrity of the skim milk. It had a negative impact on the flavor and general sensory acceptance of the treated milk. Eugenol hyperpolarized B. cereus cell membrane, decreased intracellular ATP concentration, and increased intracellular reactive oxygen species contents and extracellular malondialdehyde contents, resulting in the cell membrane of B. cereus being damaged and permeabilized, and cell morphology being changed. In addition, according to the viable count, confocal laser scanning microscopy, and spore morphology changes, eugenol reduced the germination rate of B. cereus spores. These findings suggest that eugenol can be used as a new natural antibacterial agent to control B. cereus and spores in the food production chain.

Antimicrobial Activity and Action Mechanism of Thymoquinone against Bacillus cereus and Its Spores.

In this study, thymoquinone (TQ), a natural active substance, was investigated for its antibacterial activity against Bacillus cereus, and its inhibitory effect on B. cereus in reconstituted infant formula (RIF) was evaluated. In addition, the inhibitory effect of TQ on B. cereus spore germination was explored. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of TQ against eight B. cereus strains ranged from 4.0 to 8.0 µg/mL, whereas B. cereus treated with TQ displayed a longer lag phase than the untreated control. TQ exerted a good bactericidal effect on B. cereus in Luria-Bertani broth. In addition, TQ obviously reduced the intracellular ATP concentration of B. cereus, which caused depolarization of the cell membrane, increased the intracellular reactive oxygen species level, impaired the cell morphology, and destroyed proteins or inhibited proteins synthesis. This provides a mechanism for its bacteriostatic effect. TQ also inactivated B. cereus growth in RIF. Moreover, reverse transcription-quantitative polymerase chain reaction illustrated that TQ downregulated the transcription of genes related to hemolysin, non-hemolytic enterotoxin, enterotoxin, and cytotoxin K. Meanwhile, TQ displayed the ability to inhibit the germination of B. cereus spores. These findings indicate that TQ, as an effective natural antimicrobial preservative, has potential applications in controlling food contamination and foodborne diseases caused by B. cereus.