Inactivation Of Bacillus Subtilis Spores By Combining Treatment Of High Pressure CO₂ And Temperature

The objective of this study was to investigate the inactivation of Bacillus subtilis spores by combined treatment of high pressure CO2(HPCD)and mild-temperature(60-90?)(HPCD + MT)and the inactivation mechanism.Besides,the inactivation of B.subtilis spores by combined treatment of HPCD + MT and nisin was also studied.The results and conclusions are as follows:(1)The inactivation kinetic of the B.subtilis spores by HPCD + MT at extensive pressures,temperatures and times was studied,and the effect of temperature,pressure,lower pH caused by CO2;and the molecular effect of CO2 during HPCD + MT treatment on B.subtilis spores was also investigated.B.subtilis spores suspended in sterilized distilled water was treated by HPCD + MT at 6.5-25 MPa and 44-91? for 0-120 min,and the treated spores was tested for viability and pyridine-2,6-dicarboxylic acid(DPA)release.Spore clumping was also tested using dynamic light scattering.The inactivation effect of spore by HPCD + MT was the combined effect of temperature,pressure,lower pH caused by CO2,and the molecular effect of CO2,rather than any effect alone.At the same temperature,the spores were more effectively inactivated by HPCD + MT than heat alone.The spores treated by HPCD + MT at 6.5 and 10 MPa exhibited a two-stage inactivation curve of shoulder and log-linear regions whereas the spores at 15-25 MPa exhibited a three-stage inactivation curve of shoulder,log-linear and tailing regions,suggesting a protective effect.The particle size of the spore suspensions increased with increasing the pressure,temperature and time,indicating the spores clumped during HPCD + MT treatment.0.1%Tween 80 as a surfactant inhibited the spore clumping and increased the inactivation of the spores by HPCD + MT,indicating that the spore clumping enhanced the resistance of spores to HPCD + MT and induced the protective effect.Approximately 90%DPA released after HPCD + MT,which indicatied the spores were germinated or the inner membrane(IM)was damaged during HPCD + MT treatment.(2)The germination and structure damage of B.subtilis spores by HPCD + MT treatment was investigated.The B.subtilis spores suspended in sterilized distilled water were subjected to heat at 0.1 MPa or HPCD at 6.5-20 MPa,and 64-86? for 0-60 min.The germination,DPA release,permeability of IM and cortex,and changes in the morphological and internal structures of spores were investigated.The HPCD + MT-treated spores did not lose heat resistance and their DPA release was lower than the inactivation,suggesting that spores did not germinate during HPCD + MT.The flow cytometry analysis suggested that the permeability of the IM and cortex of HPCD + MT-treated spores was increased.Furthermore,the DPA of the HPCD + MT-treated spores were released in parallel with their inactivation and the fluorescence photomicrographs showed that these treated spores were stained by propidium iodide,ensuring that the permeability of IM of spores was increased by HPCD + MT.The scanning electron microscopy photomicrographs showed that spores were crushed into debris or exhibited a hollowness on the surface,and the transmission electron microscopy photomicrographs exhibited an enlarged core,ruptured and indistinguishable IM and a loss of core materials in the HPCD + MT-treated spores,indicating that HPCD + MT damaged the structures of the spores.These findings suggested that HPCD + MT inactivated B.subtilis spores by directly damaging the structures of the spores,rather than inducing germination of the spores.(3)The damage of different structures of B.subtilis spore by HPCD + MT was studied,and the key event caused spore inactivation was also explored.The B.subtilis spores suspended in sterilized distilled water were subjected to heat at 0.1 MPa or HPCD at 20 MPa,and 84-86? for 0-60 min.the damge of the spore coat,IM,core protein and DNA was tested,and the germination and outgrowth of spores were also examined.HPCD + MT-treated spores were separated by equilibrium density gradient centrifugation,and the spores in the bottom were tested for viability and DPA content.Results showed that only 0.17%of these spores were alive,but their DPA content was 97.9%,that was some of the spores inactivated by HPCD retained the DPA,suggesting that HPCD+ MT did not inactivate spores by rupture the IM.These HPCD + MT-inactivated and DPA-retained spores were using for the following study as HPCD + MT-treated spores.The HPCD + MT-treated spore could germinate by L-valine or AGFK,and the germination,was much higher than the viability,indicating that HPCD + MT did not inactivate spore by damaging the GerA,GerB or GerK proteins.Notably,HPCD + MT treated-spore germination by dodecylamine(DDA)was higher than untreated spores,and that was due to the damage of the coat,which accelerated the penetration of DDA into the spores and act with SpoVA proteins.HPCD + MT-treated spores showed no mutants and the core protein damage was always slower than inactivation,suggesting that HPCD + MT did not inactivate spores by DNA or core protein damage.HPCD + MT treated spores became sensitive to high salts plates and their ability to maintain DPA was decreased.All the germinated HPCD + MT-treated spores could be stained by PI,and outgrowth of these germinated spores was blocked.These results indicated that HPCD + MT inactivated spores by damage the IM,which blocked the outgrowth of spores and caused their death.(4)The inactivation effects of HPCD + MT + nisin(simultaneous treatment of HPCD + MT and nisin,HPCD + MT + nisin),HPCD + MT?nisin(HPCD + MT was followed by nisin),and nisin?HPCD + MT(nisin was followed by HPCD + MT)treatments on B.subtilis spores in sterilized distilled water were compared.The spores were treated by HPCD + MT at 6.5 or 20 MPa,84-86? and 0-30 min,and the concentration of nisin was 0.02%.Treated spores were examined for the viability,the permeability of IM and DPA release,and structural damage by transmission electron microscopy.A synergistic effect of HPCD + MT + nisin treatment on inactivation of the spores was found,and the inactivation efficiency of the spores was HPCD + MT + nisin>HPCD +MT?nisin or nisin?HPCD + MT.Moreover,HPCD + MT + nisin caused higher IM permeability and DPA release of the spores than HPCD + MT.A possible action mode of nisin-enhanced inactivation of the spores was suggested as that HPCD + MT firstly damaged the coat and cortex of spores,then nisin penetrated into and acted on the IM of spores,which increased the damage to the IM of spores,and resulted in higher inactivation of the spores.