Inactivation of Bacillus subtilis spores by combined treatment of PATS and potassium sorbate
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摘要:
枯草杆菌芽孢是极难被杀灭的细菌之一，为探究山梨酸钾与压力辅助热杀菌（pressure assisted thermal sterilization，PATS）对枯草杆菌芽孢的联合作用效果，该研究以OD600值（optical density at 600 nm）、OD260值（optical density at 260 nm）、OD280值（optical density at 280 nm）、2, 6-吡啶二羧酸释放率表征芽孢内容物的释放情况，扫描电镜、流式细胞术、傅里叶红外光谱仪、激光粒度仪检测芽孢结构变化情况。结果表明，山梨酸钾强化了PATS的杀菌效果，200 MPa-75 ℃处理杀灭了2.63 lg CFU/mL芽孢，添加2 g/L山梨酸钾后，杀灭了3.24 lg CFU/mL芽孢。较单独PATS处理，添加山梨酸钾后，促进了芽孢内容物的释放，内膜通透性增加，芽孢粒径显著减小，内膜、细胞壁等结构受损程度加剧。同时，Na+ /K+-ATPase活力显著降低，膜电位平衡被破坏，基本生理代谢功能紊乱。总之，PATS结合山梨酸钾对枯草芽孢杆菌芽孢有协同杀菌作用，且随着山梨酸钾添加量或温度的升高而增强，有利于促进该技术在食品杀菌中的应用。
Abstract:Bacterial spores are formed by cells of Clostridium, Bacillus and related genera as a response to a lack of one or more nutrients. These spores are dormant and resistant to inactivation treatments, including heat, radiation, preservatives, and high-pressure. Particularly, spores can germinate and develop in food products, resulting in the food spoilage or instances of foodborne illness. Bacillus subtilis spores are inactivated in food at high temperatures exceeding 100 ℃ during the traditional food processing. Nevertheless, high temperatures can pose a significant impact on food quality. This study aims to mitigate this temperature effect for the lower sterilization temperature. Potassium sorbate was integrated with PATS (pressure assisted thermal sterilization) into inactivate Bacillus subtilis spores. The release of important intraspore substances was established by spectrophotometry testing the OD600 (optical density at 600 nm) value, OD260 (optical density at 260 nm) value, OD280 (optical density at 280 nm) value, and DPA (2,6-pyridine dicarboxylic acid) levels. And the spore structure was examined via SEM (scanning electron microscopy), flow cytometry, FTIR (Fourier transform infrared spectroscopy), and PSD (particle size distribution analysis). The addition of potassium sorbate was improved the sporicidal effect of PATS. The surviving spores plate counting demonstrated that the initial viable spore concentration was approximately 1.5×108 CFU/mL. Treatment with 200 MPa-75 ℃ inactivated 2.63 lg CFU/mLspores, and the combined treatment of 200 MPa-75 ℃ and 2 g/L potassium sorbate inactivated 3.24 lg CFU/mLspores. The untreated spore suspensions displayed OD600, OD260, and OD280 values of 1, 0.053, and 0.046, respectively. Following a spore suspension treatment of 200 MPa-75 ℃-2 g/L potassium sorbate, the OD600 value declined by 0.413, while the OD260 and OD280 values increased to 0.401 and 0.290, respectively. It was indicated that the addition of potassium sorbate was enhanced the liberation of protein and nucleic acid in spores, leading to the reduced turbidity of spore suspensions. After treatment with 200 MPa-75 ℃, the DPA release increased by 51.31%, compared with the untreated spore suspensions. Additionally, the DPA release rate increased to 75.28% after treatment with 200 MPa-75 ℃-2 g/L potassium sorbate. This result further confirmed that the combination of PATS with potassium sorbate treatment was enhanced the permeability of the spore inner membrane, which was positively correlated with the addition of potassium sorbate and temperature. SEM images demonstrated that the addition of potassium sorbate to PATS caused significant spore damage and destruction of the cell wall, cortex and inner membrane, compared with the treatment with PATS alone. Further analysis through flow cytometry and PSD revealed that the damage on the inner membrane was worsened, while particle diameter significantly decreased, and specific surface area increased considerably after combined treatment. The FTIR disclosed the peak shift and change in intensity. The spore inner membrane phospholipids were transformed from a gel state to a liquid crystal state after PATS and potassium sorbate treatment, indicating the reduced the stability, compared with the untreated spores and PATS treatment alone. Simultaneously, the Na+ /K+-ATPase activity registered a significant decrease, when PATS was used in the combination with potassium sorbate. This activity was used to disturb the balance of the inner membrane potential, as well as the fundamental physiological and metabolic functions. The findings reveal that there was the synergistic bactericidal impact on Bacillus subtilis spores at lower temperatures, compared with the conventional inactivated spores, when PATS was combined with potassium sorbate. The synergistic bactericidal effect was also augmented with the increase of temperature and potassium sorbate concentration. The primary mechanism for the inactivation of Bacillus subtilis spores was attributed to the damage of the permeability and structural integrity of inner membrane. These findings have practical applications to promote the food sterilization, in order to reduce the adverse impact of high temperatures on food quality.
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Keywords:
- potassium sorbate /
- temperature /
- scaning electron microscopy /
- Bacillus subtilis /
- spore /
- inner membrane /
- PATS
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图 1 山梨酸钾协同PATS处理对枯草杆菌芽孢灭活效果的影响
注：不同字母表示不同处理条件下差异显著（P<0.05）；CK（Control check ）表示空白对照；200 MPa-25℃表示200 MPa结合25℃处理；200 MPa-25℃表示200 MPa结合65℃处理；200 MPa-75℃表示200 MPa结合75℃处理。
Figure 1. Effects of PATS combining with potassium sorbate treatment on the inactivation of Bacillus subtilis spores
Note: Different letters indicate significant differences (P<0.05) under different treatment conditions; CK (Control check ) represent means untreated sample; 200 MPa-25 ℃ means 200 MPa combined with 25 ℃ treatments; 200 MPa-65 ℃ means 200 MPa combined with 65 ℃ treatments; 200 MPa-75 ℃ means 200 MPa combined with 75 ℃ treatments, the same below.
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图 2 山梨酸钾协同PATS处理对枯草杆菌芽孢悬浮液OD600值的影响
Figure 2. Effects of PATS combining with potassium sorbate treatment on the OD600 value of Bacillus subtilis spore suspension
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图 3 山梨酸钾协同PATS处理对枯草杆菌芽孢DPA释放率的影响
Figure 3. Effects of PATS combining with potassium sorbate treatment on DPA release from Bacillus subtilis spores
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图 4 山梨酸钾协同PATS处理对枯草杆菌芽孢紫外吸收物质释放量的影响
Figure 4. Effects of PATS combining with potassium sorbate treatment on the release of UV-absorbing substances from Bacillus subtilis spores
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图 5 山梨酸钾协同PATS处理对枯草杆菌芽孢悬浮液粒径的影响
Figure 5. Effects of potassium sorbate combining with PATS treatment on the particle diameter of Bacillus subtilis spore suspension
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图 6 山梨酸钾辅助PATS处理后芽孢形态变化
Figure 6. Changes of spore morphology after potassium sorbate assisted PATS treatment
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图 7 山梨酸钾辅助PATS处理对芽孢内膜通透性的影响
注：Count 表示发出荧光的细胞数量；M1表示荧光强度较低的阴性区域；M2表示荧光强度较高的阳性区域；FL2 表示在荧光通道2处所采集的荧光强度的对数值。
Figure 7. Effects of potassium sorbate assisted PATS treatment on the permeability of spore inner membrane
Note: Count represents the number of fluorescent cell; M1 represents negative area with low fluorescence intensity; M2 represents positive area with high fluorescence intensity; FL2 represents the logarithm of fluorescence intensity collected at fluorescence channel 2.
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图 8 山梨酸钾协同PATS处理对芽孢内膜Na+/K+-ATPase活力的影响
Figure 8. Effects of PATS combining with potassium sorbate treatment on Na+/K+-ATPase activity of spore inner membrane
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图 9 山梨酸钾协同PATS处理前后枯草杆菌芽孢3 000～2 800 cm−1范围的红外光谱图
Figure 9. Infrared spectra of Bacillus subtilis spores in the range of 3 000 to 2 800 cm−1 before and after PATS combining with potassium sorbate treatment
表 1 山梨酸钾协同PATS处理对枯草杆菌芽孢悬浮液粒径和比表面积的影响
Table 1 Effects of PATS combining with potassium sorbate treatment on the particle diameter and specific surface area of Bacillus subtilis spores
处理
TreatmentD90/μm 比表面积
Specific surface area/(m2·kg−1)未处理CK 1.39±0.10a 5 570 200 MPa-25 ℃ 2 g·L−1山梨酸钾
200 MPa-25 ℃-2 g·L−1 potassium sorbate1.25±0.01b 5 951 200 MPa-65 ℃ 2 g·L−1山梨酸钾
200 MPa-65 ℃-2 g·L−1 potassium sorbate0.914±0.04c 7 833 200 MPa-75 ℃ 0.818±0.01b 9 010 200 MPa-75 ℃ 1 g·L−1山梨酸钾
200 MPa-75 ℃-1 g·L−1 potassium sorbate0.816±0.06b 9 103 200 MPa-75 ℃ 2 g·L−1山梨酸钾
200 MPa-75 ℃-2 g·L−1 potassium sorbate0.804±0.02e 9 790 
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