[4-1015-C] Food Safety (1)

[4-1015-C-06] Evaluation Growth Characteristics of Bacterial Spores Combine Treatment with High Hydrostatic Pressure and Alkaline Electrolyzed Water

*Koki Morita¹, Taiga Kuhara¹, Yoshinori Kamitani¹, Daisuke Hamanaka¹ (1. Kagoshima University faculty of agriculture(Japan))

キーワード：Bacterial spores, high hydrostatic pressure, Alkaline electrolyzed water, Injured microbes, Gonpertz curves, Antibiotics

In our previous study, we observed that the heat resistance of bacterial spores was effectively reduced by the combining treatment with high hydrostatic pressure(HHP) and alkaline electrolyzed water(AlEW). However, it is not clear in detail the effect of bacterial spores treated with AlEW and HHP, especially behavior of injured microbes which can live but cannot growth normally with the delay of lag phase etc. Since the existence of injured microbes may cause underestimate of viable counts comparing with the actual number of survived cell, correct understanding of growth characteristic is important to evaluate the microbiological safety of food. The growth curve of the microorganism is sigmoid and is divided into four phases; lag phase, accelerated log phase, slowed log phase and stationary phase. Gompertz curves are often used as continuous equation to represent sigmoidal growth curves. Growth of injured microbes is well known to affect growth characteristics, including delay of lag phase and reduction of growth rate as compared to those of intact one. In this study, we investigated the evaluation of injury characteristics by the double culture method supplemented with antibiotics in growth media, and growth behavior of bacterial spores treated by AlEW, HHP and their combination using Gompertz equation to obtain parameters in growth curve.Bacterial spores used in this study were Bacillus subtilis NBRC3134. Bacterial spores ware formed by conventional method and suspended in sterile sterile physiological saline (PS) to treat HHP.Contacting treatment of bacterial spores with AlEW was performed by suspending in test solution in plastic tube, and centrifuged. The pellet of bacterial spores on the bottom of plastic tube were re-suspended by vigorous pipetting and vortex-shaking. This process was performed two times, and treated spores were finally suspended with PS solution. PS solution was used as a control.Spore suspension was poured into plastic bag without air for following treatment.HHP treatments were performed with 100MPa for 1 hour at 50℃.The HHP treated pouch was aseptically opened, an appropriate amount of spore suspension was inoculated trypticase soy broth (TSB) supplemented with Chloramphenicol (CP) or Penicillin (PCG) for evaluation of the injury characteristics related to synthesizes of protein(enzyme) or peptidoglycan, and incubated at 30℃ for 48 hours. The optical density of the inoculated TSB at 490nm was measured every 30 minutes.Gompertz equation was fitted to the obtained OD data by nonlinear least squares method to obtain various parameters.At the same time, the growth behavior of AlEW and HHP treated bacterial spores were evaluated by calculating the length of lag phase and their difference between control and treated sample.So far, the investigation of the changes in growth characteristics of microorganisms have been reported and, it is known that shortening and extension of lag phase, fluctuation of growth rate in log phase, and difference in maximum viable count in stationary phase are affected. In this study, normal culture media and media supplemented with antibiotics were used as recovery media for injured cells produced by high-pressure-based treatments with or without AlEW, and their growth characteristics were compared with those of intact bacteria. The period of lag phase bacterial spores was not significantly different between high-pressure-treated and untreated sample, but significant extension of lag phase was obtained by the combination with AlEW. In addition, further extensions of lag phase in growth curve were observed when a medium supplemented with CP or PCG was used. On the other hand, there were small differences between the treated and untreated samples in both the growth rate in log phase and maximum number of bacterial cell even in the case of CP or PCG supplementation. In general, extension of the lag phase in growth behavior is considered to be a process of recovery of injured bacteria. Moreover, the difference in growth rate in the log phase and the difference in the maximum number of viable cells in the stationary phase are considered to be the result of changes in the nutrient requirement of the microorganism related to the stress treatment. CP and PCG are antibiotics with features that inhibit protein and peptidoglycan synthesis, respectively. Considering the results obtained in this study, bacterial spores treated with high pressure treatment with and without AlEW require the time duration for recovering from damage associated with the synthesis of some essential biopolymers, but nutritional requirements and other phenotypic properties might not be affected.