この研究は、パルス電界（PEF）による膜透過性と生存率低下との関係を、2つのグラム陽性菌（Lactobacillus plantarum、Listeria monocytogenes）と2つのグラム陰性菌（Escherichia coli、Salmonella senftenberg 775W）で調べた。両者の関係は、パルス電界の強度および媒質のpHに依存する。膜の完全性の喪失は、蛍光色素ヨウ化プロピジウム（PI）の取り込みの増加で測定された。PEF中にPIで染色された細胞が、PEF後に染色された細胞で測定されたものよりも高い蛍光を生じた場合、非永続的/可逆的な透過化が生じたと判断した。その結果、pH 7.0で処理した場合、2つのグラム陰性菌の生存率低下は、非永続的および永続的な膜透過化の合計と相関したが、2つのグラム陽性菌の場合、生存率低下は、永続的な膜完全性の喪失と相関した；pH 7.0では、4つの細菌は可逆的な透過化を示した；しかし、透過性を逆転させることができるグラム陽性は生き残ったが、グラム陰性細胞はPEFの直後に透過性を逆転させる能力があるにもかかわらず、死滅した；したがって、再シールは必ずしもPEF処理細胞の生存に関連するわけではない、などの多くの知見を報告している。
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This study was performed to investigate the effects of pulsed electric fields on the membrane permeabilization in two gram-positive and two gram-negative bacterial strains to obtain an understanding of the mechanism of bacterial inactivation by pulsed electric fields.
The experiments were conducted with different electric field strengths, exposure times, and medium pH values (pH 4.0 and 7.0). Additionally, both gram-positive and gram-negative bacteria were investigated to discover possible differences in their reactions to pulsed electric field exposure due to their different cell membrane properties.
The uptake of the fluorescent dye propidium iodide into the cell was measured as it is a sensitive marker of cell membrane integrity. The occurence of this dye in the cell indicates loss of membrane integrity. Propidium iodide uptake was measured before and after pulsed electric field exposure to characterize whether the membrane permeabilization was reversible or not.
| ばく露 | パラメータ |
|---|---|
|
ばく露1:
1 Hz
Modulation type:
pulsed
ばく露時間:
not given in this article
|
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|
ばく露2:
2 Hz
Modulation type:
pulsed
ばく露時間:
not given in this article
|
|
|
ばく露3:
Modulation type:
single pulse
ばく露時間:
single pulse of 50 µs
|
|
|
ばく露4:
Modulation type:
single pulse
ばく露時間:
single pulses of 12,5 - 100 µs
|
|
|
ばく露5:
Modulation type:
single pulse
ばく露時間:
single pulses of 10 - 400 µs
|
|
cells were stained with PI a) before exposure b) after exposure
| 周波数 | 1 Hz |
|---|---|
| タイプ |
|
| 波形 |
|
| ばく露時間 | not given in this article |
| Modulation type | pulsed |
|---|---|
| Pulse width | 2 µs |
| Repetition frequency | 1 Hz |
| ばく露の発生源／構造 |
|
|---|---|
| ばく露装置の詳細 | cylindrical plastic tube with two polished stainless steel electrodes - 0.25 cm apart and with an area of 2.01 cm² - was used as chamber |
| 測定量 | 値 | 種別 | Method | Mass | 備考 |
|---|---|---|---|---|---|
| 電界強度 | 25 kV/cm | - | 測定値 | - | - |
Investiagtions at pH 7.0 showed positive correlation between degree of membrane permeabilization, exposure time, and electric field strength applied in all bacterial strains. In gram-negative bacteria all permeabilized cells died (both reversible and permanent permeabilized cells). In gram-positive bacteria only cells with permanent membrane permeabilization died. The membrane permeabilization was partially reversible in all bacterial strains.
Investigations at pH 4.0 revealed a relationship between loss of cell viability with permanent loss in cell membrane integrity in Listeria monocytogenes, but in Lactobacillus plantarum the degree of permeabilization was higher, and in the gram-negative bacterial strains the permeabilization much lower than the percentage of inactivated cells. The permeabilization in all bacterial strains was permanent.
These results confirm that membrane permeabilization is involved in the mechanism of bacterial inactivation by pulsed electric fields, but the nature of membrane damage and its relationship to cell death depends on the bacterial species and the medium pH value.
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