VO CONG KHANH

称号及び氏名博士（工学）

学位授与の日付

2021 年 9 月 25 日論

「Conditional generation and its mechanism of oxidative injury in文

名heated and irradiated Escherichia coli cells（大腸菌の加熱および照射細胞における酸化損傷の条件的発生とそのメカニズム）」
論文審査委員

主査

古田雅一

副査

松浦寛人

副査

宮丸広幸

論文要旨
Target number three, the goal No.12 of Sustainable development goals (SDGs –United Nations), stated that “by 2030, halve per capita global food waste at the retail andconsumer level and reduce food losses along production and supply chains, including
post-harvest losses”. The food industry is very demanding regarding food safety, and one of
the biggest problems that cause the loss of food production in supply chains is microbialcontamination. It relates to the whole supply chain: harvesting, transport, storage, andprocessing. Various chemical and physical treatments have been applied to reduce andcontrol the risk of foodborne pathogens. Thermal treatment is one of the oldest techniques
still in daily use. Depending on the target organisms, heat treatments can be classified into
pasteurization and sterilization treatment. Heat treatment has multitargets action on
membranes, DNA, RNA and protein in a microbial cell. However, on the need forsterilization or disinfection in heat-sensitive materials or food production, radiation

treatment has been promoted as an alternative treatment with a large number ofapplications in many countries except Japan. Irradiation treatment uses energy transmitted
through space in a wave pattern (including ionizing and non-ionizing radiation) to kill orinhibit bacteria. Radiation treatment can easily penetrate cell membranes, alters themolecular structure, damages cell components, and can kill/inhibit contaminatingmicroorganisms, and react with water molecules to form oxygen radicals. Both methods
have their advantages and disadvantages, but the main goal of any preservation method is
to maximize the quality of the product while achieving the highest safety level.
After the preservation treatments, bacterial cells are divided into three groups as
lethal population (dead), resistant population (survived with less severe damage) andsublethal population (injured). Bacterial injury arising from food preservation techniques
has received much attentions over the past 50 years. Injured populations show a prolonged
lag period before re-growing and re-gaining their pathogenicity under favorable conditions
and these cells pose a potential threat to food safety. The degree of injury can be influenced
by many factors, such as the stress level, processing and storage conditions, bacterial
species, and so on. The generation of oxidative stress as a secondary damage to bacterial
cells has been observed in various stresses such as heat, antibiotic, acidity, and radiation
treatments. The relevance and mechanisms of cell repair in considering the effect ofoxidative stress in injured cells is necessary to understand bacterial injury.
Identifying factors that can affect the bacterial inactivation, using E. coli as a model
microorganism, with the conditional effects of oxidative injury was a critical objective inthis study. In this study, the semi-synthetic and complex media for cell growth and recovery
were employed to evaluate the effects of heat and irradiation treatments. Furthermore, thesurvival statistics of heated and irradiated populations by adding antioxidant compounds to
the recovery process was also aimed to investigate the effects of oxidative damage onbacterial cells in those control treatments. It is necessary to explore the relationshipbetween oxidative injury and the resuscitation of microbial injure cells in the preservation
treatments in order to develop an efficient strategy to control foodborne pathogens. The
dissertation content contains 6 chapters as summarized as follows:
Chapter 1 provides general information on some of the commonly used disinfection

treatments in the food industry. This chapter also introduces the definition of bacterial
injury, characteristics of the injured cells and current knowledges regarding repair
mechanisms. Besides induced primary stress, the impact of oxidative secondary injury on
bacterial cell survival and detection methods for injured cells are introduces.
Chapter 2 presents an overview and evidence on the involvement of oxidative stressduring and after thermal treatment. The thermal treatment has been used as a method of
preventing microbial spoilage since ancient ages. After treatment, injured cells may recover
to a normal physiological state under favorable conditions which may pose a potential
threat to food safety. The presence of oxidative stress after heat treatment may contribute to
the inactivation of spoilage bacteria. In this study, we investigated the heat sensitivity ofEscherichia coli cells and the oxidative stress injury by assessing the rescue effects ofseveral reducing and reactive oxygen species (ROS) scavengers in two different conditions.
Heat resistance in E. coli cells grown in the enriched minimal M9 (EM9) medium washigher than those cells grown in Luria broth (LB) medium. In addition, the survival rate ofheat-treated cells was increased with the addition of ROS scavengers in liquid and agar
media evaluated by plate counting and the growth delay analysis as well as the mostprobable number methods. The heat sensitivity of the cells and the effects of ROS
scavengers on that depended on the growth conditions were demonstrated. Furthermore, theenumeration of the secondary injury, the resuscitation mechanisms of injured cells and thepotential of the rescuing antioxidants for bacterium were also discussed.
The repair mechanisms of heat-injured E. coli cells were discussed in the chapter 3.
In this chapter, the author investigated the effect of heat treatment on bacterial cell
physiology in terms of oxidative stress and the repair mechanisms in injured cells. The
effects of heat treatment on the activities of two antioxidative enzymes, involving in theintracellular reactive oxygen species and redox potential were examined. The activities ofcatalase and superoxide dismutase increased slightly during heating period and gradually
decreased to the baseline level within 4 hr of the post-heating incubation. The generation ofsuperoxide radicals and alteration of redox potential during heat treatment seemed to beheterogeneous in E. coli cells grown on both minimal and rich media. In addition, acomplex nutritional system may be involved in the oxidative damages in heat-treated cells
grown in the rich medium, while the repair of the membrane damage suggested to becrucial for cells grown and recovered in the minimal medium. The addition of antioxidants
to agar media also rescued many injured cells even in the presence of some of themetabolic inhibitors. In both treatments, the electron transport system seemed to criticallyaffect (both positively and negatively) to the generation of and revival from the oxidative
injury in heated cells. The results obtained in this study emphasize once again the potential
application of different antioxidants to rescues heat-damaged cells depending on therecovery medium. This finding supports the current knowledges of the inactivation actionof heat, oxidative secondary injury and the repair mechanisms in heat-treated bacterial
cells.
A new subculture method for evaluation and quantitative analysis of growth delay
time has proposed and presented in chapter 4. The microcolony formation at 30oC on EM9agar plates by individual Escherichia coli cells heated at 50oC was monitored using atime-lapse shadow image analysis system, MicroBio µ3DTM AutoScanner. The distributionof microcolony detected every half an hour for the unheated cells appeared to be consistent
with the normal distribution. Meanwhile, the heated population demonstrated the growth
delay probably resulting from cell injury and distributed in a deformed pattern with atailing. Cumulative patterns of microcolonies appearing during the post-heating cultivation
period could be expressed with three different mathematical models. The presence ofantioxidants also affects the formation time of microcolony in heated cells. This approach
can be proposed as a predictable rapid culture method to enumerate viable and repairable
injured cells in practical use.
In chapter 5, the effects of gamma and UV irradiations on the inactivation of E. coli
cells and the oxidative secondary injuries were evaluated. The direct interaction with cellcomponents of and/or indirect modification by free radicals resulting from water radiolysis
is known as the inactivation mechanism in the radiation treatment. In relation to the effectof oxidative secondary injury presented in the previous chapter, the experiments
concentrated on the conditional generation of oxidative injury population induced byionizing and non-ionizing radiation treatment. E. coli cells grown in two different media
irradiated with UV and gamma-ray treatments, then recovered in liquid and solid medium
were used to evaluate the effects of growth and recovery media to the radio-sensitizationand oxidative damage. The sensitivities to UV and γ-rays of E. coli cells depended on both

media, although the recovery medium was more important. Also, oxidative damage wasalso produced in irradiated cells and was dependent on the recovery medium. This study
suggested a different approach for the analysis of the irradiation injury mechanism and also
contributes to the understanding of the effects of oxidative stress in the application ofradiation to control pathogenic microorganisms.
Firstly, we confirmed the presence of oxidative stress as secondary injury in heated orirradiated cells by using the antioxidant agents. The rescue effect of these agents wasexamined in both liquid and solid media, which liquid media was less effective than insolid media in heat-induced cells. Reversely, secondary injury caused by UV and γ-rays
irradiation was observed at lower rates than heat treatment and the effectiveness of ROS
scavengers in irradiated cell is altered depending on the recovery medium. Oxidative status,ROS generation, growth/recovery condition and antioxidant function influence oxidative
secondary damage caused by heat and radiation treatment. In particular, membrane damage
repair is critically involved in the resuscitation of heat-injured cells, especially those grownin semi-synthetic media. In both medium, the inhibition of the electron transport chain
attenuated the viability of heat-injured cell as confirmed in the results obtained. Moreover,heat-induced oxidative secondary stress is also associated with bacterial cell inactivation.
Finally, we have introduced and proposed a novel method to enumerate the growth delay inmicrocolony formation time by using the time-lapse shadow image analysis. Interestingly,
the addition of antioxidants in agar not only rescued oxidative damage but also participatesto the colony formation in heat-injured cells.
In conclusion, this study should contribute to the understanding of the injurious
actions of heat and radiation treatments on microbial cells, as well as the significance ofoxidative stress in relation to the oxidative secondary injury and resuscitation processes ininjured bacterial cells.

審査結果の要旨
食品の損耗を防ぎ、安全で食味豊かな食材を持続的に提供することは地球規模の課題となっている。特に食品の安全性を脅かす食中毒菌の防除については食味など食品の品質を最大限に確保しながら、最小限の処理条件で行うなど、殺菌に求められる条件が厳しさを増している。特に殺菌後の食品に残存し、保存中に回復する恐れのある損傷菌の発生メカニズムを知り、適切な殺菌処理法を開発することが喫緊の課題である。本論文においては損傷菌発生の原因として酸化ストレスに注目し、代表的な物理的殺菌法である加熱殺菌と放射線殺菌に的を絞り、微生物モデルとして大腸菌を材料として以下の成果を得ている。

(1) 栄養培地及び無機合成培地を用いた場合の大腸菌の加熱、紫外線、60Coγ線感受性の違いを明らかにし、培養中に種々の抗酸化剤、活性酸素消去剤を添加した場合の感受性から微生物細胞の二次傷害としての酸化ストレスの存在を確認した。
(2）熱処理が細菌の細胞生理に及ぼす影響を、酸化ストレスと損傷菌細胞の修復機構の観点から検討し、細胞内の代表的な抗酸化酵素、カタラーゼとスーパーオキシドディスムターゼの活性は、加熱中にわずかに上昇し、加熱後 4 時間以内にベースラインレベルまで徐々に低下することを見出した。紫外線およびγ線に対する感受性は両培地に依存し、抗酸化剤添加による回復促進効果も軽微であった。以上を含めて種々の要因について検討した結果、損傷を受けた細胞の二次損傷回復過程に細胞膜や呼吸系の酸化的二次損傷と培地組成との関連性が重要であることを考察した。
(3）タイムラプスシャドウ画像解析法を用いて、目視できない寒天培地上のマイクロコロニー生長の動態解析を可能とし、殺菌処理後の回復過程の細胞増殖動態を簡便に測定する新方法を導入・提案した。
以上の研究結果は、損傷菌の回復過程における基礎的知見の蓄積に加えて、更に高度な測定、解析法の提案により、当該分野の研究の加速を期待させるものであり、食品業界が食品の微生物学的安全性を保証するための衛生管理システムを構築するためにも有用な示唆を与えるものとして今後の大きな発展を期待させるものである。これらの成果は申請者が自立して研究活動を行うに必要な能力と学識を有することを証したものである。