学 位 論 文 の 要 旨

論文題目Application of ethanolic plant extract combined with other hurdles incontrolling Escherichia coli
（植物エタノール抽出液と他の複合ハードルによる大腸菌の制御）

広島大学大学院生物圏科学研究科

Biofunctional Science and Technology 専攻
学生番号D182913

氏

Waraporn Kusalaruk

名Escherichia coli O157:H7 is a major foodborne pathogen, which causes hemorrhagic
colitis, hemolytic uremic syndrome. Besides, it has also been reported to survive in acidic
environments also in high levels of salt. In controlling E. coli O157:H7, the use of a single hurdle
might require a high concentration of antimicrobial agents or severe processing. Nowadays, theapplication of chemical preservatives is wildly not accepted due to concern about their harmful
effects on human health. Thus, consumer demand has focused on using natural antimicrobials orantimicrobials as recognition as GRAS (Generally Recognized as Safe) by FDA. Thus, this study
aimed to investigate the hurdle technology application of ethanolic plant extracts with otherhurdles to control the growth of E. coli.
In the first study, the antimicrobial activities of 22 plant extracts against pathogenic andnon-pathogenic strains of E. coli were observed. This study also determined the potential hurdle
technology application of plant extracts with glycine and NaOAc under neutral and mildly acidic
pH conditions. The results showed that only clove and rosemary extracts exhibited antimicrobialactivity against E. coli with diameter of inhibition zone (DIZ) values of 12.25 mm and 11.75 mm,respectively. Thus, only clove and rosemary extracts were observed for their minimal inhibitory
concentration (MIC) and minimal bactericidal concentration (MBC) and studied for the chemicalcomponent by GC-MS. Clove extract inhibited the antimicrobial activity against E. coli with abactericidal effect with both MIC and MBC of 0.4%, while MIC and MBC of rosemary extract
were 0.6% and 0.8%, respectively. The main components of clove and rosemary extracts were
eugenol, and glycidol, respectively. The effect of glycine or NaOAc on the antimicrobial activityof plant extracts against E. coli in neutral and mildly acidic medium was also observed. It was1

shown how various interactions ranging from absent to additive effects were observed between theplant extracts and glycine or NaOAc. Combined effects of 0.2% clove extract under neutral
condition with 0.1% NaOAc or 1.0% glycine demonstrated additive interactive antimicrobialactivity, thus FIC indices 0.53 or 0.75, respectively. Under pH 5.5, supplementations of 0.1%
NaOAc with 0.1% clove or 0.2% rosemary extracts showed additive interaction with FIC indices0.53-0.70.
In the second study, the development of hurdle technology in controlling pathogenic E.
coli O157:H7 by using plant extracts with glycine and/or NaOAc under neutral and mildly acidic
nutrient broth (NB) and phosphate-buffered saline (PBS) under different incubation temperature(15 °C and 35 °C) was studied. Treatments in NB under 15°C, and neutral pH, the combination of
0.2% clove extract + 2% glycine + 2% NaOAc showed the highest efficacy against E. coli with 3logs reduction after 48 h, while under mildly acidic conditions, treatments with 0.2% clove extract+ 2% NaOAc reduced the bacterial population by approximately 3 log units after 24 h. Under
35 °C incubation, conditions the two combined factors (0.2% clove extract with 2% glycine or 2%NaOAc) achieved about 3 log reduction after 12 h, while treatments with 0.1% clove extract + 2%glycine + 2% NaOAc completely inhibited E. coli after 24 h in neutral media. Under pH 5.5, thecombination of 0.2% clove extract with 2% NaOAc exhibited 3 log reduction of bacterial
population after 12 h incubation. The treatments with 0.2% clove extract + 2% glycine, as well astreatments with 0.1% clove extract + 2% glycine + 2% NaOAc decreased the population of E. coli
by approximately 3.0 log units after incubated 24 h, while the number of bacteria in individual
factors treatment increased after 48 h incubation.
For PBS studied, incubation at 15 °C, individual 2% glycine or NaOAc was unable tosuppress the tested bacteria. In contrast, the combination of clove extract with glycine and NaOAc
showed the highest efficacy against E. coli O157:H7 and completely reduced bacteria after 96 h.
Besides, the combination of 0.2% clove extract with 2% glycine or NaOAc showed strong
antimicrobial activity compared to individual factors used under neutral and mildly acidic
conditions. After incubation at 35 °C under neutral condition, adding an individual of 0.1% clove
extract or 2% glycine exhibited about 3 log reduction, while adding 2% NaOAc and control sample
did not change bacterial population after 96 h. The combinations of 0.1% clove + 2% glycine +2% NaOAc showed the highest reduction of E. coli O157:H7, which at population size 3.7 logs
after 24 h and complete inhibition after 48 h. For weakly acidic conditions, individual treatmentof 0.1% clove extract, 2% glycine, or 2% NaOAc slowly decreased the E. coli O157:H7 count of
about 2 log units after 96 h. However, the combination of 0.1% clove with 2% NaOAc significantly
reduced the number of E. coli O157:H7 by about 5 log units after 24 h. Based on the results of thisstudy, the survival of E. coli O157:H7 incubated at 15 °C was required higher plant extracts
concentration than incubated at 35 °C, regardless of pH condition. The reducing storage
temperature reduced the inhibitory effect of all plant extracts tested, indicating that the applicationof the tested plant extracts may protect in the case of storage at a lower temperature.
In the last part of this study, thermal processing is the most widely used to eliminate
microorganisms in the juice product. However, heat treatment can damage the nutrition and2

sensory quality of the juice. It is interesting to combine mild heat with natural antimicrobials injuice products. Thus, the objectives of this study were to investigate the efficacy of ethanolic clove
extract against pathogenic E. coli O157:H7 in different culture treatments (NB, PBS, and tomatojuice) at 35°C. Also, this study investigated the efficacy of ethanolic clove extract against
pathogenic E. coli O157:H7 in tomato juice at refrigeration temperature (4°C) and room
temperature (15°C). Furthermore, this study was conducted to evaluate the inactivation of E. coliO157:H7 by the combination of clove extract with mild heat treatment in PBS and tomato juice.
The results showed that the E. coli O157:H7 was the most sensitive to ethanolic clove
extract in PBS, followed by tomato juice and NB, respectively. Tomato juice stored at 15 °C, clove
extract exhibited higher inhibitory activity compared with at 4 °C. The combined effect of clove
extract with mild heat reduced the concentration of clove extract, temperature severity, or heating
period, resulting in the prevention of thermal damage to the juice. The combined effect of 0.05%
clove extract with mild heat treatment at 60°C for 30 minutes achieved 5 log reduction of E. coli
O157:H7. Besides, tomato juice with 0.05% clove extract was acceptable in all attributes in thesensory test. This finding could have been a good potential for improving the process of juice
production.

3