Inhibitory effect of essential oil substances against spoilage bacteria on fresh produce

국문 초록 (Abstract)

본 연구에서는 신선 채소에서 분리된 부패 미생물 (Chryseobacterium balustinum, Enterobacter sp., Pantoea agglomerans, Bacillus pumilus, Clavibacter michiganensis, Pseudomonas fluorescens, Acinetobacter calcoaceticus, Stenotrophomonas maltophilia, Klebsiella pneumonia, and Dickeya zeae, Pectobacterium carotovorum subsp. Carotovorum Pcc21)의 생육특성, 생물막(biofilm) 형성과 세포표면 특성(cell hydrophobicity, colony spreading)을 알아보았다. 연구한 부패 균주의 생물막의 형성과 세포표면 특성의 상관성이 낮게 나타났다. 식물성 항균 오일 성분의 항균 효과 연구 에서는, 부패 미생물에 대한 자연 항균물질 및 화학적 살균소독제의 항균력을 agar disc diffusion test로 검사하였는데, 다른 항균물질에 비하여 cavacrol, thymol, eugenol이 가장 높은 항균력(최소 저해 농도 각각167, 648, and 168 µg/ml) 을 나타냈다. 결합 처리할 때는 단독 처리한 것 보다 효과가 증가하는 결과를 보여주었다. 그 중에서 부패 균주에 대한, carvacrol + thymol 와 carvacrol + eugenol 결합이 가장 높은 시너지 효과를 보여 주였다. 그렇지만, 신선 채소의 있는 자연 유래 균주의 저해에 있어서는 carvacrol + thymol 와 carvacrol + thymo+ eugenol 결합 처리가 가장 좋은 항균 효과를 보여 주었다. 신선 채소에 대한 항균 실험에서는 휘발성 오일 성분의 처리가 액체 용액으로 담금 처리 보다 더 높은 항균효과를 나타내었다. 본 연구결과는 신선 채소의 새로운 살균∙소독 및 저장법의 개발에 기초자료로 활용될 수 있을 것이다.

다국어 초록 (Multilingual Abstract)

This study investigated the growth characteristics of spoilage bacteria isolated from fresh produce including the growth at various temperatures (5, 15, 25, and 35°C), the biofilm formation, cell hydrophobicity, colony spreading, and antimicrobial effects of natural antimicrobial substances against spoilage bacteria. Bacillus pumilus, Dickeya zeae, Pectobacterium carotovorum subsp. Carotovorum Pcc21, and B. pumilus (RDA-R) did not grow in the stored temperature of 5°C. The growth rate (GR) of the tested bacteria stored at 5°C was lower than in the other temperatures, except for Chryseobacterium balustinum, B. pumilus, Acinetobacter clacoaceticus (RDA-R). The primary growth models of these spoilage bacteria fit well to a modified Gompertz model (R2 > 0.90 on average). It showed low correlation between biofilm formation and hydrophobicity (r = 0.3762, r = 0.4972, before and after ethanol tretment, respectively), and no significant correlation with colony spreading (r < 0.05). Natural antimicrobial substances tested, carvacrol, thymol and eugenol showed strong antibacterial effects and resulted in average minimum inhibitory concentration (MIC) values against the spoilage bacteria were 167, 648, and 168 µg/ml, respectively. When they were combined, two combinations (carvacrol + thymol and carvacrol + eugenol) showed great synergy effects against isolated spoilage bacteria. However, two formulas (carvacrol + thymol, carvacrol + thymol+ eugenol) showed stronger antibacterial effects against natural spoilage bacteria presented on fresh vegetables. Vapor of natural antimicrobial substances showed higher effectiveness than liquid solution. Results of this study could be used for developing new sanitizing and preserving method for improving shelf life and freshness of fresh produce.

목차 (Table of Contents)

• I. Introduction 1
• II. Materials and Methods 8
• 1. Bacteria strains 8
• 2. Growth and biofilm formation characteristics 8
• 2.1 Measurement of spoilage bacteria growth 8
• 2.2 Predictive growth modeling 9
• 2.3 Biofilm formation 9
• 2.4 Bacterial hydrophobicity 10
• 2.5 Colony spreading 11
• 3. Antimicrobial test of antimicrobial substances 11
• 3.1 Preparation of antimicrobial substances 11
• 3.2 Agar disc Diffusion test 12
• 3.3 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) test 13
• 3.4 Fractional inhibitory concentration (FIC) assess 14
• 3.5 Agar disc diffusion test on fresh vegetable 15
• 4. Application of antimicrobial substances on fresh produce 15
• 4.1 Antimicrobial substances dipping method 15
• 4.2 Disc volatilization method 16
• 4.3 Disc volatilization method combined with modified atmosphere packaging (MAP) 17
• 5. Statistical analyse 18
• III. Results 19
• 1. Growth and biofilm formation characteristics 19
• 1.1 Growth of spoilage bacteria in different temperature 19
• 1.2 Modeling for confirm growth rate and lag time 25
• 1.3 Biofilm formation of spoilage bacteria 27
• 1.4 Cell hydrophobicity of spoilage bacteria 29
• 1.5 Colony spreading of spoilage bacteria 31
• 1.6 Corelation between biofilm formation and cell hydrophobicity or colony spreading 34
• 2. Antimicrobial activity of antimicrobial substances 37
• 2.1 Determination of antimicrobial activity by agar disc diffusion method 37
• 2.2 Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the antimicrobial substances 39
• 2.3 Assess of the antimicrobial effect by fractional inhibitory concentration (FIC) 45
• 2.4 Inhibitory effect of the combined antimicrobial substances against spoilage microorganisms on fresh vegetable 47
• 2.5 pH values of the antimicrobial substances 50
• 3. Application of antimicrobial substances on fresh produce 52
• 3.1 Inhibitory effect of antimicrobial substances in liquid phase (with/without surfactant) against spoilage microorganisms on fresh produce 52
• 3.2 Inhibitory effect of antimicrobial substances in vapor phase against spoilage microorganisms on fresh produce 56
• 3.3 Inhibitory effect of antimicrobial substances in the combination of vapor phase and Modified atmosphere packaging (MAP) on fresh produce 58
• IV. Discussion 61
• V. Conclusion 66
• Reference 68
• 국문 초록 79