Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
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Antibacterial Mechanism and Salad Washing Effect of Bitter Orange Extract Against Salmonella Typhimurium

광귤 추출물의 Salmonella Typhimurium에 대한 항균 메커니즘 및 샐러드 세척 효과

  • Yoon-Mi Ji (Department of Food and Nutrition, Kookmin University) ;
  • Ji-Yun Bae (Department of Food and Nutrition, Kookmin University) ;
  • Chung-Hwan Kim (Seoul Food R&D Co., Ltd.) ;
  • Se-Wook OH (Department of Food and Nutrition, Kookmin University)
  • 지윤미 (국민대학교 식품영양학과) ;
  • 배지윤 (국민대학교 식품영양학과) ;
  • 김충환 ((주)서울식연) ;
  • 오세욱 (국민대학교 식품영양학과)
  • Received : 2024.03.22
  • Accepted : 2024.06.05
  • Published : 2024.06.30
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Abstract

In this study, the antibacterial activity and mechanisms of bitter orange extract, a natural antibacterial agent, were investigated, with a focus on its potential application in washing water for controlling Salmonella Typhimurium contamination of salad, a ready-to-eat food. The minimum inhibitory concentration (MIC) of bitter orange extract against S. Typhimurium was determined using the broth dilution method. Subsequently, S. Typhimurium was exposed to various concentrations of bitter orange extract (1/16 MIC-2 MIC) and growth curves were measured. Following treatment with bitter orange extract, we investigated its antibacterial mechanism by measuring intracellular reactive oxygen species (ROS) levels, alterations in membrane potential and integrity, and nucleic acid leakage in S. Typhimurium. Additionally, salads artificially contaminated with S. Typhimurium were treated with different concentrations of bitter orange extract using the dipping method for various durations to assess the reduction effect. The MIC of bitter orange extract against S. Typhimurium was 195.313 mg/L, and bacterial growth was completely inhibited at a concentration of 1 MIC. Furthermore, an increase in bitter orange extract concentration correlated with elevated intracellular ROS levels, membrane potential disruption, membrane damage, and nucleic acid release. Importantly, salads treated with bitter orange extract exhibited a significant reduction in S. Typhimurium counts compared to the control, and prolonged treatment times resulted in further reductions in bacterial counts. Bitter orange extract was more effective than sodium hypochlorite and can be used as a safer salad wash. These findings indicate the potential treatment of salads to prevent foodborne illnesses.

본 연구에서는 천연 항균제인 광귤 추출물의 항균 활성과 항균 메커니즘을 조사해 즉석섭취식품인 샐러드에서 Salmonella Typhimurium을 제어하기 위한 세척수로써 적용 가능성을 평가하였다. 액체배지희석법으로 S. Typhimurium에 대한 광귤 추출물의 최소 억제 농도(MIC)를 구했다. 그런 다음 다양한 농도(1/16 MIC-2 MIC)에 해당하는 광귤 추출물에 S. Typhimurium을 접종하고 성장곡선을 분석해 대조군과 성장값을 비교하여 항균 활성을 확인하였다. 광귤 추출물을 처리한 후, S. Typhimurium의 세포 내 활성산소종 수준과 막 전위 및 손상도의 변화, 핵산 누출량을 측정하여 광귤 추출물의 항균 메커니즘을 확인하였다. 최종적으로 S. Typhimurium을 인위적으로 접종한 샐러드에 다양한 농도의 광귤 추출물을 다양한 시간 동안 침지 방법으로 항균 처리하여 저감화 효과를 확인했다. S. Typhimurium에 대한 광귤 추출물의 MIC는 195.313 mg/L으로, 1 MIC와 2 MIC의 광귤 추출물은 S. Typhimurium의 성장을 완전히 억제하였다. 광귤 추출물의 처리농도가 높아질수록, 세포 내 ROS 수준과 막 전위, 막 손상도 그리고 핵산 방출량은 증가하였다. 마지막으로, 세척수인 광귤 추출물의 농도가 높고 처리 시간이 길수록 샐러드의 S. Typhimurium의 수가 감소하였다. 따라서 광귤 추출물은 S. Typhimurium를 효과적으로 제어할 수 있음을 입증했다. 광귤 추출물은 차아염소산나트륨과 비교하였을 때 효과적인 항균 활성을 보이며 안전한 샐러드 세척수로 사용될 수 있다. 이는 샐러드와 같은 식품에서 광귤 추출물이 식중독 발생을 미연에 방지하는데 기여할 수 있음을 시사한다.

Keywords

References

  1. Carstens, C.K., Salazar, J.K., Darkoh, C., Multistate outbreaks of foodborne illness in the United States associated with fresh produce from 2010 to 2017. Front. microbiol., 10, 2667 (2019). 
  2. Ministry of Food and Drug Safety, (2024, January 11). Beware of food poisoning with pathogenic E. coli in summer. Retrieved from https://www.foodsafetykorea.go.kr/portal/board/boardDetail.do?menu_no=2859&bbs_no=bbs082&ntctxt_no=1074833&menu_grp=MENU_NEW05 
  3. Centers for Disease Control and Prevention, (2024, January 11). Summary of Possible Multistate Enteric (Intestinal) Disease Outbreaks in 2017-2020. Retrieved from https://www.cdc.gov/foodborne-outbreaks/php/data-research/summary-2017-2020.html 
  4. Schikora, A., Carreri, A., Charpentier, E., Hirt, H., The dark side of the salad: Salmonella typhimurium overcomes the innate immune response of Arabidopsis thaliana and shows an endopathogenic lifestyle. PLoS One, 3, e2279 (2008). 
  5. Karabiyikli, S., Degirmenci, H., Karapinar, M., Inhibitory effect of sour orange (Citrus aurantium) juice on Salmonella Typhimurium and Listeria monocytogenes. LWT-Food Sci. Technol., 55, 421-425 (2014). 
  6. Levantesi, C., Bonadonna, L., Briancesco, R., Grohmann, E., Toze, S., Tandoi, V., Salmonella in surface and drinking water: occurrence and water-mediated transmission. Food Res. Int., 45, 587-602 (2012). 
  7. Arienzo, A., Gallo, V., Tomassetti, F., Antonini, G., Implication of Sodium Hypochlorite as a Sanitizer in Ready-to-Eat Salad Processing and Advantages of the Use of Alternative Rapid Bacterial Detection Methods. Foods, 12, 3021 (2023). 
  8. Virto, R., Manas, P., Alvarez, I., Condon, S., Raso, J., Membrane damage and microbial inactivation by chlorine in the absence and presence of a chlorine-demanding substrate. Appl. Environ. Microbiol., 71, 5022-5028 (2005). 
  9. Hua, G., Reckhow, D.A., Comparison of disinfection byproduct formation from chlorine and alternative disinfectants. Water Res., 41, 1667-1678 (2007). 
  10. Seow, Y.X., Yeo, C.R., Chung, H.L., Yuk, H.G., Plant essential oils as active antimicrobial agents. Crit. Rev. Food Sci. Nutr., 54, 625-644 (2014). 
  11. Suntar, I., Khan, H., Patel, S., Celano, R., Rastrelli, L., An overview on Citrus aurantium L.: Its functions as food ingredient and therapeutic agent. Oxid. Med. Cell. Longev., 7864269 (2018). 
  12. Periyanayagam, K., Dhanalakshmi, S., Karthikeyan, V., Magesh, M., Antibacterial activity of Citrus aurantium leaf essential oil against S. aureus and MRSA. J. Drug Discov. Ther., 2, 54-60 (2014). 
  13. Degirmenci, H., Erkurt, H., Chemical profile and antioxidant potency of Citrus aurantium L. flower extracts with antibacterial effect against foodborne pathogens in rice pudding. LWT-Food Sci. Technol., 126, 109273 (2020). 
  14. Castillo, S., Heredia, N., Arechiga-Carvajal, E., Garcia, S., Citrus extracts as inhibitors of quorum sensing, biofilm formation and motility of Campylobacter jejuni. Food Biotechnol., 28, 106-122 (2014). 
  15. Andrews, J.M., Determination of minimum inhibitory concentrations. J. Antimicrob. Chemother., 48, 5-16 (2001). 
  16. Han, Y., Sun, Z., Chen, W., Antimicrobial susceptibility and antibacterial mechanism of limonene against Listeria monocytogenes. Molecules, 25, 33 (2019). 
  17. Han, L., Patil, S., Boehm, D., Milosavljevic, V., Cullen, P. J., Bourke, P., Mechanisms of inactivation by high-voltage atmospheric cold plasma differ for Escherichia coli and Staphylococcus aureus. Appl. Environ. Microbiol., 82, 450-458 (2016). 
  18. Tyagi, P., Singh, M., Kumari, H., Kumari, A., Mukhopadhyay, K., Bactericidal activity of curcumin I is associated with damaging of bacterial membrane. PLoS One, 10, e0121313 (2015). 
  19. Hyun, J.E., Moon, S.K., Lee, S.Y., Antibacterial activity and mechanism of 460-470 nm light-emitting diodes against pathogenic bacteria and spoilage bacteria at different temperatures. Food Control, 123, 107721 (2021). 
  20. O'Bryan, C.A., Crandall, P. G., Chalova, V.I., Ricke, S.C., Orange essential oils antimicrobial activities against Salmonella spp. J. Food Sci., 73, M264-M267 (2008). 
  21. Freeman, B.L., Eggett, D.L., Parker, T.L., Synergistic and antagonistic interactions of phenolic compounds found in navel oranges. J. Food Sci., 75, C570-C576 (2010). 
  22. Bacun-Druzina, V., Butorac, A., Mrvcic, J., Landeka Dragicevic, T., Stehlik-Tomas, V., Bacterial stationary-phase evolution. Food Technol. Biotechnol., 49, 13-23 (2011). 
  23. Dhakal, J., Sharma, C.S., Nannapaneni, R., McDaniel, C.D., Kim, T., Kiess, A., Effect of chlorine-induced sublethal oxidative stress on the biofilm-forming ability of Salmonella at different temperatures, nutrient conditions, and substrates. J. Food Prot., 82, 78-92 (2019). 
  24. Oparka, M., Walczak, J., Malinska, D., van Oppen, L.M.P.E., Szczepanowska, J., Koopman, W.J., Wieckowski, M.R., Quantifying ROS levels using CM-H2DCFDA and HyPer. Methods, 109, 3-11 (2016). 
  25. Ghosh, R., Hoque, N., Shanta, M.A., Nasrin, N., Asaduzzaman, M., Antioxidant, antimicrobial and cytotoxic activities of different fractions of Citrus aurantifolia peel. Dhaka Univ. J. Pharm. Sci., 19, 161-168 (2020). 
  26. Lu, K.H., Lee, H.Y., Chu, Y.L., Ho, C.T., Sheen, L.Y., Bitter orange peel extract induces endoplasmic reticulum-mediated autophagy in human hepatoma cells. J. Funct. Foods, 60, 103404 (2019). 
  27. Azhdarzadeh, F., Hojjati, M., Chemical composition and antimicrobial activity of leaf, ripe and unripe peel of bitter orange (Citrus aurantium) essential oils. Nutr. Food Sci. Res., 3, 43-50 (2016). 
  28. Liu, J., Zhu, Y., Du, G., Zhou, J., Chen, J., Response of Saccharomyces cerevisiae to D-limonene-induced oxidative stress. Appl. Microbiol. Biotechnol., 97, 6467-6475 (2013). 
  29. Thakre, A., Zore, G., Kodgire, S., Kazi, R., Mulange, S., Patil, R., .Shelar, A., Santhakumari, B., Kulkarni, M., Kharat, K., Karuppayil, S. M., Limonene inhibits Candida albicans growth by inducing apoptosis. Medical mycology, 56, 565-578 (2018). 
  30. Su, R., Guo, P., Zhang, Z., Wang, J., Guo, X., Guo, D., Wang, Y., Lu, X., Shi, C., Antibacterial activity and mechanism of linalool against Shigella sonnei and its application in lettuce. Foods, 11, 3160 (2022). 
  31. Crowley, L.C., Scott, A.P., Marfell, B.J., Boughaba, J.A., Chojnowski, G., Waterhouse, N.J., Measuring cell death by propidium iodide uptake and flow cytometry. Cold Spring Harb. Protoc., 2016, 087163 (2016). 
  32. Fortunati, E., Bianchi, V., Plasma membrane damage detected by nucleic acid leakage. Mol. Toxicol., 2, 27-38 (1989). 
  33. Hashemi, S.M.B., Amininezhad, R., Shirzadinezhad, E., Farahani, M., Yousefabad, S.H.A., The Antimicrobial and Antioxidant Effects of Citrus aurantium L. Flowers (Bahar N arang) Extract in Traditional Yoghurt Stew during Refrigerated Storage. J. Food Saf., 36, 153-161 (2016). 
  34. Liu, X., Cai, J., Chen, H., Zhong, Q., Hou, Y., Chen, W., Chen, W., Antibacterial activity and mechanism of linalool against Pseudomonas aeruginosa. Microb, Pathog., 141, 103980 (2020). 
  35. Yamada, A., Gaja, N., Ohya, S., Muraki, K., Narita, H., Ohwada, T., Imaizumi, Y., Usefulness and limitation of DiBAC4 (3), a voltage-sensitive fluorescent dye, for the measurement of membrane potentials regulated by recombinant large conductance Ca2+-activated K+ channels in HEK293 cells. Jpn. J. Pharmacol., 86, 342-350 (2001). 
  36. Strauber, H., Muller, S., Viability states of bacteria-specific mechanisms of selected probes. Cytometry Part A, 77, 623-634 (2010). 
  37. Novo, D.J., Perlmutter, N.G., Hunt, R.H., Shapiro, H.M., Multiparameter flow cytometric analysis of antibiotic effects on membrane potential, membrane permeability, and bacterial counts of Staphylococcus aureus and Micrococcus luteus. Antimicrob. Agents Chemother., 44, 827-834 (2000). 
  38. An, Q., Ren, J. N., Li, X., Fan, G., Qu, S. S., Song, Y., Li, Y., Pan, S.Y., Recent updates on bioactive properties of linalool. Food Funct., 12, 10370-10389 (2021). 
  39. Han, Y., Chen, W., Sun, Z., Antimicrobial activity and mechanism of limonene against Staphylococcus aureus. J. Food Saf., 41, e12918 (2021). 
  40. Lee, S.H., Yumnam, S., Hong, G.E., Raha, S., Venkatarame Gowda Saralamma, V.V., Lee, H. J., Heo, J.D., Lee, S.J., Lee, W.S., Kim, E.H., Park, H.S., Kim, G.S., Flavonoids of Korean Citrus aurantium L. induce apoptosis via intrinsic pathway in human hepatoblastoma HepG2 cells. Phytother. Res., 29, 1940-1949 (2015). 
  41. Takeuchi, K., Frank, J.F., Penetration of Escherichia coli O157: H7 into lettuce tissues as affected by inoculum size and temperature and the effect of chlorine treatment on cell viability. J. Food Prot., 63, 434-440 (2000). 
  42. Kondo, N., Murata, M., Isshiki, K., Efficiency of sodium hypochlorite, fumaric acid, and mild heat in killing native microflora and Escherichia coli O157: H7, Salmonella Typhimurium DT104, and Staphylococcus aureus attached to fresh-cut lettuce. J. Food Prot., 69, 323-329 (2006). 
  43. Gurtler, J.B., Two generally recognized as safe surfactants plus acidulants inactivate Salmonella, Escherichia coli O157: H7, and Listeria monocytogenes in suspension or on dip-inoculated grape tomatoes. J. Food Prot., 83, 637-643 (2020).