Listeria monocytogenes is a Gram-positive non-spore forming, facultative anaerobic, and short rod foodborne pathogen1). The pathogen can cause a listeriosis which can be severe to pregnant women, newborns, and immunocompromised people2) with 20-30% of fatality rate3). L. monocytogenes is ubiquitous in the natural environment, and has usually been isolated from foods of animal origin including meat, sausages, cheeses, and milk products3).
L. monocytogenes has frequently been isolated from slaughterhouses and meat processing plants. In Korea, a total of 290 samples including carcasses (n = 140), feces (n = 136), and the environment (n = 14) samples were collected from nine slaughterhouses, and fifteen (5.2%) of L. monocytogenes were isolated4). In 373 samples from carcasses (n = 50), pluck sets (n = 250), and the slaughterhouse environment (n = 73) of ten slaughterhouses in Finland, L. monocytogenes was detected in six slaughterhouses and 9% of the samples5). Listeria spp. was detected in seven sites (3.2%) of three slaughterhouses (27.3%) in eleven slaughterhouses in Italy, and L. monocytogenes was isolated from 7.1% of handwash basins, and 13.3% of the cold room floor in the slaughterhouses6). L. monocytogenes in the slaughterhouse environment can easily migrate to raw meat processed at the slaughterhouses and can cause food poisoning7,8), and they can cross-contaminate processed-meat products. Thus, antimicrobials have been applied to control L. monocytogenes in the processed meat, but consumers prefer to use natural antimicrobials than synthesized antimicrobials9).
Therefore, the objective of this study was to evaluate activities of natural antimicrobials to control the growth of L. monocytogenes isolated from domestic slaughterhouses environment.
Materials and Methods
Preparation of natural antimicrobials
Grapefruit seed extract (Quinabra, Eldorado, Brazil), citrus fruit extract (Quinabra), ginger extract (Serim Food, Bucheon, Gyeonggi, Korea), pear extract (Serim Food), jujube extract (Sanjung Food, Eumseong, Chung-cheong bukdo, Korea), Japanese apricot concentrate (Sanjung Food), omija extract (Sanjung Food), and balloon flower extract (Sanjung Food) were purchased from online stores. Each antimicrobials was diluted with distilled water to final concentration of 8 μg/mL.
Inoculum preparation
Fifteen L. monocytogenes strains SMFM-SI-1, SMFM-SI- 2, SMFM-SI-3, SMFM-SI-4, SMFM-SI-5, SMFM-SI-6, SMFM-SI-7, SMFM-SI-8, SMFM-SI-9, SMFM-SI-10, SMFMSI- 11, SMFM-SI-12, SMFM-SI-13, SMFM-SI-14, and SMFM-SI-15 which were isolated from slaughterhouses in Korea4) were used in this study. Each L. monocytogenes strain was cultured in 10 mL of tryptic soy broth with 0.6% yeast extract (TSBYE; Becton, Dickinson and Company, Sparks, MD, USA) at 30°C for 24 h. One hundred microliter of each culture was inoculated to 10 mL of TSBYE, and incubated again at 30oC for 24 h. The cultures were centrifuged at 1,912 × g and 4°C for 15 min, and the cell pellets were washed twice with phosphate buffered saline (PBS, pH 7.4; 8.0 g of NaCl, 0.2 g of KH2PO4, 1.5 g of Na2HPO4, and 0.2 g of KCl in 1 L of distilled water). These suspensions were mixed, and its optical density was measured at 600 nm with UV-spectrophotometer (OPTIZEN 2120UV, Mecasys Co., Ltd., Daejeon, Korea). The OD600 was adjusted to 0.1 (8 log CFU/mL) to be an inoculum.
Determination of minimum bactericidal concentration (MBC)
MBCs were determined by the conventional two-fold dilution method10,11) with some modifications. Three hundred sixty microliters of each antimicrobial extract solution was added to the 96-well cell culture plate (SPL Life Sciences Co., Ltd., Pocheon, Gyeonggi, Korea). One hundred eighty microliter aliquots of TSBYE were placed to the other wells, and two-fold dilution of the antimicrobial extracts was performed to have 4.0, 2.0, 1.0, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015, 0.008, 0.004, 0.002, and 0.001 μg/mL of concentrations. Twenty microliter aliquots of L. monocytogenes inoculum were then inoculated into each well of the 96-well plates to have 7 Log CFU/mL of L. monocytogenes. To have 3 Log CFU/mL of L. monocytogenes, the inoculum was diluted with PBS and inoculated. The wells containing TSBYE and L. monocytogenes were positive controls, and the wells containing TSBYE and antimicrobials were negative controls. The plate was incubated at 30oC for 24 h. A loopful of the culture in the plate was then streaked onto the Palcam agar (Becton, Dickinson and Company), and the Palcam agar plates were incubated at 30oC for 48 h. After incubation, colony formation on the plates was confirmed.
Results and Discussion
The MBCs of grapefruit seed extract, citrus fruit extract, balloon flower extract, ginger extract, Japanese apricot concentrate, jujube extract, omija extract, and pear extract for L. monocytogenes isolated from slaughterhouses were investigated. L. monocytogenes was not destroyed at 7 log CFU/mL by ginger extract, pear extract, Japanese apricot concentrate, balloon flower extract, jujube extract, and omija extract regardless of concentration (Fig. 1). The result for ginger extract was similar to the result of previous studies, which reported that the ginger extract had no antimicrobial effect on L. monocytogenes at 10~100%12), and water and 50% ethanol extracts of ginger at concentration up to 10% also did not show any antimicrobial activity against L. monocytogenes13). However, the antimicrobial effects against Escherichia coli and Salmonella Typhi were shown by Ekwenye et al.14), and the effect was improved as the extract was prepared by ethanol, compared to water. Omija seeds extracts were found to be antibacterial against Lactobacillus plantarum, Bacillus subtilis, Staphylococcus aureus, E. coli, and Salmonella Typhimurium15), and Kim et al. also reported that omija extract has strong antibacterial activity against B. subtilis, S. aureus, E. coli, and Pseudomonas aeruginosa16). However, in our study the extract did not show antilisterial activity. Japanese apricot extract also showed strong antimicrobial activity on B. subtilis, E. coli, and P. aeruginosa, and the jujube extract showed a slightly weaker antimicrobial activity against these kinds of bacteria16), but these extracts do not have antilisterial activities.
The MBCs of grapefruit seed extract and citrus fruits extract against L. monocytogenes at 7 Log CFU/mL were 0.002 μg/mL and MBC to L. monocytogenes at 3 Log CFU/ mL was 0.001 μg/mL (Fig. 2).
In conclusion, grapefruit seed extract and citrus fruits extract can be used as antimicrobials to destroy L. monocytogenes isolated from slaughterhouse environment.
국문요약
본 연구에서는 도축장에서 분리된 Listeria monocytogenes 를 저해할 수 있는 식물성 항균 물질을 탐색하고, 각 항 균 물질의 최소살균농도(MBC)를 확인하였다. 잠재적인 항 균 물질로 자몽종자 추출물, 감귤류 추출물, 생강 추출물, 배 추출물, 매실 농축액, 도라지 추출물, 대추 추출물, 오 미자 추출물을 선정하였고, 대상 균주로는 국내 도축장에 서 분리한 15 개의 L. monocytogenes 균주를 혼합하여 사 용하였다. MBC 확인 결과, 최대 4.0 μg/mL 농도의 생강 추출물, 배 추출물, 매실 농축액, 도라지 추출물, 대추 추 출물, 오미자 추출물은 고농도의 L. monocytogenes (7 Log CFU/mL)에 대해 균 저해 효과가 전혀 나타나지 않았다. 반면, 7 Log CFU/mL의 L. monocytogenes에 대한 자몽종 자 추출물과 감귤류 추출물의 MBC는 0.002 μg/mL인 것 으로 확인되었다. 3 Log CFU/mL의 L. monocytogenes에 대 한 MBC를 시험해 본 결과, 자몽종자 추출물과 감귤류 추 출물의 MBC는 0.001 μg/mL로 나타났다. 이상의 결과로 볼 때 자몽종자 추출물과 감귤류 추출물은 L. monocytogenes 를 제어하기 위해 적용될 수 있을 것으로 판단된다.
