A Quantitative Microbial Risk Assessment Model for Salt Effects on the Inhibiting Salmonella enteritidis

Abstract

최근 살모넬라는 신선편의식품에서 발생된 주요 식중독 원인균으로 보고되었다. 감염된 식품을 섭취함으로써 발생될 수 있는 위해도를 평가하는 작업은 식중독 위해분석시 가장 중요한 과정이다. 이 연구의 주요 목적은 항균활성이 예상되는 죽염을 이용하여 살모넬라의 억제효과를 미생물학적 위해성 평가 기법으로 분석하는 것이다. 위해성 평가를 수행하기 위해서는 오염된 식품과 임상적 살모넬라 식중독 질환 발병율 사이의 정량적인 자료가 필요하다. 그러나 현재 이러한 임상 자료는 부족한 실정이다. 따라서 본 연구에서는 이러한 현실적인 단점을 실험적인 방법으로 보완하여 위해성 평가를 수행하였다. 첫째, 노출량 평가 부분에서는, 신선편의식품에 소금을 첨가하여 살모넬라 성장 억제 정도를 측정하고 예측모델을 개발하였다. 1차 연구에서는 (1) 토마토 추출물에 다양한 종류의 소금(죽염, 천일염 및 NaCl)과 pH 및 저장온도를 추가하여 다양한 환경적 영향에 따른 살모넬라의 성장 변화를 측정하였다; (2) 성장곡선을 모델링하기 위하여 Gompertz 방정식에 적용하였다; (3) 실험적 환경조건하의 실제 식품에서의 살모넬라의 예측 성장율을 Response Surface Model을 이용하여 개발 후 모델 검증을 실시하였다. 모델 검증 결과 본 예측 모델은 토마토 추출물에 다양한 온도와 염도 조건을 적용하여 살모넬라의 성장을 예측하는 것이 가능한 것으로 판단되었다. 두 번째, 용량반응평가에서는 동물모델을 이용하여 소금에 따른 살모넬라 감염 관계를 평가하였다. 7주령 수컷 Balb/c 생쥐에게 각각 다른 농도의 살모넬라를 경구 투여한 후, NaCl과 죽염을 1.75, 4.0% 농도로 음용수에 혼합하여 자유로이 공급하였다. 경구투여 후 일정 기간별로 해부하여 조직의 감염정도를 측정하였다. 죽염 투여군의 경우 NaCl 투여군에 비하여 살모넬라 감염 가능성이 저하되었다. 용량반응평가 결과 동물모델에서의 식중독 감염은 급성독성 단계에서 용량반응 모델 적용이 가능한 것으로 평가되었다. 실험적 제한점에도 불구하고, 본 연구는 미생물학적 위해성 평가에 필요한 자료와 도구로 이용 가능하며 위해성 관리와 결정 단계에서도 참고 자료로 활용 가능할 것으로 생각된다.; Salmonella is one of the most important causes of foodborne illness associated with fresh-cut vegetables and fruits. The assessment of the real risk associated with the consumption of these products is needed and the most appropriate method to achieve this goal is the risk analysis process which links pathogens in food to the public health program. The main aim of this dissertation was to quantitatively assess the risk of salmonellosis inhibited by the consumption of bamboo salt. A data gap that is routinely identified in risk assessment is the lack of quantitative data on pathogens contaminated food and human salmonellosis. Therefore, we only developed two steps of the quantitative microbial risk assessment. The first aims of this study, the exposure assessment, we developed the predictive modeling of salts for inhibiting Salmonella enteritidis growth in vitro: (a) to investigate the single and combined effect of different salts (bamboo salt, sea salt and NaCl) with pH and storage temperature on the survival of S. enteritidis in tomato extracts; (b) to evaluate the Gompertz model and equation for modeling survival curves; (c) to develop and validate a Response Surface Model (RSM) for the response of S. enteritidis in a real food system at different combinations of the controlling factors. Tomato extracts were inoculated with S. enteritidis supplemented with three concentrations of different salts (bamboo salt, sea salt and NaCl; 0, 30, 50 mg/mL) and stored at different temperatures (4, 10, 15, 20, 25 ℃). The products’ pH was adjusted from 5.0 to 7.0. At each combination of the environmental factors, the bacterial counts were modeled as a function of time in order to estimate the maximum specific growth rate (μ_(max)) and lag time (LT) of the pathogen. In the exposure assessment, the Gompertz equation was applied to calculate and depict the growth curve for each experimental temperature and corresponding μ_(max) and LT in the primary model. In the secondary model, the Response Surface Model (RSM) was applied with the estimated lag time and maximum specific growth rates as functions of temperature, pH and salt concentrations. The coefficients of variations and RMSE for μ_(max) and LT agreed well with the observed growth data. The results of these experiments and observations led us to suggest that the bamboo salt has antimicrobial activity against the growth of S. entertidis in tomato extracts, and the models developed in this research would be useful in predicting S. enteritidis in tomato extract. In the second part, we studied dose-response relationships in S. enteritidis infected with salts in vivo. The aims were to investigate the quantitative relationship between inoculation doses and physiological responses to infection by enteropathongeic bacteria and to assess the in vivo antimicrobial effect of the bamboo salt against S. enteritidis. Seven weeks old, male BALB/c mice were exposed orally to different dose of S. enteritidis after neutralization of gastric acid by sodium bicarbonate. Mortality was observed in animals exposed to doses of 106 cfu/mice or more. In S. enteritidis-infected mice, the spleen and the liver were found to be the main target organs which were positive for salmonellosis. The mice model suggests that the bamboo salt has the antimicrobial effect by the excretion carrier of Salmonella. The Beta-Poisson model agrees better with experimental data in this dose range than the exponential model and the quantity of salmonellosis was H-BS > L-NC > L-BS > H-NC group, respectively. In summary, we demonstrated that bamboo salt has the antimicrobial activity for S. enteritidis in vivo and in vitro study. In addition, this study provides a toll that can actually be used as a risk evaluation toll and a helping device in the decision-making process and the risk management.