초록 ▼

IV. 연구개발 결과 및 활용에 대한 건의
1. 오염원 추적기술 활용
Fingerprinting에 의한 genotype 확인은 동종의 오염미생물을 세분화해주므로 작업장내 오염원 추적에 매우 유용하다. 생산제품의 특성과 작업조건에 적합한 주요 오염미생물을 선정하고 분자생물학적인 분석을 한다면, 작업장 오염의 근본원인을 진단하고 이를 예방하는 조치가 가능하여 식품안전관리에 크게 기여할 것이다. 업계에서는 HACCP 컨설팅, 현장 위해분석 및 진단에 즉시 활용 가능할 것으로 보인다.
2. 오염발생예측 시스템 활용
○

IV. 연구개발 결과 및 활용에 대한 건의
1. 오염원 추적기술 활용
Fingerprinting에 의한 genotype 확인은 동종의 오염미생물을 세분화해주므로 작업장내 오염원 추적에 매우 유용하다. 생산제품의 특성과 작업조건에 적합한 주요 오염미생물을 선정하고 분자생물학적인 분석을 한다면, 작업장 오염의 근본원인을 진단하고 이를 예방하는 조치가 가능하여 식품안전관리에 크게 기여할 것이다. 업계에서는 HACCP 컨설팅, 현장 위해분석 및 진단에 즉시 활용 가능할 것으로 보인다.
2. 오염발생예측 시스템 활용
○ 작업조건에 따른 controlling factor 선정과 자료입력으로 최종제품에서의 위해 미생물 오염수준 예측이 가능하며, 입력 자료의 정확성 제고에 따라 개발된 모델에서 산출되는 결과 값의 신뢰성도 높아진다.
○ 축산식품 생산현장의 작업조건별 변수를 선정하고, 입력자료를 산출하여 본 연구에서 개발된 오염발생예측 시스템에 적용하면, 최종생산품에서의 오염수준 및 위해발생 가능성을 예측할 수 있다.
○ Sensitivity 분석과 scenario 분석을 수행하면, 공정상의 영향력 있는 부분 혹
은 critical control point 선정과 위생관리기준 설정에 과학적인 자료를 제공하므로,
HACCP system의 유용성 평가에 활용 가능하다.
3. 기업화 추진방안
○ Fingerprinting을 이용한 오염원 추적기술은 안전관리 팀을 운영하는 기업체가 자사 작업장의 위생상태, 오염의 흐름 진단 및 평가에 활용하도록 권장한다.
○ MHEM 모델의 핵심기술을 프로그래밍 언어(C 언어 등)를 이용 산업용 소프트웨어로 개발한다.

Abstract ▼

1. Patterns of contamination and tracing the sources.
This study was performed to characterize the patterns of contamination and to trace the fundamental sources of contamination of Listeria spp. and Salmonella spp. in pork processing plants using randomly amplified polymorphic DNA (RAPD). Sample

1. Patterns of contamination and tracing the sources.
This study was performed to characterize the patterns of contamination and to trace the fundamental sources of contamination of Listeria spp. and Salmonella spp. in pork processing plants using randomly amplified polymorphic DNA (RAPD). Samples were collected from carcass, pork on processing, surfaces of equipment and environment from two small plants (80~100 carcasses processing/day) and two large plants (600~800 carcasses).
1) Survey on the procedure condition in pork processing plant
The results of survey showed that processing time was a minimum of 15 minutes and usually 25 minutes, but it took two hours in some case. The temperature of inner pork carcass appeared 5.4℃ as a mean value, including a minimum of 1.0℃ and maximum of 8.1℃. The processing room temperature was 18℃ as a mean value, including minimum 15℃ and maximum 25℃. Processing time, processing room temperature which showed high variability were selected as controlling factors.
2) Isolation rate of Listeria spp. and Salmonella spp.
A total of 782 samples were collected from carcass, pork during procedure, surfaces of hands, equipment and environment. Of the 244 Listeria spp. solates, L. innocua, L. monocytogenes, and L. welshimeri was 26.91%, 14.7%, 2.4% respectively. Listeria spp. were found in the carcass 22.7%, pork on processing 35.3%, final pork 51.5%, equipments 46.3%, and environments 54.7%. Serotype 1 was a dominant type (94.8%) of the L. monocytogenes isolates. Isolation rate of Salmonella spp. was 2.0% which was too low to characterize patterns of contamination and to conduct DNA fingerprinting to trace the contamination source.
The contamination of equipments and environments before operation due to ineffective washing and disinfection activity, easily disseminated microorganisms through out the processing line during procedure and affected the safety of final pork.
When we compared HACCP plants and non-HACCP plants, the contamination rate of Listeria spp. in final pork was 16.7% in HACCP plant and 38.9% in non-HACCP plant.
This high prevalence required more stringent HACCP audit and HACCP system to be used widely.
3) Tracing the source of Listeria spp. using RAPD
Contamination of microorganisms such as Listeria and Salmonella is a threat in pork processing plants, because no killing steps during the procedure. Random amplified polymorphic DNA (RAPD) analysis performed to investigate the origin and routes of Listeria contamination, showed 35 composite types of L. monocytogenes and 55 composite types of L. innocua. It was found that the first source of Listeria contamination was incoming pork carcass contaminated during transporation from slaughterhouse to processing plant. It required more strict inspection for incoming carcasses. The second source of contamination was the remaining Listeria strains throughout the processing environments. The remaining strains of L. monocytogenes and L. innocua throughout the processing line revealed that the sanitation standard operating procedure should be implemented to minimize the risk of colonization in the workplace. When we used L. innocua together with L. monocytogenes during RAPD genotyping, we could get useful information to figure out the patterns of contamination.
2. Exposure assessment and development of Microbial Hazard Exposure Model
This study was performed to develop a model system which can be utilized to predict and estimate a real-time prediction of the contamination level of Salmonella spp. and L. monocytogenes in pork processing plants by the application of Microbiological Risk Assessment.
1) Development of primary models
(1) Construction of framework model
On the base of CF, framework was designed as a series of unit operations that included the initial contamination level of cold storage pork after slaughtering (node 1), the growth and cross-contamination during processing (node 2) and the growth and the contamination level of cold storage pork after packing.
(2) Transfer rate and response surface regression model
Adherence rate to pork and to contact surfaces (stainless steel and Conveyor belt) and probability rate of survival and growth were examined for measuring the transfer rate for Salmonella spp. and L. monocytogenes in pork processing plant. Adherence rates were tested from pork to pork and from pork to contact surfaces. Probability rate of survival and growth were examined for carcasses, pork on procedure, and the composite of pork, employee, pork contact surface.
Based on this results, we could determine time and temperature as an independent variables and design a surface regression model using a transfer rate as a dependent variable.
(3) Estimation of survival and growth
Model was developed to predict the survival and growth of Salmonella spp., and L. monocytogenes in pork. Experimental conditions for model development was full 5-by-7 factorial arrangements of temperature (0, 5, 10, 15, and 20℃) and time (0, 1, 2, 3, 18, 48, and 120 hr). Gompertz value and Growth kinetic were calculated based on growth data. From them, Response surface analysis equation for predictive growth was produced.
2) Development of Microbial Hazard Exposure Model
(1) Exposure simulation model and development of software hard core
Software hard cores for the estimation of hazardous microbial contamination level in pork processing plant constitute three nodes in accordance with framework model. The initial input variables included the microbial contamination level during cold storage of pork carcass, the temperature of the processing room, and processing time. The output was the prediction of contamination level during cold storage after packing. This hard cores included transfer rate model, growth predictive model, and probability distribution functions. Input variables were constructed to Excel spreadsheet as an exposure simulation model (2) Application of Microbial Hazard Exposure Model in pork processing plant The MHEM simulated with @RISK and compared the contamination level of Salmonella spp. with L. monocytogenes for initial contamination of carcass, processing procedure, and storage steps in pork processing plant. Contamination level for L. monocytogenes during procedure was fluctuated but not for the Salmonella. This results showed that L. monocytogenes could grow better than Salmonella spp. Sensitivity analysis showed that Salmonella spp. had low correlation coefficients and did not show much difference of the parameters. But L. monocytogenes had high correlation coefficients at the initial contamination level, cold storage time, and processing time. Therefore, these factors were considered as Critical Control Point. We compared the contamination level of Salmonella spp. and L. monocytogenes between HACCP and non-HACCP plant.
The level of Salmonella contamination was not much different between them, but L. monocytogenes was contaminated less in HACCP plant.
(3) Evaluation of Microbial Hazard Exposure Model
MHEM was good enough for real-time estimation of contamination level of Salmonella spp., and L. monocytogenes in pork processing plants using only initial contamination level, processing room temperature, and processing time.
Also, this tool could be applied for the risk evaluation of the hazard factors identified during procedure and their control.
3. Development of molecular typing, detection and enumeration method
We compared molecular typing methods for the differentiation of Listeria spp and Salmonella spp. The results typed by RAPD were compared with those obtained by ERIC, ribotyping and SSCP. As for Listeria spp., the discriminatory power of RAPD typing was the best of the methods examined. RAPD typing with two different primers for 13 Listeria spp. reference strains did not differentiate all 13 reference strains. Composite of two separate RAPD (lis 11 and primer 6) results or RAPD (lis 11)/ribotyping-PCR differentiated all 13 Listeria spp. reference strains. As for Salmonella spp., the RAPD had the greatest discriminatory power of the methods examined, and was the single most discriminatory. RAPD typing did not differentiate all 57 reference strains. Combination of two different typing methods generally increased the discrimination of salmonella strains. Discrimination using a combination of RAPD (primer 1 or primer A) and ERIC was the most discriminatory and could differentiate all 57 Salmonella strains. For the specific detection of Listeria monocytogenes, we chose hlyA gene of L. monocytogenes encoding 58 kDa listeriolysin O which has been tested for the specific detection of L. monocytogenes in various foods. For the specific detection of Salmonella spp., invA was chosen. Competitive PCR was used to develop a direct enumeration of L. monocytogenes and Salonella spp. in pork. Known amount of competitor DNA was coamplified with L. monocytogenes or Salmonella spp. total DNA isolated from artificially inoculated pork. Since the relative amounts of cPCR products generated from amplification reflect the relative initial levels of the two different kinds of DNA, the colony forming unit (cfu) of microorganism could be easily estimated by comparing the intensity of the two bands after electrophoresis. The hlyA gene was cloned and was 148-bp deleted to produce L. monocytogenes competitor DNA. As for the L. monocytogenes, the detection limit achieved was 860 cfu per 0.1 g of pork. The invA gene was cloned and was 82-bp deleted to produce Salmonella spp. competitor DNA. As for the Salmonella spp., the detection limit was 700 cfu per 0.1 g of pork.

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 요 약 문 ... 3
• Summary ... 11
• Contents ... 17
• 목 차 ... 19
• 제1장 서 론 ... 23
• 제1절 연구개발의 필요성 ... 23
• 제2절 연구개발의 목표 ... 24
• 제3절 연구개발의 내용 ... 26
• 제2장 국내외 기술개발 현황 ... 29
• 제1절 해외의 관련연구 ... 29
• 1. MRA/Exposure assessment ... 29
• 2. MRA/QRA 최근 연구동향 ... 30
• 3. Molecular typing과 정량화 분석 기술 ... 31
• 제2절 국내의 관련연구 ... 32
• 제3절 현 기술상태의 취약성 ... 32
• 제4절 본 연구결과의 의미 ... 33
• 제3장 작업장의 오염발생 특성 및 오염원 분석 ... 35
• 제1절 공정조건 분석 ... 36
• 1. 포장돈육 작업공정 분석 ... 36
• 2. 작업환경 분석 ... 39
• 제2절 실험재료 및 방법 ... 41
• 1. 실험대상 작업장 선정 ... 41
• 2. 시료채취 및 방법 ... 42
• 3. Listeria 분리 및 동정 ... 42
• 4. Salmonella 분리 및 동정 ... 43
• 5. DNA 분리 정제 ... 43
• 6. RAPD-polymerase chain reaction(PCR) 조건 ... 46
• 제3절 오염발생 특성분석 ... 47
• 1. 포장돈육 가공장에서 Listeria spp.의 분리율 ... 47
• 2. 혈청형별 Listeria monocytogenes의 분포 ... 49
• 3. 포장돈육 가공장에서 Salmonella spp.의 분리율 ... 52
• 4. 작업전·중의 Listeria spp. 오염발생 양상 ... 56
• 5. HACCP 지정전·후의 Listeria spp. 오염수준 비교 ... 62
• 제4절 포장돈육 가공장의 오염원 추적 ... 69
• 1. L. monocytogenes의 RAPD typing ... 69
• 2. Listeria innocua의 RAPD typing ... 77
• 3. RAPD typing 결과를 이용한 가공장내 오염원 추적 ... 83
• 제4장 Exposure assessment와 오염발생 예측 모델 개발 ... 89
• 제1절 Frame-work 모델 작성 ... 90
• 1. 입력변수 선정 ... 90
• 2. Frame-work 모델 구성 ... 90
• 3. Frame-work 모델 활용 ... 91
• 제2절 Controlling factor에서의 오염전이율(TR) 분석 ... 94
• 1. 서론 ... 94
• 2. 실험적 접근방법 ... 94
• 3. 오염부착율 및 전이율 분석과 예측모델 개발 ... 107
• 제3절 공정내 위해미생물 생존 및 잔류 분석 ... 145
• 1. 서론 ... 145
• 2. 실험적 접근 방법 ... 145
• 3. 결과 ... 150
• 제5장 오염발생 예측 시스템(Microbial Hazard Exposure Model)개발 및 평가 ... 167
• 제1절 오염발생 예측용 소프트웨어 핵심기술 개발 ... 167
• 1. 모델 핵심기술 구성 개요 ... 167
• 2. 확률분포 모델 선정 ... 167
• 3. 핵심기술 구성 ... 169
• 제2절 포장돈육 가공장에 대한 Microbial Hazard Exposure Model 적용 ... 174
• 1. 적용 방법론 ... 174
• 2. 모델을 이용한 포장돈육공정의 Salmonella, L. monocytogenes 오염수준 평가 ... 175
• 제3절 Microbial Hazard Exposure Model 평가 ... 204
• 1. 모델 검증 ... 204
• 2. 모델 평가 ... 207
• 3. 모델 활용 ... 208
• 제6장 위해미생물의 분류, 신속검출 및 정량화 기술 개발 ... 211
• 제1절 위해미생물의 molecular typing을 위한 fingerprinting 기술보완 ... 212
• 1. 재료 및 방법 ... 212
• 2. Listeria의 특성분석을 위한 molecular typing 방법의 상호보완 ... 218
• 3. Salmonella의 특성분석을 위한 molecular typing 방법의 상호보완 ... 226
• 제2절 위해미생물의 신속검출 및 정량화 기술개발 ... 236
• 1. 재료 및 방법 ... 236
• 2. 돈육에서의 L. monocytogenes 검출 ... 241
• 3. 돈육에서의 오염된 Salmonella 검출 ... 245
• 4. Competitive PCR을 이용한 돈육에서 L. monocytogenes 정량 ... 251
• 5. Competitive PCR을 이용한 돈육의 Salmonella 정량 ... 253
• 제7장 목표달성도 및 관련분야에의 기여도 ... 255
• 제8장 연구개발결과의 활용계획 ... 259
• 제9장 연구개발과정에서 수집한 해외과학 기술정보 ... 261
• 제10장 참고문헌 ... 262
• 끝페이지 ... 272