초록

○ 연구결과
- NIR 및 CE를 이용한 농산물의 원산지 판정방법 개발
- 전자코를 이용한 농산물의 판별기술 및 NIR을 이용한 육류의 신선도 및 저장성 평가기술 개발
- PCR을 이용한 축산물 및 축산가공식품으로부터 Listeria, Escherichia 및 Salmonella 신속검출법 개발
- 농산물의 안전성 평가를 위한 중금속/항생제 신속계측 바이오센서 시스템 개발
- 농산물 중의 잔류농약 신속․다성분 분석기술 개발

Abstract ▼

IV. Results and Opinion for Future Use
1. Development of the discrimination methods for geographical origin of agricultural products by NIR and CE
The price of agricultural products cultivated in Korea is much higher than that imported from foreign countries. To prevent the forgery of geograph

IV. Results and Opinion for Future Use
1. Development of the discrimination methods for geographical origin of agricultural products by NIR and CE
The price of agricultural products cultivated in Korea is much higher than that imported from foreign countries. To prevent the forgery of geographical origin for agricultural products, it is urgent to establish the database for discrimination of geographical origin. Capillary electrophoresis (CE) is a recent technique and has developed very fast. Such rapid development is due to several factors, but especially to the speed and versatility of CE. Thus, it is possible that CE is a rapid and accurate discrimination method for agricultural products. This study is to produce faster and simpler protocols for agricultural products by CE and to investigate the possibility of CE as a tool to discriminate the geographical origin. The optimal analytical conditions for job's-tears (Coix lachrymajobi var. mayuen) and angelicae gigantis radix (Angelica gigas) were established using CE at the 1st year of the study. The average correct discrimination percentage of domestic or foreign job'stears and angelicae gigantis radix were 82 and 86%, respectively. But safflower seed (Carthamus tinctorius) and ginseng radix (Panax ginseng) powder were not analysed using CE. At the 2nd year of the study, optimal analytical conditions for astragalus roots (Astragalus membranaceus), cnidii rhizoma (Ligusticum wallichi) and bracken (Pteridium aquilinum) were settled by CE. The mean values of accuracy for correct discrimination of geographical origin of astragaus roots, cnidii rhizoma and bracken were 80, 62 and 84%, respectively. Red pepper (Capsicum anuum) powder was not analysed using CE. Burdock (Aretrium lappa), carrot (Dancus carota) and lotus root (Nelumbo nucifera) were analysed by CE at the 3rd year of the study. The optimal analytic conditions were established and the average correct discrimination percentage of domestic or foreign origin burdock was 55%. In conclusion, our results raise the possibility that CE could be potential methods for discrimination of geographical origin of agricultural products.
For the development of the discrimination methods for geographical origin of agricultural products by NIR, the following works were undertaken. First, the standard material preparation for main items of agricultural products was done because this is the most important and fundamental point to secure standard materials in this study. Hence, we prepared standard materials from samples collected in main places of production and import harbors. Second, the database construction of each item by NIR analysis was done by constructing the database on each item by NIR analysis, followed by the preparation of the model (calibration equation) of each item in the database. Finally, the validation of each calibration equation was done by applying unknown samples to each calibration equation. We checked the possibility of the discrimination of geographical origin. From now on, it will be possible to accurately manage labeling geographical origins of the agricultural products by applying this research on the spot.
2. Development of the rapid detection method for Listeria, Escherichia and Salmonella from livestock products and foods by PCR
Recently, microbial food safety issues have been emerged as a major public health concern worldwide. Developments for detection of food-borne pathogens such as E. coli O157:H7, L. monocytogenes and Salmonella spp. are highly required for prevention and control. For decades, various molecular diagnostic techniques have been developed to monitor the food-borne pathogens for public health protection.
Despite the potential of false-positive, PCR-based detection method has been used as a powerful tool because of very high specificity and simplicity. Thus, we have put great efforts to establish the multiplex conditions for both simplifying the sample preparation and increasing the sensitivity and specificity in terms of saving time and labor. In this study, we first tried to optimize a simple step for extraction-free PCR template preparation and reliable PCR condition in model food, and then, tested applicability of this process to the field samples for the purpose of monitoring the microbial hazard in the market and industry.
Briefly, centrifugation at 16,000 ×g for 5 min and partial heat treatment at 98℃ for 10 min were mainly used for the simple preparation of PCR templates. To eliminate possibility of false positive, more than two primer sets were used for PCR amplification. Both E. coli O157:H7 and L. monocytogenes were detected at the level of 100 initial cell counts after enrichment without genomic DNA extraction in the culture and the food sample, respectively. Compared to E. coli O157:H7, detection sensitivity of L. monocytogenes was low at the level of 103 cells. Thus, to improve the sensitivity of L. monocytogenes, we applied cell wall destabilizer to the sample preparation step. As the result of PCR amplification treated with destabilizer, the limit of detection was lowered 103 to 101 and 102 cells in the pure culture and the food samples, respectively. Sensitivity of Salmonella detection with heat treatment before PCR was 102 cells per g of food sample or 1 pg of DNA template.
Feasibility test for the field trial was carried out on the basis of empirically optimized PCR method. Five representative samples such as raw chicken meat, raw pork, zocbal, sundae, and raw beef were aseptically collected from the different local markets, and treated to screen both E. coli O157:H7 and L. monocytogenes.
According to the PCR amplified with species-specific primers, L. monocytogenes was detected in two pork samples but E. coli O157:H7 was not detected in any sample.
Ten imported foods and 9 domestic foods were tested for the contamination of Salmonella but none was contaminated with Salmonella. These results suggested PCR method can be used for rapid, sensitive and species-specific detection to monitor the food-borne pathogens in the market.
Based on these results, PCR-based method is a powerful tool for sensitive and specific detection of the food-borne pathogens, if time to results and the potential of positive-false can be reduced. As explained above, DNA extraction-free PCR assay for detection of E. coli O157:H7 and L. monocytogenes in meat and meat products can meet these demands. As the results of these application with model foods, E.coli O157:H7 and L. monocytogenes were detectable as low as 100 initial CFU per ml (g) after enrichment for 8 and 24 hr, respectively. Thus, the protocol developed in this work seems to have a significant impact for the rapid and easy detection of E.coli O157:H7 and L. monocytogenes in foods. Conclusively, this simple method can help safety management to monitor the pathogen contaminated foods at low cost.
3. Development of the biosensor systems for the detection of heavy metals and antibiotics in agricultural products
The development of biosensor system for heavy metal detection was done as follows. Urease-based inhibition sensors which measure the inhibition of urease activity by heavy metals were developed. First, the properties of the free urease comprising the effects of enzyme amount and substrate concentration on enzyme activity were examined, followed by the development of a cuvette assay based on spectrophotometric principle. The inhibition characteristics of the free urease at varying concentrations of the single and dual heavy metal ions including Hg(II), Cu(II) and Pb(II) were determined, which confirmed the possibility of detection on heavy metals through the measurement of inhibition degree of urease. In the next stage, the biosensor system which exploited an ammonium ion-selective electrode as the transducer was developed by installing Immobilon cellulose nitrate membrane immobilized with urease onto the electrode and connecting the resulting sensing electrode to a PH/mV meter, followed by the measurements of its basic properties and inhibition characteristics with heavy metal ions. As a whole, the biosensor system showed the sensitivity as comparable as that of the cuvette assay. Also, the set-up of hardware structure on a computer interfacing system for processing biosensor signals was done and the operating software was devised, followed by the examination of its operating characteristics.
The development of biosensor system for antibiotics detection was done as follows. Chemiluminescent immunosensor systems which measure chloramphenicol (CAP) were prepared in batch and flow basis. First, an anti-CAP antibody and analyte-HRP conjugate (tracer) for a direct competitive immune reaction were secured or chemically synthesized. The antibody immobilization was done by soaking various membranes into the antibody solution and the best performance in biosensor response was found in case of Biodyne A and B membrane. A batch type biosensor system using a fiber-optic probe and Radiance Spectrometer of Avantes Co., and a flow type biosensor system using a home-made flow cell and R955 photomultiplier of Hamamatsu Photonics were constructed. After the optimization of reaction conditions, the responses of the immunosensors according to analytes were determined, resulting in the detection limit around 10-7 M. A potentiometric biosensor system measuring penicillin compounds was developed using penicillinase (β-lactamase) and a flat-bottomed pH electrode as the biological component and transducer, followed by the elucidation of biosensor response concerning the amount of enzyme for immobilization, ionic strength, pH and substrate types. The detection limit of the penicillin sensor could be extented to 1 μM. Also, a computer interfacing system for processing the optoelectronic signals from the immunosensors was also developed.
The PC-controll for the biosensor systems was made possible and a flow type urease inhibition sensor system was developed exploiting a micro-column enzyme reactor packed with Chitopearl beads, followed by the elucidation of biosensor performance concerning the amount of enzyme for immobilization, flow rate and length effect of the micro-column. The sample pre-treatments for the biosensors and the conventional methods were done. To the analytical samples, heavy metals, CAP or penicillin compounds were added at varying amount. The model samples were analyzed for the corresponding contaminants by both methods and the analytical results were compared. On the whole, the analytical results obtained with the biosensor methods were matched well with those of the conventional methods. For the dissemination of the research output, a part of our results was presented at IFT Annual Meeting. Also, the automation device and software for the developed biosensors were constructed and the bench-scale controls for the measuring procedures were done.
4. Development of the new, rapid, environmentally sound and economical multi-residue pesticide analysis technique of agricultural products The optimum SPME condition was selected with 85 nm poly acrylated fiber for 10 min of extraction and 30 min of desorption at 35℃ by automated vibration mode as agitation. SPME coupled with GC and HPLC procedures have been developed for 50 kinds of pesticides and were determined by ECD or NPD and UVD or FLD.
Sample (25g) was homogenized with 50 ml of acetonitrile and 20 ml of the upper layer was blended with 2g of NaCl and distilled water or pH 8 phosphate buffer.
Three milliliters of the upper layer acetonitrile for GC or 2 ml of it for HPLC was dried for 20 min at 50℃ under air stream. Dried extract was dissolved with 1.5 ml of 20% acetone/DW for GC or with 1 ml of 20% acetonitrile for HPLC. After SPME, the fiber was injected into GC or HPLC. Recoveries of SPME ranged 40∼170% in GC/NPD and 60∼138% in HPLC/FL. Organophosphorus and carbamate pesticides were ranged 70∼130% in GC and HPLC, indicating SPME could typically be applicable in pesticide residue monitoring. At in-situ test, tolclofos-methyl was detected in ginseng with GC analysis with enough sensitivity and repeatability which are same level of traditional residue analysis. Carbendazim was detected in sedum at 3% difference compared with traditional analysis by HPLC. Total analysis time of SPME-GC/HPLC was less than 2 hr, which saved a lot of time compared with traditional method of 6∼8 hr. Solvent consumption was also reduced to 50.5 ml compare with 500∼1000 ml of traditional method.
Two same samples spiked with or without pesticides were simultaneously analyzed by optimized SPME (i.e., 100 μm polymethylsiloxane fiber, 50 min adsorption and 10 min desorption (270℃ for GC), pH=7). Most of pesticides were extracted by SPME coating fiber, but a few pyrethroid and organophosphorus pesticides were not detected by SPME-GC/ECD-NPD dual detection system. The behaviour of the analytical responses of pesticides in agricultural products ranged between 0.01 and 1.0 ppm, and showed good linearity with regression coefficient for > 0.990). The detection limits for most pesticides were approximately 0.001 ppm.
This method can be employed for screening studies in which pesticides known to be capable of analysis by SPME-GC-HPLC are involved. A typical example is the monitoring of the residue level of particular compounds used in a treatment of a given crop versus time, and their parents and decay.
An immunoassay method for a widely used herbicide 2,4-D, as described below, was developed. Monoclonal antibody (MAb) against 2,4-D was produced by hybridoma method. Myeloma cells were fused with spleen cells of BALB/c mice, immunized with hapten conjugated via carboxyl group to keyhole limpet hemocyanin (KLH). To evaluate the obtained MAb (MAb 2,4-D-121-57, 2,4-D-121-46), competitive indirect ELISA procedure was developed. For the purpose of increasing the sensitivity and selectivity of ELISA, several kinds coating antigens conjugated with proteins such as bovine serum albumin, ovalbumin, corn albumin, and thyroglobulin were employed. Among them, 2,4-D-OVA was the most sensitive among other conjugates. MAb 2,4-D-121-57 was specially reacted with 2,4-D (IC₅₀=4.89 ng/ml) but much less with its analogs instead of methylester 2,4-D (15.6%).
When 2,4-D was spiked in tap water, orange juice and barley with different concentration, recovery ratio was 98∼102, 89∼100 and 92∼108%, respectively.
Experimental immunoaffinity column was made with coupling of antibody to affi-gel 10 for the purification of 2,4-D in samples. Column was initially washed with 10 ml PBS, loaded with sample solution, and washed with 10 ml distilled water and 10 ml PBS. Then, 2,4-D bound to antibody was eluted with 5 ml methanol. The best doseresponse curve presented in this study was almost linear in the concentration range of 0.1∼1000 ng/ml. In heterologous ELISA system using 3,4-D or 3,4-DPA for coating Ag, the sensitivity increased more (IC₅₀ 2 ng/ml 2,4-D). This immunoassay was potentially a valuable analytical tool for the rapid and sensitive determination of 2,4-D from various samples.
5. Habitat discrimination of agricutural product using electronic nose and study on the evaluation for freshness and storage stability of meat using NIR
It would be a reduction in the overload of analysis work due to expansion of imported agricultural products as a usage of electronic nose. The discrimination of origin on especially locally produced or imported agricultural products such as black rice, jujube, sorghum, Ganoderma lucidum, sesame, Angelica gigas Nakai, Cnidium officinale, Paeomia albiflora, black bean, and bokryoung was investigated by using electronic nose. The electronic nose was composed of metal oxide sensors and the other is GC based on SAW detector. Volatile compounds of raw materials were described by the sensitivities (Rgas/Rair) of sensors. Principal component analysis (PCA) was carried out using the data obtained from six metal oxide sensors. The first principal component analysis was shown with well separating two different origins.
The number of 57 from 69 species of black rices recognized as locally produced one (83.3%) and 11 from 13 species one (imported black rice) was correctly discriminated. Also commercial electronic nose (e-nose 5000) that was combined with metal oxide sensor and conducting polymer sensor showed 92.75% (locally produced black rice) and 92.31% (imported one) of discrimination. The origin of jujube recognized as locally produced one (89.16%) and the importrd one was correctly discriminated as 81.40%. The origin of sorghum recognized as locally produced one (90.9%) and the imported one was correctly discriminated as 89.2%. That of Ganoderma lucidum recognized as locally produced one (93.3%) and the importrd one was correctly discriminated as 90.0%. That of sesame recognized as locally produced one (96.30%) and the imported one was correctly discriminated as 97.22%. The origin of black bean produced was not correctly discriminated. For GC based on SAW detector, the obtained chromatogram was expressed using VaporPrintTM image program. The results of pattern analysis of Angelica gigas Nakai, Cnidium officinale and Paeomia albiflora showed a difference between two origin of samples.
As the research output for the development of an objective, non-destructive and fast measuring sensor of meat freshness, the following results were obtained.
Counting microorganisms, chemical methods and physical methods were selected for indices of freshness, and electric conductivity, biogenic amine, D-glucose, L-lactate, VBN and K value were selected for the indices of storage condition. Also, the evaluation of possibility on measuring freshness indices and storage condition indices with NIRS was processed. Statistical methods and neural network were used for developing measuring models for each freshness and storage condition, followed by the design of an NIRS probe holder to be worked properly during meat measurement. Visual C++ 6.0 was used to develop program of the system and a notebook computer was used for data input, output and storage. By the developed system, calibration data and validation data were evaluated as r2=0.989 with SEC=1.78 and r2=0.963 with SEP=2.285, respectively.
6. Opinion for future use
Currently, we have 36 quantitative achievement including papers, presentation at scientific meetings, application to patentship, seminars and workshops. In the future, we will persue this activity further to disseminate our research output. Also, the NIR and electronic nose technique have already been utilized by National Agricultural Products Quality Management Service in habitat discrimination. The SPME technique is also planned for a future safety analysis in large-scale planting area, etc. As the contents and quality of the other research output seem to be good, they might be exploited through the technical transfer when required by relevant institutions, etc.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 4
• SUMMARY ... 14
• CONTENTS ... 26
• 목차 ... 28
• 제 1 장 연구개발과제의 개요 ... 30
• 제 1 절 연구개발의 목적 및 범위 ... 30
• 제 2 절 연구개발의 필요성 ... 31
• 제 2 장 국내외 기술개발 현황 ... 34
• 제 3 장 연구개발수행 내용 및 결과 ... 37
• 제 1 절 NIR 및 CE를 이용한 농산물의 원산지 판정방법 개발(제1세부과제) ... 37
• I. CE를 이용한 농산물의 원산지 판정방법 개발 (주관연구기관) ... 37
• II. NIR을 이용한 농산물의 원산지 판정방법 개발 (위탁연구기관) ... 83
• 제 2 절 PCR을 이용한 축산물 및 축산가공식품으로부터 Listeria, Escherichia 및 Salmonella의 신속검출법 개발(제2세부과제) ... 93
• I. PCR을 이용한 축산물 및 축산가공식품으로부터 Listeria 및 Escherichia의 신속검출법 개발 (주관연구기관) ... 93
• II. PCR에 의한 Salmonella enterica의 신속검출법 개발 (위탁연구기관) ... 132
• 제 3 절 농산물의 안전성 평가를 위한 중금속 및 항생제 신속계측 바이오센서 시스템 개발(제3세부과제) ... 149
• I. 농산물의 안전성 평가를 위한 중금속 및 항생제 신속계측 바이오센서 개발 (주관연구기관) ... 149
• II. 센서반응에 대한 컴퓨터 접속시스템 및 제어연구 (위탁연구기관) ... 222
• 제 4 절 농산물 중의 잔류농약 신속 다성분 분석기술 개발(제1협동과제) ... 244
• I. 농산물 중의 잔류농약 신속 다성분 분석기술 개발 (협동연구기관) ... 244
• II. Solid Phase Microextraction 방법을 이용한 신속분석법 개발(위탁연구기관) ... 283
• III. 농산물의 잔류농약 신속판별을 위한 면역분석기술 개발(위탁연구기관) ... 336
• 제 5 절 전자코를 이용한 농산물의 판별기술과 NIR을 이용한 육류의 신선도 및 저장성 평가 (제2협동과제) ... 381
• I. 전자코를 이용한 농산물의 판별기술 (협동연구기관) ... 381
• II. NIR을 이용한 육류의 신선도 및 저장성 평가 (위탁연구기관) ... 414
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 455
• 제 1 절 연구개발목표의 달성도 ... 455
• 제 2 절 관련분야에의 기여도 ... 456
• 제 5 장 연구개발결과의 활용계획 ... 457
• 제 1 절 기존 활용실적 ... 457
• 제 2 절 활용계획 ... 460
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 461
• 제 7 장 참고문헌 ... 462
• 끝페이지 ... 464