초록 ▼

Ⅳ. 연구개발결과
제 1협동에서는 육제품 중 주요 식중독균 3종과 부패균 3종의 성장패턴을 분석하였다. 육제품 제조조건인 소금(0∼1.75%), 아질산염(0∼120 ppm), 온도(4∼15℃), 포장형태(호기/혐기)에 따라 총 345,300점을 분석하였다. 분석 결과를 토대로 제 1세부에서는 식중독균과 부패균의 성장예측모델과 이를 기반한 전산프로그램을 개발하였다. 제 2협동에서는 육제품 제조공정 중 유해균을 분리하고 이를 펄스장 겔 전기영동법(PFGE)과 rep-PCR 방법으로 분석하여 유해균 간의 유래 상관성을 구명하였다.

Ⅳ. 연구개발결과
제 1협동에서는 육제품 중 주요 식중독균 3종과 부패균 3종의 성장패턴을 분석하였다. 육제품 제조조건인 소금(0∼1.75%), 아질산염(0∼120 ppm), 온도(4∼15℃), 포장형태(호기/혐기)에 따라 총 345,300점을 분석하였다. 분석 결과를 토대로 제 1세부에서는 식중독균과 부패균의 성장예측모델과 이를 기반한 전산프로그램을 개발하였다. 제 2협동에서는 육제품 제조공정 중 유해균을 분리하고 이를 펄스장 겔 전기영동법(PFGE)과 rep-PCR 방법으로 분석하여 유해균 간의 유래 상관성을 구명하였다. 또한 대장균군 6종에 대해 PFGE의 최적 조건을 확립하였다. 기존 기술과 비교시 분석시간을 4시간, 사용효소를 1～2개 감소할 수 있다. 제 3협동에서는 관능 및 품질 평가 수행 후 육제품 제조에 적절한 저염 농도를 소금 0.75∼1%, 아질산염 30 ppm으로 설정하고, 저염 제품의 경우 15～24일 사이의 유통기한을 제안하였다.

Abstract ▼

○ Development of microbial predictive growth models and establishment of safety guideline on low sodium meat products

Intake of sodium has been dramatically increased all over the world since few decades. Excessive intake of sodium caused various diseases such as hypertension (Lewis K. and Da

○ Development of microbial predictive growth models and establishment of safety guideline on low sodium meat products

Intake of sodium has been dramatically increased all over the world since few decades. Excessive intake of sodium caused various diseases such as hypertension (Lewis K. and Dahl M. D., 1972; Fries, 1976; Law et al., 1991a; Law et al., 1991b and Law et al., 1991c). Sodium chloride is one of the most frequently used ingredient during meat processing. It affects the flavor, texture and shelf life of meat products. Sodium chloride also plays an important role in the texture of meat products. It improves the water and fat binding properties of meat products resulting in the formation of a desirable gel texture upon cooking (Terrell, 1983). The preservative effect of sodium chloride is primarily due to its ability to lower water activity (Marsh, 1983; Sofos, 1984). Sodium chloride intake is may critical affect on health problems. To reduce these ciritical issues, the contents of sodium chloride in processed meat products should be reduced (Ruusunen M. and Puolanne E., 2005). However, the reduction of sodium chloride contents may caused the acceleration of putrefactive bacteria. Bjorkroth et al. reported that many microorganisms in meat products can cause spoilage of meat and food-borne disease. Due to its chemical compositions and biological characteristics, meats are highly perishable. Meat has high water contents (70%), which promotes the growth of many microorganisms that could accelerate the spoilage of meat and food-borne disease (Bjorkroth et al., 1998). Food-borne pathogens such as Listeria spp., Salmonella spp., Escherichia coli and Campylobacter spp. are known as a major factor of meat spoilage (Uyttendaele et al., 1999; Gibbons et al., 1996). Also, LAB (Lactic acid bacteria) species including Lactobacillus (Lb.), Leuconostoc (Leu.), Pediococcus (P.), and Streptococcus (S.) were identified as the major cause of spoilage in vacuum-packaged sausages and other processed meats (von Holy et al., 1991; Holley, 1997; Bjorkroth et al., 1998; Nychas et al., 2008). The hetero-fermentative Lactobacilli such as Lb. curvatus and Lb. sakei, and Leuconostoc species in meat products were caused a gas, off-flavor, slime, decrease pH, and discoloration (Borch et al., 1996). Therefore, microbiological analysis of commercial meat products is needed.
Therefore processed meat products formulated with low concentrations of NaNO₂ and NaCl have been produced, but low concentrations of the additives may allow bacterial growth in the products. This study developed probabilistic models and kinetic models to predict bacterial growth responses in Bologna sausage formulated with low concentrations of NaNO₂ and NaCl. The growth patterns for spoilage bacteria (Lactobacillus spp., Enterococcus spp., Pseudomonas spp.) and foodborne pathogenic bacteria (L. monocytogenes, Salmonella spp., S. aureus) on nutrient broth and Bologna sausage were investigated at different storage temperature. Probabilistic models were developed to describe the antimicrobial function of NaNO₂ (0-210 ppm) in combination with NaCl (0-1.75%) on spoilage bacteria growth and foodborne pathogenic bacteria growth under aerobic and vacuum conditions at different storage temperature (4-15℃). Growth (value of 1) or no growth (value of 0) was determined every 24 h by turbidity. The growth response data were analyzed by logistic regression to select significant variables (P <0.05) for spoilage bacteria growth and foodborne pathogenic bacteria growth inhibition, and these variables were used to generate a probabilistic model. Also, kinetic models were developed to predict kinetic behavior of L. monocytogenes, Salmonella spp., and Pseudomonas spp. in Bologna sausage fomulated with low-NaNO₂ (0 and 10 ppm) and NaCl (1.0, 1.25 and 1.5%). The bacterial cell counts were enumerated during storage at 4, 10, and 15°C for up to 60 days under aerobic and anaerobic condition. The modified Gompertz model was fitted to the growth data to calculate _maximum specific growth rate (μ_max; log CFU/g/h) and lag phase duration (LPD; h). The parameters were further analyzed with the square root model (μ _max) and a polynomial model (LPD) as a function of temperature and NaCl. Using the results of the further development prediction model, this study developed Foodborne bacteria Animal products Modeling Equipment (FAME) predictive model software, FAME software was able to see the simple bacterial growth for non- professional people.
According to the results of probabilistic models, a single application of NaNO₂ or NaCl significantly (P <0.05) inhibited Lactobacillus spp., Enterococcus spp., and L. monocytogenes at 4℃ - 15℃ under aerobic and vacuum conditions. However, growth of Pseudomonas spp., Salmonella spp. and S. aureus was not inhibited in the case of a single NaNO₂. The combination of NaNO₂ and NaCl more effectively (P <0.05) inhibited spoilage bacteria (Lactobacillus spp., Enterococcus spp., and Pseudomonas spp.) and foodborne pathogenic bacteria (L. monocytogenes, Salmonella spp., and S. aureus) growth than single application of NaNO₂ or NaCl under both aerobic and vacuum condition. The performance of the developed probabilistic model was evaluated by comparing the predicted growth responses to the observed responses obtained from experiment using bologna sausage under aerobic and anaerobic conditions. This shows that the developed probabilistic model is appropriate for describing the antimicrobial effect of NaNO₂ on inhibiting spoilage bacteria and foodborne pathogenic bacteria in combination with NaCl in processed meat products. A concordance percentage between observed and predicted growth responses was average about 90%. Also, results of kinetic models were similar to growth data of probabilistic models. Growth was observed (P <0.05) only under aerobic storage condition. In bologna sausage at 0 and 10 ppm of NaNO₂, μ_max of L. monocytogenes and Pseudomonas spp. decreased, but LPD increased as NaCl concentration increased. However, μ_max of Salmonella spp. growth was higher (P <0.05) at 0 ppm than at 10 ppm of NaNO₂ and the antimicrobial effect of NaNO₂ became more obvious as sodium chloride concentration increased. By using these results of developed models, FAME software was developed to be used as basic data for the expiration date and also evaluated for products, livestock products, food safety accident to prevent in advance, and to ensure the safety of the distribution. In conclusion, it was effective to control the growth of spoilage bacteria and foodborne pathogenic bacteria when using a combination of NaCl and NaNO₂ in meat product under aerobic and anaerobic condition in meat product. Also, FAME sould be useful for extended applications.
The aim of the study was also to investigate genetic diversity of the pathogens and putrefactive bacteria strains from pork meat and pork meat products and to analyze the genetic relatedness during each processing steps of pork products. To isolate the pathogens and putrefactive bacteria from the samples, bacteriological analyses of the pork products were conducted. 30 raw pork meat samples, semi processed pork meat and pork meat products were purchased from meat factories and local markets in Korea. In this study, we isolated and identified a total of 531 meat-borne bacteria by 16S rRNA gene sequencing and MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry). Lactobacillus sakei (139 isolates, 25%) was the most dominant LAB associated with all samples, regardless of the origin or packaging. The next predominant meat-borne bacteria were Enterobacteriaceae which include Hafnia alvei (73 isolates, 13%), Staphylococcus saprophyticus (24 isolates, 4.4%) and Citrobacter braakii (20 isolates, 3.7%). To obtain more accurate genetic relatedness, 137 Lb. sakei isolates were analyzed by molecular typing by PFGE (Pulsed-field gel electrophoresis) and REP (Repetitive extragenic palindromic elements)-PCR methods. In PFGE results, the isolates were categorized into 39 groups, while in REP-PCR assay, 38 groups were generated. The similarity level of generated bacterial groups were calculated by the Pearson correlation and UPGMA (Unweighted pair group method with arithmetic mean) method. 69 Enterobacteriaceae included in 3 Hafnia alvei, 35 Citrobacter sp., 14 Enterobacter sp., 8 Proteus sp., 8 Morganella morganii, 4 Raoultella sp. and 2 Serratia sp. were analyzed to obtain the genetic relatedness in pork products. Identification of putrefactive bacteria in pork meat and pork meat products is fundamental for understanding of spoilage. Our results may provide the prevalence of different species in different processing conditions and would be helpful for the rapid and accurate analysis of spoilage in pork products. Molecular typing of Lb. sakei and Enterobacteriaceae strains in pork products will contribute the collection of genetic database of meat-borne strains. In this study, we developed a novel rapid and simple but very accurate experimental protocol of molecular analysis of pork products. Furthermore, our results will provide a critical data for setting up the shelf-life of meat products and high throughput assay on handling of microbiological deterioration in meat products.
Recently, consumers which purpose healthy life have avoided the consumption of meat products containing excessive sodium despite nutritional value of meat. The addition of sodium chloride and sodium nitrite to prepare of meat product affects extracting myofibrillar protein, texture, flavor, antimicrobial, antioxidation and sensory properties. This research was also conducted to develop healthy meat product by optimal combination reducing sodium chloride and sodium nitrite, and the obtained results contribute to the revitalization of meat industry. In experiment I, emulsion sausages and patties were prepared with sodium chloride(0, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, and 2.00%). The reduced sodium chloride caused increase in the pH value of meat products as a dose dependent manner. On the other hand, cooking loss, protein solubility, water holding capacity, texture properties and sensory properties of treatments that contain less than 1.00%(sausage) or 0.75%(patty) showed significantly lower values(p<0.05). Thus, the minimum amount of sodium chloride for maintaining quality possible was evaluated by each 1.00%(sausage) and 0.75%(patty). In experiment II, low-sodium chloride emulsion sausage(1.00%) and patty(0.75%) were prepared with sodium nitrite(0, 30, 60, 90, and 120 ppm). The redness values of the non-add sodium nitrite treatment(a*; 4.54) was significantly lower than other treatments; but, similar values was observed in the other treatments(a*; 8.82-9.22). The cooking loss, protein solubility, water holding capacity and texture properties were not affected. Color and overall acceptance score that are parts of sensory evaluation were significantly the lowest in the non-added sodium nitrite treatment(p<0.05). In conclusion, adding 30 ppm sodium nitrite is efficient redness and sensory evaluation of low-sodium meat products. In experiment III, the aim of this study was to evaluate the effects of reducing sodium chloride and sodium nitrite levels on quality characteristics and shelf-stability of meat products. All treatments were prepared with follwed fomulation: emulsion sausages(60% pork, 20% fat and 20% ice) and patties(80% pork, 15% fat and 5% ice). And each treatment was differently formulated by adding sodium chloride and sodium nitrite levels [HSHN : 1.75% sodium chloride and 160 ppm(sausage) or 110 ppm(patties) sodium nitrite; HS : only 1.75% sodium chloride; MSLN : 1.50% sodium chloride and 30 ppm sodium nitrite; LSLN : 1.00%(sausages) or 0.75%(patties) sodium chloride and 30 ppm sodium nitrite, AKC : 1.00%(sausages) or 0.75%(patties) sodium chloride, 0.25% potassium chloride and 0.40% celery powder]. The samples were analyzed for pH, color, cooking loss, protein solubility, texture, 2-thiobarbituric acid reactive substances(TBARS), volatile basic nitrogen(VBN) and total plate count(TPC) etc. The results of this study show that sodium nitrite with in low-sodium meat products had no influence on cooking loss, protein solubility and texture. With increasing storage period, all treatments showed the decrease in residual nitrite. In addition, 30 ppm of sodium nitrite considerably prevented the oxidation and protein deterioration on low-sodium meat product during storage for 30 days. However, total plate count on except for HSHN treatments showed more than 5 log CFU/g during storage for 30 days. In conclusion, low-sodium meat products should be shelf life of less than 24 days set by consider safety factor about safety of consumers.