고추냉이무로부터 추출한 Isothiocyanates의 항균 활성과 김치 유통기한 연장에 관한 연구 : A Study on the Antimicrobial Activity and Shelf-life Extension of Kimchi by Isothiocyanates Extracted from Horseradish (Armoracia rusticana)
- 주제(키워드) 도움말 고추냉이 , Isothiocyanates , 김치 , ITCs , horseradish (Armoracia rusticana)
- 발행기관 강릉원주대학교 일반대학원
- 지도교수 도움말 신일식
- 발행년도 2024
- 학위수여년월 2024. 8
- 학위명 박사
- 학과 및 전공 도움말 일반대학원 해양식품공학과
- 실제URI http://www.dcollection.net/handler/kangnung/000000011848
- UCI I804:42001-000000011848
- 본문언어 한국어
초록/요약 도움말
Isothiocyanates (ITCs) extracted from horseradish (Armoracia rusticana) root is a well-recognized antimicrobial agent but, application of ITCs to food system is limited due to its high volatility and pungent flavor. This study, therefore, anti-microbial activity, effects on shelf-life of bread, fish and Kimchi of ITCs that was performed to control the volatility and pungent flavor of ITCs by patch and encapsulation were investigated. The anti-inflammatory activity of ITCs in vito and in vivo, also, was investigated. The concentration of ITCs extracted from horseradish was 641.062 μg/mL, and the main components were allyl isothiocyanate (471,952 µg/mL) and phenethyl isothiocyanate (154,418 µg/mL). ITCs extracted from horseradish showed the strong anti-bacterial activity against both Gram-positive and Gram-negative bacteria, and especially showed strong antibacterial activity against Vibrio vulnificus and Vibrio paraehaemolyticus that are pathogenic bacteria in seafoods. Minimal bactericidal concentration (MBC) of ITCs against food-borne pathogenic and putrefactive bacteria were range of 125~500 ppm, and were 125 ppm against V. parahaemolyticus and V. vulnificus. MBC of Bacillus cereus which is spore forming bacteria, however, was 500 ppm. MBC of ITCs against Saccharomyces cerevisiae and Saccharomycopsis fibuligera which is putrefactive yeasts were 250 and 500 ppm, respectively. MBC of ITCs against putrefactive molds were 125 ppm with lowest concentration of all MBC against tested microorganisms. Antibacterial activity of isothiocyanates (ITCs) extracted from horseradish (Armoracia rusticana) root was determined against 4 strains of antibiotic-resistant bacteria, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), multidrug-resistant Acinetobacter baumanii (MRAB), and multidrug-resistant Pseudomonas aeruginosa (MRPA), and 2 strains of normal pathogenic bacteria, Acinetobacter baumanii, and Pseudomonas aeruginosa by disk diffusion assay and broth microdilution method. The diameter of the inhibition zones of ITCs (10,000 mg/mL) against MRSA, VRSA, MRAB, MRPA, A. baumanii, and Ps. aeruginosa were 85, 10, 18, 11, 55, and 85 mm, respectively. The minimum inhibitory concentrations (MIC) of ITCs against antibiotic-resistant bacteria, MRSA, VRSA, MRAB, and MRPA were 640, 80, 160, and 160 mg/mL, respectively. The minimum bactericidal concentrations (MBC) of ITCs against antibiotic-resistant bacteria, MRSA, VRSA, MRAB, and MRPA were 640, 640, 320, and 160 mg/mL, respectively. The MBC of ITCs against A. baumanii, and Ps. aeruginosa were 40 and 40 mg/mL, respectively. From these results, ITCs were effective against all tested antibiotic-resistant bacteria with the most effective against MRPA followed by MRAB. Therefore, ITCs extracted from horseradish root may be candidate for antibacterial agent against antibiotic-resistant bacteria. Effects of antimicrobial patch made by polyethylene (PE) and polypropylene (PP) on shelf-life of bread were measured. The growth of molds was observed on bread used antimicrobial patch made by PP, but was not observed on bread used polyethylene (PE) at 10 days of storage. To develop natural preservative for seafood, we studied about the effects of ITCs extracted from horseradish on the shelf-life of two kinds of round fish, mackerel and croaker, and two kinds of sliced raw fish (sashimi), salmon and flatfish at 4℃. Total viable cell number in croaker without ITCs patch reached to 106 colony form unit (CFU)/g (microbiological value of initial putrefaction) at 2nd day after storage. However, total viable cell number in croaker with ITCs patch contain 200 μL and 300 μL of ITCs (5,000 μg/mL) reached to 1106 CFU/g at 3rd day after storage, respectively. Total viable cell number of mackerel without ITCs patch reached to 106 CFU/g at 3rd day, but total viable cell number of mackerel with ITCs patch contain 200 μL and 300 μL of ITCs (5,000 μg/mL) reached to 106 CFU/g at 7th day after storage, respectively. Total viable cell number of flatfish sashimi without ITCs patch reached to 106 CFU/g at 3rd day after storage, but total viable cell numbers of flatfish sashimi with ITCs patch contain 200 μL and 300 μL of ITCs (5,000 μg/mL) reached to 106 CFU/g at 3rd day and 4th day after storage respectively. Total viable cell number of salmon sashimi without ITCs patch reached to 106 CFU/g at 4th day after storage, but total viable cell numbers of salmon sashimi with ITCs patch contain 200 μL and 300 μL of ITCs (5,000 μg/mL) reached to 106 CFU/g at 6th day and 8th day after storage, respectively. These results suggest that AIT extracted from horseradish have strong antibacterial activity and may be a candidate of natural preservative for extending of seafood shelf-life. The pH of control (mackerel without ITCs patch) was changed slightly throughout the storage period. The initial pH of control was 6.15, and rise to 7.33 at 30th day of storage. The pH of mackerel with ITCs patch contain 300 μL of ITCs (5,000 μg/mL) was 6.45 at 30th day after storage and that was lower than that of control. The initial TVBN value of mackerel was 8.42 mg%. The TVBN value of control was increased rapidly from 13th day after storage, and reached to 30 mg% (initial stage of decomposition value) at 26th day of storage. The TVBN value of mackerel with ITCs patch contain 200 μL and 300 μL of ITCs (5,000 μg/mL), however, were 16.54 mg% and 12.36 mg%, respectively at 30th day after storage. Thiobarbituric acid (TBA) value of control was also increased rapidly from 13th day of storage, and reached to 8.059 mgMA/Kg at 30th day after storage. The TVB-N value of mackerel with ITCs patch contain 200 μL and 300 μL of ITCs (5,000 μg/mL), however, were 5.785 and 4.689 mgMA/Kg, respectively at 30th day after storage. The sensory score of control was reached to 9.4 and 12.0 at 10th day and 13th day after storage, respectively. The sensory score of ITCs patch contain 300 μL of ITCs (5,000 μg/mL) was reached to 6.23, 7.94 and 10.0 at 13th day, 17th day and 21th day after storage, respectively. From above results, the shelf-life of ITCs patch contain 300 μL of ITCs (5,000 μg/mL) was about 10 days longer than that of control. The yield of microcapsule (63.4%) and ITCs (74.8%) were highest when the chitosan solution (2.5% chitosan in 0.1 M lactic acid) and dextrin were added 1% and 10% to Gum Arabic (GA)-ITCs emulsion, respectively. The ITCs amount released from microcapsule with ITCs of 12,315±263 µg/mL was 4,495±168 µg/mL during 30 days, and 150±6 µg/mL (mean value) of ITCs per day was released from microcapsules. The pH of control (Kimchi without ITCs microcapsules) was dropped from pH 6.8 (initial pH) to pH 5.2 in a week after storage, and dropped to pH 4.2 after 6 weeks at 4℃. The pH of Kimchi with ITCs microcapsules however, were dropped more slowly than that of control Kimchi. Especially, The pH of Kimchi with 5% of ITCs microcapsules sustained above pH 5.0 after 6 weeks at 4℃. Total viable cell number of Kimchies with ITCs microcapsules were increased more slowly than that of control Kimchi. Total viable cell number of control was reached to about 108 CFU/g but total viable cell number of Kimchies with ITCs microcapsules was 105-6 CFU/g at 2nd week after storage, The cell number of Leuconostoc mesenteroides, lactic acid bacteria that is relate to taste delicious coolness of Kimchi, was increased rapidly at early in the storage. and decreased from 12th day after storage in control Kimchi and Kimchi with ITCs microcapsules. The cell number of Leu. mesenteroides in control Kimchi and Kimchi with ITCs microcapsules were no significant differences, although the cell number of Leu. mesenteroides in Kimchi with ITCs microcapsules was decreased more rapidly than that of control Kimchi. The cell number of Lactobacillus plantrum and Lactobacillus brevis, lactic acid bacteria that are relate to production of acid during fermentation of Kimchi, were reached to 108 CFU/g at 28th day after storage. but the cell number of Lac. plantrum and Lac. brevis was 105 CFU/g at the same time after storage. From above results, the time that taste of Kimchi turn to sour will be delayed by addition of ITCs microcapsules to Kimchi. The inhibition rate of ITCs against prostaglandin E2 (PGE2) was depend on concentration of ITCs, and 82.7% and 77.0% at 3.0 mg/L and 2.5 mg/L, respectively. The inhibition rate of ITCs by oral administration against paw oedema in rat induced by carrageenan was depend on concentration of ITCs after 3 hrs from administration, the inhibition rate of ITCs were 13%, 36% and 66% at 5, 10 and 20 mg/L of ITCs, respectively. The inhibition rate of ITCs by spreading against ear oedema in rat induced by carrageenan was depend on concentration of ITCs, the inhibition rate of ITCs were 34.9%, 60.1% and 78.7% at 1, 2 and 4 mg/L of ITCs, respectively. From above results, it was founded that ITCs has strong anti-inflammatory activity.
more목차 도움말
Contents
List of Tables 1
List of Figures 2
Abstract 4
Ⅰ. 서론 8
Ⅱ. 재료 및 방법 14
1. Horseradish로부터 Isothiocyanates(ITCs)의 추출 및 동정 14
1.1. 시료 14
1.2. ITCs의 추출 14
1.3. ITCs의 gas chromatography에 의한 농도 측정 14
2. ITCs의 항균 활성 측정 17
2.1. 공시 균주 17
2.1.1. 병원성 세균 17
2.1.2. 부패성 세균 17
2.1.3. 부패성 효모 17
2.1.4. 부패성 곰팡이 17
2.1.5. 항생제 내성균 20
2.2. 항균 활성 측정 20
2.2.1. Paper disk diffusion assay 20
2.2.2. 최저증식억제농도(Minimum Inhibitory Concentration, MIC) 측정 20
2.2.3. 최저살균농도(Minimum Bactericidal Concentration, MBC) 측정 22
3. ITCs patch의 항균 활성 측정 22
3.1. 항균 patch의 제작 22
3.2. 빵의 shelf-life 연장 효과 측정 22
3.3. 생선 어패류의 Shelf-life 연장 효과 측정 22
3.3.1. 미생물학적 평가 22
3.3.2. 화학적 평가 22
3.3.3. 관능 평가 23
4. ITCs 함유 microcapsule의 항균 활성 측정 27
4.1. ITCs 함유 microcapsule의 제조 27
4.1.1. Gum Arabic (GA)-ITCs emulsion의 제조 27
4.1.2. Chitosan solution과 dextrin의 첨가 비율에 따른 microcapsule과 ITCs 수율의 최적 조건 결정 27
4.2. Microcapsule의 수율 및 ITCs 회수율 측정 27
4.2.1. Microcapsule의 수율 27
4.2.2. Microcapsule의 ITCs 회수율 28
4.2.3. Microcapsule의 형태 28
4.3. Microcapsule로부터 방출되는 ITCs의 함량 측정 28
4.4. 김치의 shelf-life 연장 효과 측정 28
4.4.1. 일반세균수 측정 29
4.4.2. 젖산균 29
4.4.3 관능 평가 29
5. ITCs의 in vitro 항염증 활성 측정 30
5.1. 세포주 30
5.2. 대조구 30
5.3. 세포 독성 시험 30
5.4. Prostaglandin E2 (PGE2) 생성 억제 시험 30
6. ITCs의 in vivo 항염증 활성 측정 31
6.1. 실험 동물 31
6.2. in vivo 부종 시험 I 31
6.2.1. 시험 물질 투여 31
6.2.2. 대조구 32
6.2.3. 시험군의 구성 및 투여 액량 32
6.2.4. 시험 방법 32
6.2.5. 평가 기준 33
6.3. In vivo 부종시험 II : Croton-oil induced ear oedema 33
6.3.1. 시험 물질 투여 33
6.3.2. 대조구 33
6.3.3. 염증 유발 물질 34
6.3.4. 시험군의 구성 및 투여 액량 34
6.3.5. 시험 방법 34
6.3.6. 평가 기준 35
7. 통계 처리 35
Ⅲ. 결과 및 고찰 35
1. Horseradish로부터 Isothiocyanates(ITCs)의 추출 및 동정 36
2. ITCs의 항균 활성 36
2.1. Paper disk method에 의한 항균 활성 36
2.2. 병원성 미생물에 대한 ITCs의 최저살균농도(MBC) 36
2.3. 항생제 내성균에 대한 ITCs의 항균 활성 43
2.3.1. Paper disk method 43
2.3.2. 항생제 내성균에 대한 ITCs의 MIC와 MBC 43
3. ITCs patch의 항균 활성 52
3.1. 빵의 shelf-life 연장 효과 52
3.2. 생선 및 생선회의 shelf-life 연장 효과 52
3.2.1. 미생물학적 평가 52
3.2.2. 화학적 평가 53
3.3. 관능 평가 62
4. ITCs 함유 microcapsule의 항균 활성 65
4.1. Chitosan solution과 dextrin의 첨가 비율에 따른 microcapsule의 수율 65
4.2. Chitosan solution과 dextrin의 첨가 비율에 따른 ITCs의 회수율 65
4.3. Microcapsule의 형태 68
4.4. Microcapsule로부터 방출되는 ITCs의 함량 68
5. ITCs 함유 microcapsule의 김치 유통기한 연장 효과 71
5.1. 김치의 pH 변화 71
5.2. 김치의 생균수 변화 71
5.3. 유산균수 변화 71
5.4. 관능 검사 72
6. ITCs의 항염증 활성 79
6.1. ITCs의 RAW264.7 세포에 대한 독성 79
6.2. ITCs의 PGE2 생성의 억제 효과 79
6.3. ITCs의 Rat 발 부종 억제 효과 79
6.4. ITCs의 Rat 귀 부종 억제 효과 83
Ⅳ. 결론 85
참고문헌 90
감사의 글 93
