유기농 배추와 세척탈수 천일염으로 제조된 김치의 항암 및 항비만 효과 = Kimchi prepared with organically cultivated baechu cabbage and washed-dehydrated solar salt enhance the anticancer and antiobesity effects

국문 초록 (Abstract)

본 연구는 유기농 배추와 세척·탈수 천일염을 이용하여 제조된 김치의 대장암 억제 효과 및 항비만 효과에 대해 연구하였다.
첫 번째로 HT-29 인체 유래 대장암 세포에서 유기농 배추로 제조된 김치의 항암 기능성 효과를 확인하였다. 일반 배추로 제조된 김치(SK), 암탁배추로 제조된 김치(AmK), 유기농 배추로 제조된 김치(AK)로 나누어 MTT assay, RT-qPCR 및 western blotting을 이용하여 분석하였다. 그 결과, AK는 Bim, Bax, Bak, caspase-8, -9, -3 및 p53과 같은 세포 사멸 관련 유전자의 mRNA 발현을 유의적으로 증가시켰지만 Bcl-xL 및 Bcl-2와 같은 anti-apoptotic 유전자의 mRNA 발현을 감소시켰다. 또한, AK의 단백질 발현도 mRNA 발현과 비슷한 경향을 보였으며 특히caspase-3의 발현은 유의적으로 증가시키고 Bcl-2의 발현은 현저히 감소시켰다(p<0.05). 따라서, 유기농 배추로 제조된 김치(AK)는 세포 사멸 유전자를 활성화시켜 HT-29 세포의 증식을 현저하게 억제하는 것을 확인할 수 있었다.
두 번째로 AOM/DSS로 대장암을 유도한 C57BL/6마우스에서 SK, AmK, AK의 대장암 예방 효과를 확인하였다. AK 군은 control 군에 비해 체중의 감소를 억제하고 간과 비장의 무게 증가를 억제하며, 대장의 길이가 증가하였고 종양 수는 감소하였다. H&E staining을 통한 조직의 병리학적 관찰 결과 조직의 침윤이나 종양 생성도 현저하게 줄어든 것을 확인하였다. 또한 대장의 apoptosis와 cell cycle 관련 유전자(p53, Bid, Bak, Bax, caspase-3 및 caspase-9)의 mRNA 및 단백질 발현을 크게 증가시켰으며 anti-apoptotic 유전자(Bcl-2, Bcl-xL)의 mRNA 및 단백질 발현 수준을 감소시켰다. 대사체 분석 결과, 배추의 차이로 인해 AK가 SK, AmK보다 활성 성분(pheophorbide A, sphondin)이 높게 나타나는 것을 알 수 있었다. In vitro와 in vivo에서, 김치 제조시 유기농 배추를 사용한다면 대장암 예방 효과가 우수한 기능성 김치를 제조할 수 있을 것이라고 생각한다.
세 번째로 in vitro에서 소금의 종류를 달리하여 제조한 김치의 대장암 억제 효과를 확인하였다. HT-29 세포에서 대장암 억제 효과를 확인하기 위해 일반 천일염으로 제조한 김치(CSK), 여과해수 천일염으로 제조한 김치(FSK), 탈수 천일염으로 제조한 김치(DSK), 세척·탈수 천일염으로 제조한 김치(WDSK)으로 구성하여 실험하였다. 그 결과, WDSK 군의 이화학적 특성 및 관능평가에서 우수하게 나타났다. HT-29 세포에서 실시한 MTT, RT-qPCR 결과 전반적으로 WDSK가 가장 우수한 효과를 나타냈다. WDSK는 다른 김치에 비해 apoptosis 및 세포 주기 관련 유전자(Bax, Bim, caspase-3, caspase-9 및 p21)의 mRNA 발현의 발현이 유의적으로 증가하였다. 또한, WDSK 군은 Con 군에 비해 Bcl-2 단백질 발현을 크게 감소시켰으며 Bax, caspase-3, caspase-9, p53의 단백질 발현은 유의적으로 증가하였다. ICP-OES 결과, WDS는 다른 세 개의 천일염(CS, FS, DS)과 비교했을 때, 미네랄 성분이 다르게 나타났다. 특히, WDS는 Mg(9.3g/kg)과 S(4.7g/kg)의 함량 낮아 WDSK의 발효 특성, 맛, 그리고 기능성을 향상시킬 수 있다.
마지막으로 in vivo에서 소금의 종류를 달리하여 제조한 김치의 대장암 억제 효과 및 항비만 효과를 확인하였다. 정제염으로 제조한 김치(PSK), 일반 천일염으로 제조한 김치(CSK), 세척·탈수 천일염을 제조한 김치(WDSK), 세척·탈수 천일염과 Lactobacillus plantarum를 스타터로 첨가하여 제조한 김치(WDSK-S)로 실험하였다. WDSK와 WDSK-S는 다른 김치에 비해 발효 특성과 관능평가에서 우수하게 나타났다. C57BL/6 마우스에 고지방 식이(HFD)로 비만을 유도하고 AOM/DSS로 대장암을 함께 유도하였다. WDSK 군과 WDSK-S 군은 대조군에 비해 부고환 지방/체중 비율, 간/체중 비율, 대장 길이/체중 비율, 대장의 종양의 수가 감소하였고 대장의 길이는 증가하였다. H&E staining을 통한 조직의 병리학적 관찰 결과 WDSK 군과 WDSK-S 군의 간, 부고환 지방 및 대장 조직의 손상을 현저하게 완화시켰다. WDSK 군과 WDSK-S 군은 apoptosis와 세포 주기 관련 유전자의 mRNA 및 단백질 발현을 조절하여 대장암을 억제하였다. 또한, WDSK 군과 WDSK-S 군은 adipogenesis, lipogenesis 및 lipolysis 관련 유전자의 mRNA와 단백질 발현을 조절하여 비만을 억제하였다. 마우스 분변에서 장내 미생물 변화를 next-generation sequencing(NGS)를 통해 확인한 결과 WDSK 군의 Bacteroidetes는 증가하였고 Firmicutes는 감소하였다. 따라서, 세척·탈수 천일염을 이용하여 김치를 제조한다면 발효 품질, 맛뿐만 아니라 대장암 억제 효과와 항비만 효과가 있는 김치를 제조할 수 있으며 다른 발효식품에도 사용이 가능할 것으로 생각된다.

다국어 초록 (Multilingual Abstract)

The overall objective of this dissertation was to develop new functional kimchi based on different functional ingredients, and investigate the anti-colorectal cancer and antiobesity effects of kimchi. The whole research consists of study 1 and study 2.
Study 1 contains chapter 1 and chapter 2, focuses on the functionality of kimchi developed based on different types of baechu cabbage, and determine the health benefits of organically cultivated (OC) baechu cabbage prepared kimchi against colorectal cancer. Three types of kimchi were prepared for study 1: standardized kimchi brined with general commercial baechu cabbage (SK), Amtak baechu cabbage brined kimchi (AmK), and anticancer kimchi brined with OC baechu cabbage (AK). In chapter 1, kimchi-induced apoptosis in HT-29 human colon carcinoma cells was investigated. MTT assay, RT-qPCR, and western blotting analysis were performed. Results show that AK significantly increases mRNA expression of apoptosis-related genes Bim, Bax, Bak, caspase-8, -9, -3, and p53 and suppresses Bcl-xL and Bcl-2 expressions. Additionally, AK strongly up-regulates protein expressions of caspase-3 but strikingly reduces the protein expression of Bcl-2 (p < 0.05). The in vitro data suggest that AK can markedly inhibits the proliferation of HT-29 cells via activation of apoptosis.
The in vivo part of study 1 (chapter 2) determined the anti-colorectal cancer effect of SK, AmK, and AK through AOM/DSS-induced colorectal cancer in C57BL/6 mice. AK treated group markedly suppresses body weight loss, mitigate the increases in liver and spleen weight, while prevents colon length shortening and tumor numbers in mice. Hematoxylin and eosin (H&E) staining demonstrate that AK treated group protects the colon and spleen tissues from pathological damage. Results of RT-qPCR and western blotting display that AK treated group significantly increases the expressions of p53, Bid, Bak, Bax, caspase-3, and caspase-9, while reduces Bcl-2 and Bcl-xL levels. Additionally, the metabolites analysis result indicate that AK contains higher amounts of bioactive compounds (pheophorbide A and sphondin) than that in SK and AmK, which may be due to the OC baechu cabbage, and it seems to contribute to the anti-colorectal cancer activity of AK. Taken together, study 1 indicates that AK can effetely prevent colorectal cancer development by promoting apoptosis in vitro and in vivo, suggest that OC baechu cabbage can be used as an ideal ingredient for preparing new functional kimchi with potential anticancer effects.
Study 2 contains chapter 3 and chapter 4, focuses on the functionalities of kimchi prepared with different kinds of salt, and verified the anti-colorectal cancer and antiobesity effects of kimchi prepared with washed-dehydrated solar salt (WDS) in vitro and in vivo. In chapter 3, a cellular system of HT-29 human colon carcinoma cells was utilized to investigate the anticancer effect of kimchi. Conventionally manufactured solar salt brined kimchi (CSK), filtered sea water solar salt brined kimchi (FSK), dehydrated solar salt brined kimchi (DSK), and washed-dehydrated solar salt (WDS) brined kimchi (WDSK) were prepared. Results indicate that WDSK exhibits the best fermented quality and organoleptic characteristics. WDSK exhibits significant growth inhibition of HT-29 cells in MTT assay. mRNA and protein expressions of apoptosis and cell cycle arrest related factors reveal that WDSK meaningfully increases the mRNA expressions of Bax, Bim, caspases-3, caspases-9, and p21 as compared to other kimchi samples. In addition, WDSK treatment strongly decreases the Bcl-2 protein expression, as compared to control group (no kimchi treatment) and meaningfully increases the protein expressions of Bax, caspases-3, caspases-9, and p53. Inductively coupled plasma atomic emission spectrometer (ICP-OES) reveals that WDS possesses a different mineral composition than other three solar salts; notably, the lower Mg (9.3 g/kg) and S (4.7 g/kg) content of WDS may cause better taste, fermented characteristics, and functionality of WDSK.
The in vivo part of study 2 (chapter 4) aimed to determine the anti-colorectal cancer and antiobesity effects of kimchi prepared with different salt. Purified salt brined kimchi (PSK), CSK, WDS brined kimchi (WDSK), and WDS brined kimchi with a starter of Lactobacillus plantarum (WDSK-S) were prepared. An AOM/DSS-induced colorectal cancer model in C57BL/6 mice fed with high-fat diet (HFD) was constructed. WDSK and WDSK-S exhibit better fermented and organoleptic characteristics. WDSK and WDSK-S consumed mice exhibit lower epididymis fat/body weight ratio, liver/body weight ratio, colon length/weight ratio, colon polyp numbers, and higher colon length as compared to control groups. H&E staining results demonstrate that WDSK and WDSK-S treated notably alleviate pathological damage in liver, epididymal fat, and colon tissues. Additionally, WDSK and WDSK-S groups strongly inhibit the development of colorectal cancer by regulating apoptosis and cell cycle arrest pathways in both mRNA and protein levels. Meanwhile, those two groups demonstrate significantly obesity inhibitory effect through adipogenesis, lipogenesis, and lipolysis signaling pathways. Furthermore, next-generation sequencing (NGS) of mice fecal indicated the gut microbiota changes in each group, WDSK group up-regulate Bacteroidetes and down-regulate Firmicutes levels. In summary, study 2 indicate that WDS could increase the fermented quality and taste of kimchi, and WDS brined kimchi has potential anti-colorectal cancer and antiobesity effects. Hence, WDS can be employed as an idealized salt in kimchi fermentation, also in other fermented food product.

목차 (Table of Contents)

• TABLE OF CONTENTS
• ACKNOWLEDGEMENTS i
• TABLE OF CONTENTS ii
• LIST OF TABLES vii
• LIST OF FIGURES viii
• ABSTRACT xi
• CHAPTER 1.
• Kimchi Markedly Induces Apoptosis in HT-29 Human Colon Carcinoma Cells
• Ⅰ. INTRODUCTION 2
• Ⅱ. MATERIALS AND METHODS 5
• 1. Kimchi preparation 5
• 2. Kimchi extract preparation 6
• 3. Determination of kimchi pH and acidity 6
• 4. Determination of kimchi total bacteria and lactic acid bacteria counts 8
• 5. Kimchi sensory evaluation 8
• 6. Cell culture 9
• 7. MTT assay 9
• 8. RT-qPCR analysis 10
• 9. Western blotting analysis 12
• 10. Statistical analysis 13
• Ⅲ. RESULTS 13
• 1. Kimchi physicochemical characteristics analysis 13
• 2. Anti-proliferation effect of kimchi on HT-29 cells 17
• 3. mRNA expression levels of Bcl-2 family 17
• 4. mRNA expression levels of caspases family 20
• 5. mRNA expression levels of p53 and p21 20
• 6. Protein expression levels of apoptotic factors and p21 24
• Ⅳ. DISCUSSION 24
• Ⅴ. CONCLUSION 30
• CHAPTER 2.
• Organically Cultivated Baechu Cabbage Prepared Kimchi Enhancecs Anticancer Effect on AOM/DSS-Induced Colorectal Cancer in C57BL/6 Mice
• Ⅰ. INTRODUCTION 32
• Ⅱ. MATERIALS AND METHODS 36
• 1. Kimchi preparation 36
• 2. Animal study 37
• 3. Sample collection 38
• 4. Histopathological studies 38
• 5. Analyses of colon mRNA expression of cancer-related genes 39
• 6. Analyses of colon protein expression of cancer-related factors 40
• 7. Metabolomics analyses for kimchi metabolites 40
• 8. Data processing of metabolomics 41
• 9. Statistical analysis 42
• Ⅲ. RESULTS 44
• 1. Mice body weight, liver weight, spleen weight, colon length, colon length/weight, and tumor count 44
• 2. Histopathology studies of colon and spleen morphology 47
• 3. Effects of kimchi samples on colon mRNA expression 49
• 4. Effects of kimchi samples on colon protein expression 49
• 5. Metabolite profiling of kimchi 52
• 6. Qualitative analysis of kimchi 52
• Ⅳ. DISCUSSION 55
• Ⅴ. CONCLUSION 63
• CHAPTER 3.
• Lower Mg and S Contents in Solar Salt Increased Taste and Anticancer Effect on HT-29 Colon Carcinoma Cells of Kimchi
• Ⅰ. INTRODUCTION 65
• Ⅱ. MATERIALS AND METHODS 68
• 1. Kimchi preparation 68
• 2. Measurement of kimchi pH and acidity 68
• 3. Kimchi sensory evaluation 69
• 4. Preparation of kimchi extract 69
• 5. Cell culture 70
• 6. MTT assay 70
• 7. RT-qPCR analysis 71
• 8. Western blotting 72
• 9. Mineral composition analysis of solar salt (ICP-OES) 73
• 10. Statistical analysis 73
• Ⅲ. RESULTS 73
• 1. Physicochemical characteristic of kimchi 73
• 2. Antiproliferative effect of kimchi (MTT assay) 77
• 3. Expression analysis of apoptosis related factors in HT-29 cells 77
• 4. Expression analysis of cell cycle arrest related factors in HT-29 cells 80
• 5. Solar salt mineral composition analysis (ICP-OES) 81
• Ⅳ. DISCUSSION 84
• Ⅴ. CONCLUSION 91
• CHAPTER 4.
• Kimchi Prepared with Washed-Dehydrated Solar Salt Ameliorated the Development of Colorectal Cancer and Obesity in AOM/DSS-Induced C57BL/6 Mice Fed a High Fat Diet
• Ⅰ. INTRODUCTION 93
• Ⅱ. MATERIALS AND METHODS 99
• 1. Kimchi preparation 99
• 2. Measurement of kimchi fermentation characteristics 101
• 3. Sensory evaluation of kimchi 102
• 4. Animal study 102
• 5. Mice tissues and fecal sample collection 106
• 6. Histopathological study 106
• 7. RT-qPCR analysis on mice liver, epididymal fat, and colon tissues 107
• 8. Western blotting analysis on mice liver, epididymal fat, and colon tissues 109
• 9. Next-generation sequencing (NGS) analysis of mice feces 110
• 10. Statistical analysis 111
• Ⅲ. RESULTS 112
• 1. Physicochemical characteristics of kimchi 112
• 2. Sensory evaluation of kimchi 113
• 3. Kimchi effect on mice body weight, liver/body weight, and spleen/body weight 116
• 4. Histopathology analysis of liver tissue 118
• 5. The mRNA and protein expressions of pro-inflammatory factors in liver tissue 120
• 6. Histopathology analysis of epididymal fat tissue 123
• 7. The mRNA and protein expressions of adipogenic, lipogenic, and lipolysis factors in epididymal fat tissue 124
• 8. Kimchi effect on the colon weight, colon weight/colon length, and polyp numbers 129
• 9. Histopathology analysis of colon tissue 131
• 10. The mRNA and protein expressions of apoptosis and cell cycle arrest related factors in colon tissue 133
• 11. The influence of kimchi on mice faecal microbiota 137
• Ⅳ. DISCUSSION 138
• Ⅴ. CONCLUSION 150
• REFFERENCES (chapters 1-4) 151
• ABSTRACT IN KOREAN 163