Title Page
Contents
ABSTRACT 21
Ⅰ. INTRODUCTION 24
Ⅱ. LITERATURE REVIEW 27
1. Non-alcoholic fatty liver 27
1.1. Definition 27
1.2. Pathogenesis 27
1.3. Occurrence of NAFL in Korea 28
1.4. Treatment 28
2. Postbiotics 29
2.1. Definition 29
2.2. Functional property 29
2.3. Bioactive compounds 30
3. Whey bioconversion 33
Ⅲ. MATERIALS AND METHODS 34
1. Analysis for effects of whey solution, whey products, skim milk, and skim milk products on NAFL in the mice model 34
1.1. Preparation of whey solution, skim milk, WT, ST, BWP, and BSP 34
1.2. Use of animals and experimental design 36
1.3. Serum biochemistry analysis 37
1.4. Gross examination of the liver 37
1.5. Pathological analysis of the liver 40
1.6. Quantification of gene expression in the liver tissue 40
1.7. Gut microbiome analysis 44
2. Selection of peptide fraction of BWP for alleviation NAFL 45
2.1. Preparation of peptide fraction of BWP 45
2.2. Analysis of lipase inhibitory activity 45
2.3. Analysis of α-glucosidase inhibitory activity 47
3. Analysis for effects of peptide fraction of BWP on NAFL in the mice model 48
3.1. Use of animals and experimental design 48
3.2. Serum biochemistry analysis 51
3.3. Gross examination of the liver 51
3.4. Pathological analysis of the liver 51
3.5. Quantification of protein expression in the liver tissue 51
4. Analysis for effects of peptide fraction of BWP on the ER stress-induced HepG2 cell model 55
4.1. Analysis of cell viability 55
4.2. Induction of ER stress in the HepG2 cells 56
4.3. Quantification of gene expression 57
4.4. Staining of lipid droplets in the HepG2 cells 59
5. Statistical analysis 60
Ⅳ. RESULTS AND DISCUSSION 64
1. Effect of whey solution, whey products, skim milk, and skim milk products on NAFL in the mice model 64
1.1. Weight change and liver weight in mice 64
1.2. Changes in lipid and liver enzyme in the mice serum 69
1.3. Comparison of gross morphology of the liver in mice 93
1.4. Comparison of microscopical liver pathology in mice 97
1.5. Gene expression levels in the liver tissue 102
1.6. Changes in the gut microbiome 109
2. Screening of lipase and α-glucosidase inhibitors from peptide fraction of BWP 127
3. Effect of peptide fraction of BWP on NAFL in the mice model 130
3.1. Weight change and liver weight in mice 130
3.2. Changes in lipid and liver enzyme in the mice serum 133
3.3. Comparison of gross morphology of the liver in mice 142
3.4. Comparison of microscopical liver pathology in mice 142
3.5. Protein expression levels in the liver tissue 146
4. Effect of peptide fraction of BWP on ER stress in the HepG2 cells 154
4.1. Viability following treatment with peptide fraction of BWPs on HepG2 cells 154
4.2. Gene expression levels in ER stress-induced HepG2 cells 156
4.3. Changes in lipid deposition on HepG2 cells 171
Ⅴ. SUMMARY AND CONCLUSION 173
Ⅵ. REFERENCES 179
ABSTRACT IN KOREAN 200
Table 1. Example of bioactive short chain fatty acids (SCFAs) in fermented foods 31
Table 2. Example of bioactive peptides in fermented foods 32
Table 3. Grouping of experimental animals for whey solution, whey products [mixture (WT) of whey solution and tyndallized probiotics and... 38
Table 4. Sequences of primers used to amplify genes related to inflammation and lipid metabolism via quantitative real-time polymerase chain reaction 43
Table 5. Grouping of experimental animals for peptide fraction of bioconverted whey product (BWP) on high fat diet (HF)-induced... 49
Table 6. Primary antibody information used in this study 54
Table 7. Sequences of primers used to amplify genes related to endoplasmic reticulum stress associated-apoptosis, lipogenesis, and inflammation metabolism via quantitative real-time polymerase chain reaction 61
Table 8. Taxonomic abundance ratio (%) of phylum level for the gut microbiome after treatment with bioconverted whey product (BWP) for 10 weeks 112
Table 9. Taxonomic abundance ratio (%) of genus level for the gut microbiome after treatment with bioconverted whey product (BWP) for 10 weeks 115
Table 10. Taxonomic abundance ratio weeks of species level for the gut microbiome after treatment with bioconverted whey product (BWP) for 10 weeks 117
Table 11. α-diversity index among groups after treatment with bioconverted whey product (BWP) for 10 weeks 124
Table 12. Recovery rates and lipase and α-glucosidase inhibitory activities of peptide fractions of bioconverted whey product by Pediococcus pentosaceus KI13 128
Table 13. Recovery rates and lipase and α-glucosidase inhibitory activities of peptide fractions of bioconverted whey product by Weissella cibaria KI35 129
Figure 1. Scheme of the 1st animal experiment of this study.[이미지참조] 39
Figure 2. Scheme of the 2nd animal experiment of this study.[이미지참조] 50
Figure 3. Body weight gain (g) of mice after treatment with whey solution, mixture (WT) of whey solution and tyndallized probiotics, and... 66
Figure 4. Liver weight (g) of mice after treatment with whey solution, mixture (WT) of whey solution and tyndallized probiotics, and... 67
Figure 5. Body weight gain (g) of mice after treatment with skim milk, mixture (ST) of skim milk and tyndallized probiotics, and bioconverted... 71
Figure 6. Liver weight (g) of mice after treatment with skim milk, mixture (ST) of skim milk and tyndallized probiotics, and bioconverted skim milk... 72
Figure 7. Levels of total cholesterol (T-chol) in the mice serum after treatment with whey solution, mixture (WT) of whey solution and... 73
Figure 8. Levels of low-density lipoprotein cholesterol (LDL-C) in the mice serum after treatment with whey solution, mixture (WT) of whey... 74
Figure 9. Levels of high-density lipoprotein cholesterol (HDL-C) in the mice serum after treatment with whey solution, mixture (WT) of whey... 75
Figure 10. Levels of triglyceride (TG) in the mice serum after treatment with whey solution, mixture (WT) of whey solution and tyndallized... 77
Figure 11. Levels of glucose (GLU) in the mice serum after treatment with whey solution, mixture (WT) of whey solution and tyndallized... 78
Figure 12. Levels of alanine aminotransferase (ALT) in the mice serum after treatment with whey solution, mixture (WT) of whey solution and... 81
Figure 13. Levels of aspartate aminotransferase (AST) in the mice serum after treatment with whey solution, mixture (WT) of whey solution and... 82
Figure 14. Levels of total cholesterol (T-chol) in the mice serum after treatment with skim milk, mixture (ST) of skim milk and tyndallized... 84
Figure 15. Levels of low-density lipoprotein cholesterol (LDL-C) in the mice serum after treatment with skim milk, mixture (ST) of skim milk... 85
Figure 16. Levels of high-density lipoprotein cholesterol (HDL-C) in the mice serum after treatment with skim milk, mixture (ST) of skim milk... 87
Figure 17. Levels of triglyceride (TG) in the mice serum after treatment with skim milk, mixture (ST) of skim milk and tyndallized probiotics,... 88
Figure 18. Levels of glucose (GLU) in the mice serum after treatment with skim milk, mixture (ST) of skim milk and tyndallized probiotics,... 89
Figure 19. Levels of alanine aminotransferase (ALT) in the mice serum after treatment with skim milk, mixture (ST) of skim milk and... 91
Figure 20. Levels of aspartate aminotransferase (AST) in the mice serum after treatment with skim milk, mixture (ST) of skim milk and... 92
Figure 21. Representative image of the liver in mice after treatment with whey solution, mixture (WT) of whey solution and tyndallized probiotics, and bioconverted whey product (BWP) for 10 weeks. 94
Figure 22. Representative image of the liver in mice after treatment with skim milk, mixture (ST) of skim milk and tyndallized probiotics, and bioconverted skim milk product (BSP) for 10 weeks. 98
Figure 23. Representative microscopic image of the liver in mice after treatment with bioconverted whey product (BWP) for 10 weeks (X200). 100
Figure 24. Steatosis grade of the liver in mice after treatment with bioconverted whey product (BWP) for 10 weeks. 101
Figure 25. Gene expression level of sterol regulatory element binding protein-1c (Srebp-1c) in the liver tissue after treatment with bioconverted... 104
Figure 26. Gene expression level of peroxisome proliferator activated receptor-γ (Ppar-γ) in the liver tissue after treatment with bioconverted... 105
Figure 27. Gene expression level of fatty acid synthase (Fas) in the liver tissue after treatment with bioconverted whey product (BWP) for 10 weeks. 107
Figure 28. Gene expression level of carnitine palmitoyltransferase-2 (Cpt-2) in the liver tissue after treatment with bioconverted whey product... 108
Figure 29. Depiction of the representative microbial phylum among groups after treatment with bioconverted whey product (BWP) for 10 weeks. 111
Figure 30. Heat map of the spearman's rank correlation coefficient between the relative abundance of the intestinal bacteria at the phylum level and the non-alcoholic fatty liver-related indicators. 119
Figure 31. Heat map of the spearman's rank correlation coefficient between the relative abundance of the intestinal bacteria at the genus level and the non-alcoholic fatty liver-related indicators. 120
Figure 32. Heat map of the spearman's rank correlation coefficient between the relative abundance of the intestinal bacteria at the species level and the non-alcoholic fatty liver-related indicators. 121
Figure 33. Principal coordinate analysis of the gut microbiota among groups after treatment with bioconverted whey product (BWP) for 10 weeks. 125
Figure 34. Unweighted pair group method with arithmetic mean algorithm hierarchical clustering among groups after treatment with bioconverted whey... 126
Figure 35. Body weight gain (g) of mice after treatment with peptide fraction of bioconverted whey product (BWP) for 10 weeks. 131
Figure 36. Liver weight (g) of mice after treatment with peptide fraction of bioconverted whey product (BWP) for 10 weeks. 132
Figure 37. Levels of total cholesterol (T-chol) in the mice serum after treatment with peptide fraction of bioconverted whey product (BWP) for... 134
Figure 38. Levels of low-density lipoprotein cholesterol (LDL-C) in the mice serum after treatment with peptide fraction of bioconverted whey... 135
Figure 39. Levels of high-density lipoprotein cholesterol (HDL-C) in the mice serum after treatment with peptide fraction of bioconverted whey... 136
Figure 40. Levels of triglyceride (TG) in the mice serum after treatment with peptide fraction of bioconverted whey product (BWP) for 10 weeks. 137
Figure 41. Levels of glucose (GLU) in the mice serum after treatment with peptide fraction of bioconverted whey product (BWP) for 10 weeks. 139
Figure 42. Levels of alanine aminotransferase (ALT) in the mice serum after treatment with peptide fraction of bioconverted whey product... 140
Figure 43. Levels of aspartate aminotransferase (AST) in the mice serum after treatment with peptide fraction of bioconverted whey product... 141
Figure 44. Representative image of the liver in mice after treatment with peptide fraction of bioconverted whey product (BWP) for 10 weeks. 143
Figure 45. Representative microscopic image of the liver in mice after treatment with peptide fraction of bioconverted whey product (BWP) for 10 weeks (X200). 144
Figure 46. Steatosis grade of the liver in mice after treatment with peptide fraction of bioconverted whey product (BWP) for 10 weeks. 145
Figure 47. Protein expression level of tumor necrosis factor-α (TNF-α) in the liver tissue after treatment with peptide fraction of bioconverted whey... 148
Figure 48. Protein expression level of cyclooxygenase-2 (COX-2) in the liver tissue after treatment with peptide fraction of bioconverted whey... 149
Figure 49. Protein expression level of interleukin-6 (IL-6) in the liver tissue after treatment with peptide fraction of bioconverted whey product... 150
Figure 50. Protein expression level of peroxisome proliferator activated receptor-γ (PPAR-γ) in the liver tissue after treatment with peptide... 151
Figure 51. Protein expression level of interleukin-1β (IL-1β) in the liver tissue after treatment with peptide fraction of bioconverted whey product... 152
Figure 52. Protein expression level of sterol regulatory element binding protein-1 (SREBP-1) in the liver tissue after treatment with peptide... 153
Figure 53. Viability of HepG2 cells exposed to peptide fraction of bioconverted whey product (BWP). 155
Figure 54. Gene expression level of protein kinase R-like endoplasmic reticulum kinase (PERK) in the HepG2 cells after treatment with palmitic... 158
Figure 55. Gene expression level of activating transcription factor-4 (ATF-4) in the HepG2 cells after treatment with palmitic acid (PA) and... 159
Figure 56. Gene expression level of C/EBP homologous protein (CHOP) in the HepG2 cells after treatment with palmitic acid (PA) and peptide... 160
Figure 57. Gene expression level of activating transcription factor-6 (ATF-6) in the HepG2 cells after treatment with palmitic acid (PA) and... 161
Figure 58. Gene expression level of inositol-requiring enzyme-1α (IRE-1α) in the HepG2 cells after treatment with palmitic acid (PA) and... 162
Figure 59. Gene expression level of c-Jun NH2-terminal kinase-1 (JNK-1) in the HepG2 cells after treatment with palmitic acid (PA) and peptide... 164
Figure 60. Gene expression level of tumor necrosis factor-α (TNF-α) in the HepG2 cells after treatment with palmitic acid (PA) and peptide... 165
Figure 61. Gene expression level of cyclooxygenase-2 (COX-2) in the HepG2 cells after treatment with palmitic acid (PA) and peptide fraction... 166
Figure 62. Gene expression level of liver X receptor-α (LXR-α) in the HepG2 cells after treatment with palmitic acid (PA) and peptide fraction... 168
Figure 63. Gene expression level of liver X receptor-β (LXR-β) in the HepG2 cells after treatment with palmitic acid (PA) and peptide fraction... 169
Figure 64. Gene expression level of sterol regulatory element binding protein-1c (SREBP-1c) in the HepG2 cells after treatment with palmitic... 170
Figure 65. Representative microscopic images of lipid accumulation in palmitic acid (PA)-induced HepG2 cells (X40). 172