Title Page
Contents
Abstract 13
Chapter 1. Literature review 15
1.1. Deer antler velvet 15
1.1.1. Uses and efficacy of deer antler velvet 15
1.1.2. Chemical constituents of deer antler velvet 16
1.1.3. Pharmacological properties of deer antler velvet 16
1.1.4. Research for domestically deer antler velvet 17
1.2. Probiotics 22
1.2.1. Definitions of probiotics and health-promoting effects. 22
1.2.2. Fermented foods by probiotics. 23
1.2.3. Probiotics and immunity 23
1.3. Caenorhabditis elegans 25
1.3.1. Caenorhabditis elegans as a model system to study prolongevity. 25
1.3.2. Description of the main pathways 26
1.4. Cyclophosphamide-induced immunosuppressed mice 28
1.4.1. Immune promoting effects 28
1.4.2. Cyclophosphamide 29
Chapter 2. Material and methods 32
2.1. Bacterial strains 32
2.2. Bile and acid tolerance 32
2.3. C. elegans longevity assay 33
2.4. Antler velvet extract fermentation 33
2.5. Total acidity, pH, and bacterial growth 34
2.6. Total nitrogen, amino acid, and protein quantification 34
2.7. Gamma-aminobutyric acid analysis 35
2.8. Sialic acid analysis 36
2.9. Metabolite analysis 36
2.10. C. elegans killing assay 38
2.11. Isolation of mouse splenocytes 38
2.12. Measurement of cell cytotoxicity and proliferation 39
2.13. Cy-induced immunosuppression mouse model 39
2.14. RNA isolation and quantitative real-time reverse-transcription-polymerase chain reaction (RT-qPCR) 40
2.15. Statistical analyses 40
Chapter 3. Results and discussion 42
3.1. Resistance of LAB strains to artificial stomach conditions 42
3.2. The ability of LAB strains to adhere to epithelial cells 45
3.3. Probiotic candidates for fermentation 47
3.4. Selection of LAB strains using C. elegans 50
3.5. Proteolysis in fermented antler velvet 52
3.6. GABA contents 54
3.7. Sialic acid contents 56
3.8. Select optimal strains 58
3.9. Comparison of Volatile Metabolomic Profiles 60
3.10. Metabolite changes in four sample types 63
3.11. C. elegans life span 67
3.12. GFP in C.elegans 69
3.13. Effects and mechanisms of prolongevity induced by FAV in C. elegans 71
3.14. Effect of FAV extract on C. elegans infected with pathogens 73
3.15. Cytoprotective effect of FAV on mouse splenocytes 76
3.16. Conclusion 80
Reference 81
Summary in Korean 97
Table 1. Chemical constituents are present in the deer antler velvet. 19
Table 2. Primer sequences for quantitative real-time PCR. 41
Table 3. Lactobacillus strains evaluated by the present study. 48
Table 4. Changes of pH, acidity and LAB proliferation during the fermentation of antler velvet extract. 49
Table 5. Effect of Fermentation (48h) with Lactobacillus Strains on Nutrient Content of antler velvet extract. 53
Figure 1. An image of dry slices of deer antler velvet from three countries (Korea, Russia, and New Zealand). 21
Figure 2. Schematic diagram of the most common signaling pathways in C. elegans affected by probiotic strains. 27
Figure 3. Polarization of M1 and M2 macrophages. 30
Figure 4. Mechanism of action of the alkylating agents. 31
Figure 5. Evaluation of acid resistance to Lactobacillus strains. 43
Figure 6. Evaluation of bile resistance to Lactobacillus strains. 44
Figure 7. Effect of adhesion ability of Lactobacillus strains. 46
Figure 8. Anti-aging effects of C. elegans by exposure to Lactobacillus spp. 51
Figure 9. Effect of fermentation with probiotic Lactobacillus strains on the GABA content of antler velvet extract. 55
Figure 10. The sialic acid content of antler velvet fermented (48 h) with probiotic Lactobacillus. 57
Figure 11. The intersection of top 3 strains of 3 experiments. 59
Figure 12. PCA plot displaying the bacterial communities in each type of sample. 61
Figure 13. Heatmap and cluster analysis of metabolomic profiles for samples, and the color ranges represent the relative abundances of metabolites. 62
Figure 14. Amino acid profiles distinguishing non-fermented, and probiotic fermented antler velvet. 65
Figure 15. Compound profiles distinguishing non-fermented, and probiotic fermented antler velvet. 66
Figure 16. Lifespan analysis of L4-stage CF512 worms (fer-15;fem-1) fed FAV or E. coli OP50. 68
Figure 17. Induction of PMK-1::GFP exposed to bacteria strain 70
Figure 18. The mRNA expression changes of genes ageing and immunity genes by antler velvet feeding was evaluated by the real-time PCR. 72
Figure 19. Survival analysis of C. elegans using 10mg/ml FAV (4 or 7) in pathogen. 74
Figure 20. Survival analysis of C. elegans using FAV-Co in pathogen. 75
Figure 21. Splenocytes were treated with AV, FAV (0, 0.1, 0.5 and 1 mg/mL) and/or Cy (1.6mg/mL). 78
Figure 22. mRNA expression of the following cytokines as immunostimulatory indicators. 79