Title Page
국문요약
ABSTRACT
Contents
1. INTRODUCTION 27
1.1. Bacillus subtilis (B. subtilis) 27
1.1.1. Biotechnological applications of B. subtilis 27
1.1.2. B. subtilis Endospores 29
1.2. Biosurfactant 31
1.2.1. Definition and types of biosurfactants 31
1.2.2. Application of biosurfactants 33
1.3. Citrus peel 36
1.4. Tissue engineering 39
1.5. In-Vitro Skin Permeation Test 43
1.5.1. Skin Permeation Test 46
1.5.2. Application of surfactant in skin penetration testing 47
1.6. Process Validation 49
1.6.1. Validation 49
1.6.2. Process Validation 50
1.7. Objective of this research 52
2. MATERIALS AND METHODS 55
2.1. Bacterial Strain 55
2.1.1. Bacterial Strain 55
2.1.2. Manufacturing of Cell bank about Bacillus Strain 55
2.2. Experimental Design 56
2.2.1. Culture Medium composition 56
2.2.2. Screening of culture conditions for B. subtilis strain received from KCCM 58
2.2.3. Screening of culture conditions for Bacillus strain received from JBRI 58
2.3. Fermenter System 60
2.3.1. Design of foam fractionation culture system 60
2.3.2. Fermentation on the Lab Jar fermenter 61
2.3.3. Fermentation on the large-scale fermenter 62
2.4. Separation and Extraction of biosurfactant 63
2.4.1. Separation of biosurfactant 63
2.4.2. Extraction of biosurfactant 63
2.5. Screening of biosurfactant production 65
2.5.1. Calculation of Emulsification index(EI24%) 65
2.5.2. Contact angle 65
2.5.3. Bromothymol Blue (BTB) assay 66
2.5.4. BTB-CPC assay 66
2.6. Analysis of biosurfactant 68
2.6.1. HPLC 68
2.7. Efficacy analysis of biosurfactant 69
2.7.1. Antimicrobial activity analysis 69
2.7.2. Moisturizing activity analysis 70
2.7.3. Antioxidant activity 70
2.7.4. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay 73
2.8. Cell culture and tissue formation 75
2.8.1. Cell culture condition 75
2.8.2. Artificial skin construction 75
2.8.3. Analysis of artificial skin structure 77
2.9. Process and Product validation 78
2.9.1. Manufacturing batch record 78
2.9.2. MSDS(Material Safety Data Sheet) and COA(Certificate of Analysis) 78
2.9.3. Economic analysis of biosurfactant 79
2.10. In-Vitro Skin Permeation Test 80
2.10.1. Design of In-Vitro Skin Permeation system 80
2.10.2. Skin Permeation Test of biosurfactant 81
2.11. Statistical analysis 82
3. Results and Discussion 83
3.1. Bacterial Strain 83
3.1.1. Bacterial Strain 83
3.1.2. Manufacturing of Cell bank about Bacillus Strain 83
3.1.3. Discussion 87
3.2. Experimental Design 88
3.2.1. Screening of culture conditions for biosurfactant production from Bacillus strain received from KCCM 88
3.2.2. Screening of culture conditions for biosurfactant production from Bacillus strain received from JBRI 90
3.2.3. Discussion 97
3.3. Fermenter System 100
3.3.1. Design of foam fractionation culture system 100
3.3.2. Fermentation of Bacillus strain received from KCCM 102
3.3.3. Fermentation of Bacillus strain received from JBRI 103
3.3.4. Discussion 115
3.4. Screening of biosurfactant production 118
3.4.1. Calculation of Emulsification index (EI24%) 118
3.4.2. Contact angle 120
3.4.3. Bromothymol Blue assay 122
3.4.4. BTB-CPC assay 124
3.4.5. Discussion 126
3.5. Analysis of biosurfactant 128
3.5.1. HPLC 128
3.5.2. Discussion 134
3.6. Efficacy analysis of biosurfactant 136
3.6.1. Antimicrobial activity 136
3.6.2. Antioxidant activity 138
3.6.3. Moisturizing activity 143
3.6.4. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay 145
3.6.5. Discussion 147
3.7. Cell culture and tissue formation 148
3.7.1. Artificial skin construction 148
3.7.2. Discussion 150
3.8. Process and Product validation 151
3.8.1. Manufacturing batch record 151
3.8.2. Material Safety Data Sheet (MSDS) of biosurfactant from Bacillus sub. 151
3.8.3. Economic analysis of biosurfactant 152
3.8.4. Discussion 156
3.9. Skin Permeability assay 158
3.9.1. Design of In-Vitro Skin Permeation system 158
3.9.2. Skin Permeation Test of biosurfactant 161
3.9.3. Discussion 164
4. Conclusion 165
5. Reference 166
Appendix 1. Manufacturing Batch Record of Citrus peel fermentation with B. subtilis 174
Table 1. The composition of MSM1 and MSM2 media 57
Table 2. Primer sequences for qRT-PCR analysis 73
Table 3. Information on B. subtilis strains used in the experiment 86
Table 4. Experiment design for bacillus strain received from KCCM 92
Table 5. Experiment design for bacillus strain received from JBRI 95
Figure 1.7.1. Overview of research process in this study 54
Figure 3.1.1. Information of B. subtilis ACTT 21322 stain 85
Figure 3.2.1. Experiment design results of bacillus strain purchased from KCCM (a), EI24% photo (b), contact angle photo (c), EI24% and contact angle... 93
Figure 3.2.2. Experimental design variable statistical analysis results of bacillus strains purchased from KCCM: Pareto chart (A), Main effect plot (b) 94
Figure 3.2.3. Experiment al design results of bacillus strain received from JBRI (a), EI24% photo (b), contact angle photo (c), EI24% and contact angle... 96
Figure 3.3.1. Overview of Fermenter system and schematic diagram of the Fermenter set-up 101
Figure 3.3.2. OD₆₀₀ trend of B. subtilis strains in 1L fermenter 107
Figure 3.3.3. DO pattern trend of B. subtilis 043 strains in 1L fermenter 108
Figure 3.3.4. Large-scale fermenter process flow design and photos of the actual installed fermenter 113
Figure 3.3.5. OD₆₀₀ trend of B. subtilis in 1L and 300L fermenter process 114
Figure 3.3.6. GAP analysis between 1L and 300L fermenter process and biosurfactant production process for B. subtilis using citrus peel 117
Figure 3.4.1. Emulsification index(EI24%) measurement results and photos of samples from the citrus peel Bacillus fermentation process over time 119
Figure 3.4.2. Contact angle measurement results and photos of samples from the citrus peel Bacillus fermentation process over time 121
Figure 3.4.3. BTB assay results of samples from the citrus peel Bacillus fermentation process over time. 123
Figure 3.4.4. BTB-CPC assay results of samples from the citrus peel Bacillus fermentation process over time. 125
Figure 3.5.1. Preliminary tests for selecting HPLC analysis conditions. 131
Figure 3.5.2. HPLC analysis of Fermentation samples 132
Figure 3.5.3. Results of surfactin content analysis in samples 133
Figure 3.6.1. Antimicrobial activity results of surfactant from bacillus fermentation with citrus peel 137
Figure 3.6.2. DPPH, ABTS assay results of fermentation broth from bacillus fermentation with citrus peel 140
Figure 3.6.3. Total Phenolic Content, Flavonoid Content assay results of fermentation broth from bacillus fermentation with citrus peel 141
Figure 3.6.4. Cell cytotoxicity and DCF-DA assay results of fermentation broth from bacillus fermentation with citrus peel on the artificial skin 142
Figure 3.6.5. HA assay result of fermentation broth from bacillus fermentation with citrus peel on the artificial skin 144
Figure 3.6.6. qRT-PCR assay result of fermentation broth from bacillus fermentation with citrus peel on the artificial skin 146
Figure 3.7.1. 6well transwell device for artificial skin and MTT and H&E staining results 149
Figure 3.8.1. Manufacturing batch record of citrus peel fermentation with B. subtilis 153
Figure 3.8.2. MSDS, COA of citrus peel fermentation with B. subtilis 154
Figure 3.8.3. Economic analysis of citrus peel fermentation with B. subtilis 155
Figure 3.9.1. Overview of In-Vitro skin permeation system and illustration of the set-up 160
Figure 3.9.2. Verification of biosurfactant permeability improvement ability in In-Vitro Skin Permeation system 163