Candida rugopelliculosa 에 의해 발효된 해양미세조류 Pavlova lutheri 유래 펩타이드의 근섬유아세포 분화 유도기전
- Alternative Title
- A peptide derived from microalga, Pavlova lutheri, fermented by Candida rugopelliculosa induces myofibroblasts differentiation in human dermal fibroblasts
- Abstract
- Up to date, many kinds of substances have been developed from the fermented of organisms and their products that have beneficial health effects. Especially, fermentation using proteolytic yeast Candidia rugopelliculosa is an effective method in hydrolyzing proteins by breaking the chains into small peptide chains. These natural peptides can regulate body functions or conditions and thereby ultimately may influence human health as potential bioactive substances; which expected to be provided by a safe, reliable, and consistent oral delivery system.
Microalga, Pavlova lutheri is one of the largest producers of biomass in the marine environments, which contains high protein and also unconventional source producing a wide variety of chemically active metabolites. In this study, P. lutheri was mass cultured and proteolytically fermented by C. rugopelliculosa. Therefore, using central composite experimental design, the optimal culture and fermentation conditions were examined by the ascent path and the optimality was further investigated according to the response surface methology. Under the optimized conditions, P. lutheri protein was hydrolyzed, and the hydrolysate was purified and characterized in order to obtain a bioactive peptide. For the selection of active fractions during the purification steps, ROS scavenging assay was performed as a marker of antioxidant activity and the ability to induce the myofibroblast differentiation was checked as the main marker of damaged tissue repair. Following several purification steps, the peptide responsible for the antioxidant and myofibroblast inducing activities was isolated and identified as MPGPLSPL (793.01 Da). This study further demonstrates the FPP (fermented P. lutheri peptide) induced differentiation of fibroblast to myofibroblast via increased α-SMA and TGF-β1 expression. The underlying mechanism of FPP induced differentiation was identified as delayed expression of p-smad2 for 4 days instead of cytokine TGF-β1 inducement at 10 min. Following a different pathway to cytokine TGF-β1 induced α-SMA, FPP enhances the α-SMA expression through inducing the p-smad2 expression via activating Gi/o and p38 pathways. Therefore in this study it was found that FPP induced the differentiation of fibroblasts to myofibroblasts and may prove to be an attractive therapeutic agent for wound healing.
- Issued Date
- 2011
- Awarded Date
- 2011. 8
- Type
- Dissertation
- Keyword
- fermentation microalga
- Publisher
- 부경대학교
- Alternative Author(s)
- BoMi Ryu
- Affiliation
- 화학과
- Department
- 대학원 화학과
- Advisor
- Kim Se-Kwon
- Table Of Contents
- Chapter 1. Characterization of cultivation and fermentation by Candidia rugopelliculosa of marine microalga, Pavlova lutheri (Pavlovophyceae) 1
1. INTRODICTION 2
2. MATERIALS AND METHODS 7
2.1. Materials 7
2.2. Growth characteristics of microalga in the laboratory condition 7
2.2.1. Microalga strains and culture conditions 7
2.2.2. Experimental design and growth optimization 8
2.2.3. Growth measurements 10
2.2.4. Mass culture and breakdown the diatoms membrane 10
2.3. Proximate composition of P. lutheri 13
2.4. Amino acid analysis of P. lutheri 13
2.5. Fermentation by C. rugopelliculosa with laboratory cultures experiments 14
2.5.1. Microalgal strains and culture conditions 14
2.5.2. Experimental design and fermentate optimization 14
2.5.3. Determination of turbidity removal 15
2.6. Measurement of free radicals scavenging activity by electron spin resonance (ESR) 17
2.6.1. DPPH radical assay 17
2.6.2. Hydroxyl radical assay 17
2.6.3. Superoxide radical assay 18
2.7. Cell culture and viability determination 18
2.8. Intracellular reactive oxygen species (ROS) measurement 19
2.9. RNA isolation and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis 19
3. RESULTS 22
3.1. Optimum growth conditions of P.lutheri 22
3.1.1. Deduction of response surface method equation 22
3.2. Mass culture and proximate composition of P. lutheri hydrolysate 40
3.3. Amino acid analysis of P. lutheri hydrolysate 40
3.4. Optimum fermentation by C. rugopelliculosa conditions of P.lutheri 43
3.4.1. Deduction of response surface method equation 43
3.5. Mass culture and proximate composition of fermented P. lutheri hydrolysate 56
3.6. Amino acid analysis of fermented P. lutheri hydrolysate 56
3.7. Antioxidant properties of fermented P. lutheri hydrolysate 60
3.7.1. Scavenging of free radical by fermented P. lutheri hydrolysate 60
3.7.2. Scavenging of ROS formation by fermented P. lutheri hydrolysate 60
3.8. Effects of myofibroblast markers by fermented P. lutheri hydrolysate 65
4. DISCUSSION 68
Chapter 2. Purification and isolation of a bioactive peptide from proteolytic fermentation of microalga, Pavlova lutheri and its effect on myofibroblast differentiation 74
1. INTRODUCTION 75
2. MATERIALS AND METHODS 79
2.1. Materials 79
2.2. Purification of active peptide from fermented P. lutheri hydrolysate 79
2.2.1. Ion exchange chromatography 79
2.2.2. High-performance liquid chromatography (HPLC) 80
2.2.3. Determination of amino acid sequence 81
2.3. Cell culture and viability determination 81
2.4. Intracellular reactive oxygen species (ROS) measurement 82
2.5. RNA isolation and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis 82
2.6. Antibodies, growth factors, and inhibitors used in western blot analysis 84
2.7. Western blot analysis 84
2.8. Immunostaining and microscopy 85
2.9. Intracellular calcium (Ca2+) measurement 86
2.10. Collagen gel contraction 87
2.11. Statistical analysis 87
3. RESULTS 88
3.1. Preparation and identification of fermented P. lutheri peptide (FPP) and its properties 88
3.2. Effects of FPP and TGF-β1 on myofibroblast differentiation markers 102
3.3. Effects of FPP on smad-2 in myofibroblast differentiation though a Gi/o signaling 108
3.4. Effects of FPP on myofibroblast differentiation though a p38 dependent pathway 112
3.5. Effects of FPP on oxidation stress under peroxide stress 117
3.6. Effects of FPP on myofibroblast differentiation though a TGF-β1 dependent pathway 120
3.7. Effects of FPP on gel contraction from fibroblasts to myofibroblasts 125
4. DISCUSSION 129
CONCLUSION 135
REFERENCES 139
- Degree
- Doctor
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Candida rugopelliculosa 에 의해 발효된 해양미세조류 Pavlova lutheri 유래 펩타이드의 근섬유아세포 분화 유도기전.pdf
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