제주대학교 Repository

Acid-processing and fermentation of Hizikia fusiforme and bioactivities of fucoidan from the processed H. fusiforme

Metadata Downloads
Abstract
Hizikia fusiforme is the most popular edible seaweed in Asian countries such as Korea, China, and Japan. H. fusiforme is a rich and potential natural resource could be used as an ingredient in functional food and medicine, industries. Many reports support that H. fusiforme contains various natural bioactive compounds and possess the potential to develop functional food and medicine. However, the arsenic (As) in H. Fusiforme is a serious threat to food safety and it restricts the on the application of H. Fusiforme. Therefore, in the present study, we remove of the arsenic from H. fusiforme by acid-processing and fermentation, and isolation of bioactive compound from the fermented H. fusiforme as well as evaluation of its bioactivities. In the present study, H. fusiforme was processed by citric acid (CA) and hydrochloric acid (HA). The results indicated that CA-processed H. fusiforme contains a low As amount. In addition, the optimal CA process condition has been investigated by response surface methodology experiment. Finally, the CA-processing condition has been decided as washing suing the water contain 0.4% of CA for 120 min at 60℃, and 97.11% As of H. fusiforme had been removed after processing at the optimal condition. In order to further remove of the As from H. fusiforme, the CA-processed H. fusiforme was fermented by different of probiotics. The results indicated that the H. fusiforme fermented by Lactobacillus rhamnosus possesses the lowest As content and highest reducing capacity. Therefore, Lactobacillus rhamnosus was selected as the strain to fermentation of CA-processed H. fusiforme. In addition, the chemical composition and bioactivities of non-fermented and fermented H. fusiforme were investigated. The results displayed that the bioactivities of H. fusiforme were increased after fermentation and the main composition of fermented H. fusiforme is polysaccharides. The crude polysaccharides (HFF-PS) of fermented H. fusiforme (HFF) were separated by ethanol precipitation. HFF-PS contains high amount polysaccharides (60.63%) and sulfated content (17.18%), as well as possesses strong antioxidant and anti-inflammatory activity. Thus, it has been further purified. After purification, an active fraction (HFF-PS-F5) was separated from the crude polysaccharides (HFF-PS) from fermented H. fusiforme. HFF-PS-F5 contains 91.68% sulfated polysaccharides, which comprise fucose (53.84%), galactose (19.08%), glucose (2.22%), and mannose (24.86%). In addition, the characteristics of HFF-PS-F5 had been determined by Fourier-transform infrared (FT-IR) and HPGPC. The results indicated that HFF-PS-F5 is the fucoidan with an average molecular weight as 213.33 kDa. Protective effect of the fucoidan (HFF-PS-F5) isolated from HFF-PS against hydrogen peroxide (H2O2)-induced oxidative stress in vitro in Vero cells and in vivo in zebrafish had been investigated. HFF-PS-F5 significantly reduced cytotoxicity induced by H2O2 in Vero cells. In addition, HFF-PS-F5 reduced intracellular ROS level and apoptosis body formation induced by H2O2. The Western blot results demonstrate that HFF-PS-F5 against oxidative stress by improving the expression of endogenous antioxidant enzymes including catalase and superoxidase dismutase (SOD) via regulating Nrf2 pathways. In addition, HFF-PS-F5 has strongly protective against H2O2-stimulated oxidative stress in vivo zebrafish that demonstrated in improving survival rate, decreasing heart beating rate, and reducing ROS generation, cell death and lipid peroxidation. These results suggest that HFF-PS-F5 may use as a beneficial antioxidant ingredient in medical and cosmetic industries.
Author(s)
Yong Ri Cui
Issued Date
2020
Awarded Date
2020. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/common/orgView/000000009475
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
Jeon, You Jin
Table Of Contents
CONTENT
SUMMARY. i
LIST OF TABLE. iii
LIST OF FIGURE. iv
NTRODUCTION . 1
Part Ⅰ. 6
Abstract 7
1. Materials and methods. 8
1.1. Reagents and chemicals 8
1.2. Processing H. fusiforme by acid washing . 8
1.3. Measurement of As content of acid-processed H. fusiforme 11
1.4. Processing H. fusiforme by CA-washing with different conditions . 11
1.5. Optimizing the CA-processing condition by response surface methodology (RSM) 12
2. Results and discussion . 14
2.1. As content of H. fusiforme processed by different conditions 14
2.2. As content of H. fusiforme processed by CA with different conditions. 14
2.3. Processing optimization using RSM. 20
3. Summary 25
Part Ⅱ. 26
Abstract 27
1. Materials and methods. 28
1.1. Reagents and chemicals 28
1.2. Activation of probiotics 28
1.3. Fermentation of CA-processed H. fusiforme 28
1.4. Measurement of the reducing capacities of fermented H. fusiforme samples 30
1.5. Fermentation of CA-processed H. fusiforme by Lactobacillus rhamnosus in different times 30
1.6. Preparation of non-fermented and fermented samples . 30
1.7. Chemical analysis . 32
1.8. Measurement of As content of fermented acid-processed H. fusiforme. 32
1.9. Measurement of DPPH radical scavenging activity . 32
1.10. Measurement of hydrogen peroxide scavenging activity . 32
1.11. Determination of the cytotoxicity on Vero cells. 33
1.12. Determination of the protective effect of HF and HFF against H2O2-induced intracellular ROS generation in Vero cells 33
1.13. Measurement of the protective effect of HF and HFF against H2O2-induced cytotoxicity in Vero cells. 33
1.14. Measurement of NO production and cell viability in LPS-stimulated RAW cells 34
1.15. Statistical analysis. 34
2. Results and discussion . 36
2.1. The reducing capacities of fermented H. fusiforme 36
2.2. Chemical and element composition of processed H. fusiforme 36
2.3. Chemical composition of HF and HFF. 40
2.4. Antioxidant activities of HF and HFF 40
2.5. Protective effects of HF and HFF against H2O2-induced oxidative stress in Vero cells. 43
2.6. The effect of HF and HFF on LPS-induced NO generation and cytotoxicity in RAW 264.7 macrophages 43
3. Summary 46
Part Ⅲ. 47
Abstract 48
1. Materials and methods. 49
1.1. Reagents and chemicals 49
1.2. Separation of crude polysaccharides from HFF 49
1.3. Isolation fucoidan from HFF-PS. 51
1.4. Chemical analysis . 51
1.5. Fourier-transform infrared (FT-IR) analysis. 51
1.6. Molecular weight analysis 52
1.7. Measurement of DPPH radical scavenging activity . 52
1.8. Measurement of hydrogen peroxide scavenging activity . 52
1.9. Determination of the cytotoxicity on Vero cells. 53
1.10. Determination of the protective effect of HFF-PS against H2O2-induced intracellular ROS generation in Vero cells 53
1.11. Measurement of the protective effect of HFF-PS against H2O2-induced
cytotoxicity in Vero cells. 53
1.12. Measurement of NO production and cell viability in LPS-stimulated RAW 264.7 cells 54
1.13. Statistical analysis. 54
2. Results and discussion . 55
2.1. Chemical composition of HFF-PS 55
2.2. Antioxidant activity of HFF-PS 55
2.3. Protective effect of HFF-PS against H2O2-induced oxidative stress in Vero cells 55
2.4. The effect of HFF-PS on LPS-induced NO generation and cytotoxicity in RAW 264.7 acrophages 60
2.5. Total carbohydrate, phenolic, and sulfate content of fractions separated from HFF-PS 62
2.6. Monosaccharide composition of fractions separated from HFF-PS. 62
2.7. DPPH radical and hydrogen peroxide scavenging activity of fractions separated from HFF-PS 65
2.8. FT-IR analysis of HFF-PS-F5. 65
2.9. Molecular weight analysis of HFF-PS-F5 65
3. Summary 69
Part Ⅳ. 70
Abstract 71
1. Materials and methods. 72
1.1. Chemicals and reagents. 72
1.2. Cell culture 72
1.3. Determination of the protective effect of HFF-PS-F5 against H2O2-induced intracellular ROS generation in Vero cells 72
1.4. Measurement of the protective effect of HFF-PSF5 against H2O2-induced cytotoxicity in Vero cells. 73
1.5. Nuclear staining with Hoechst 33342 . 73
1.6. Western blot analysis 73
1.7. Origin and maintenance of zebrafish 74
1.8. Application of HFF-PS-F5 and H2O2 to zebrafish embryos. 74
1.9. Measurement of heart-beating rate, ROS generation, cell death, and lipid peroxidation in zebrafish 74
1.10. Statistical analysis. 75
2. Results and discussion . 76
2.1. Protective effect of HFF-PS-F5 against H2O2-induced oxidative stress in Vero cells 76
2.2. Protective effect of HFF-PS-F5 against H2O2-induced apoptosis 76
2.3. HFF-PS-F5 improves the expression of catalase and SOD via regulating Nrf 2 pathway in H2O2-induced Vero cells. 79
2.4. HFF-PS-F5 improves survival rate and reduces heart-beating rate in H2O2-induced zebrafish . 81
2.5. Protective effect of HFF-PS-F5 against H2O2-induced ROS generation, cell death, and lipid peroxidation in zebrafish 81
3. Summary 86
Section 2: Anti-inflammatory effect of HFF-PS-F5 in vitro in RAW 264.7 macrophages and in vivo in zebrafish 87
Abstract 87

1. Materials and methods. 88
1.1. Reagents and chemicals 88
1.2. Cell culture 88
1.3. Measurement of NO production and cell viability . 88
1.4. Measurement of PGE2 and pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) production 89
1.5. Western blot analysis 89
1.6. Application of HFF-PS-F5 and LPS to zebrafish embryos 90
1.7. Determination of heart-beating rate, ROS generation, cell death, and NO generation in zebrafish. 90
1.8. Statistical analysis. 90
2. Results and discussion . 92
2.1. The effect of HFF-PS-F5 on LPS-induced NO generation and cytotoxicity in RAW 264.7 macrophages 92
2.2. HFF-PS-F5 decreased PGE2 and pro-inflammatory cytokines release in LPS-induced RAW 264.7 macrophages 92
2.3. HFF-PS-F5 inhibited the expression of iNOS and COX-2 in LPS-induced RAW 264.7 macrophages 95
2.4. HFF-PS-F5 inhibited NF-κB in LPS-induced RAW 264.7 macrophages 95
2.5. HFF-PS-F4 improved survival rate and reduced heart-beating rate in LPS-induced zebrafish. 98
2.6. Protective effect of HFF-PS-F4 against LPS-induced ROS generation, cell death, and NO production in zebrafish 98
3. Summary 103
CONCLUSION 104
REFERENCES 105
감사의 글 119
Degree
Doctor
Publisher
제주대학교 대학원
Citation
Yong Ri Cui. (2020). Acid-processing and fermentation of Hizikia fusiforme and bioactivities of fucoidan from the processed H. fusiforme
Appears in Collections:
General Graduate School > Marine Life Sciences
Authorize & License
  • AuthorizeOpen
Files in This Item:

Items in Repository are protected by copyright, with all rights reserved, unless otherwise indicated.