Anti-microbial and anti-oxidant mechanism study of a novel peptide YD1 from Bacillus amyloliquefaciens
- Issued Date
- 2017
- Abstract
- The current study reports that many fermented foods possess both nutritive and non-nutritive components, which have the modulating potential to particular molecular function relevant to well-being and health of the consumer. However, around 90% of naturally fermented foods and alcoholic beverage in different regions of the world are still relied on traditionally home made production. Foods and beverages are fermented naturally contain both functional and non-functional microorganism. During the fermentation functional microorganisms convert the chemical constitutions of materials of animal/plant sources; thereby enhancing the bio-availability of nutrients, sensory quality of the food, imparting bio-preservative effects and betterment of food safety, toxic components degradation, and anti-nutritive factors, producing antimicrobial and antioxidant compounds, stimulation the probiotic functions, and to increase the defenses of, with some bioactive health promoting compounds.
In this dissertation, we report the isolation, purification, and complete biochemical characterization of a novel peptide YD1 from the strain Bacillus amyloliquefaciens CBSYD1 isolated from Korean traditional fermented food kimchi. In addition, we report that the probiotic characteristics of Bacillus amyloliquefaciens CBSYD1. Moreover, upon on confirmation of the novelty of peptide YD1, we studied the antimicrobial and antioxidant mechanism study of YD1.
Newly isolated the strain Bacillus amyloliquefaciens CBSYD1 displayed high probiotic nature in in-vitro studies. The growth of the strain was adapted to the bile-salt condition, and a clear halos-zone was observed in a bile-salt plate assay and viable in different conditions of the digestive track.
Antimicrobial peptides (AMPs), low-molecular-weight peptides with broad-spectrum antimicrobial effects, are the most promising candidates for the novel antimicrobials. An influential cationic glycine-rich AMP YD1 (molecular-weight ~1.0 kDa) was purified from the strain Bacillus amyloliquefaciens CBSYD1 isolated from conventional Korean fermented food kimchi. The YD1 is a potential candidate for the treatment of multidrug-resistant (MDR) bacteria infection. The 16S rRNA sequence analysis showed that CBSYD1 was identified as 99.79% similar to Bacillus amyloliquefaciens subsp. plantarum FZB42. The amino acid sequence was determined by Edman degradation method, and residues of YD1 were found to be Ala-Pro-Lys-Gly-Val-Gln-Gly-Pro-Asn-Gly. After experiencing computational and sequence analysis using different biological servers suggested that YD1 possesses entirely novel amino acid sequence. YD1 exhibited antimicrobial effects against both Gram-positive and negative bacteria. The minimal inhibitory concentrations (MIC) of pure YD1 for methicillin-resistant vancomycin-resistant enterococci (VRE), Staphylococcus aureus B15 (MRSA), and Escherichia coli KCTC1923 (E. coli), concentration ranged from 8 to 64 µg/mL, representing higher potency over commercial reference antibiotics. The antimicrobial mechanism of action of YD1 was measured to involve cell-penetrating translocation inside the bacterial cell as well as interaction with the DNA leading ultimately to bacterial cell death. Analogously, Q-G-P-N-G is the likely predictable cell-penetrating motif for YD1.
The YD1 treatment on RAW 264.7 cells increased the translational and transcriptional activities of NF-E2-related factor-2 (Nrf-2) corresponding to the enhanced levels of heme oxygenase-1 (HO-1). Furthermore, the YD1-treated group showed higher levels of antioxidant enzymes compared to the oxidative stress group. YD1 demonstrated a high antioxidative activity by decreasing reactive oxygen species (ROS) and nitric oxide (NO) generation in RAW 264.7 cells along.
This study suggests that YD1 and the strain could be a natural antioxidant, antimicrobial agent and a probiotic candidate respectively. YD1 could be a promising antimicrobial candidate for the therapeutic application. Also, suggest that peptide YD1 from probiotics like Bacillus amyloliquefaciens CBSYD1 could be used as natural foods and in preventing the oxidation reaction in food processing.
|최근 연구 보고들에 따르면 많은 발효식품들이 영양 성분들과 비영양 성분들을 두루 함유하고 있으며 이 성분들은 소비자의 웰빙과 건강에 관련된 특정 분자들의 기능을 조절할 수 있는 능력이 있다. 한편 세계 각지의 자연 발효식품들 및 술들의 90%는 전통적인 방식으로 가정에서 생산되고 있다. 자연적으로 발효된 식품과 술은 기능성 및 비기능성 미생물들을 함유하고 있다. 발효 과정 중에 미생물들은 동식물에서 유래된 물질들의 화학적 조성을 바꿈으로서 영양분들의 생이용성 및 식품의 관능적 품질 향상, 생물학적 보존 효과 발생, 식품 안전도 향상, 독성분 분해, 항영양요인 제거, 향균 및 항산화 화합물 생성, 프로바이오틱 기능 촉진 및 일부 생활성(bio-active) 건강증진 화합물들의 방어능력 향상에 기여한다.
본 논문은 전통적인 한국발효식품인 김치로부터 분리된 Bacillus amyloliquefaciens CBSYD1 균주에서 유래된 새로운 펩타이드 YD1의 추출, 분리정제 및 전반적인 생화학적 특성들과 함께Bacillus amyloliquefaciens CBSYD1의 프로바이오틱 특성들을 보고하고 있다. 또한 새로운 펩타이드인 YD1를 발견하고 YD1의 항균 및 항산화 기전을 연구하였다.
새로 분리된 Bacillus amyloliquefaciens CBSYD1 균주는 in-vitro 연구에서 높은 프로바이오틱 특성을 보였다. 이 균주는 여러 담즙염 조건들 하에서 배양되었는데, 담즙염 플레이트 에세이에서 투명환(clear halos-zone)을 보여주었고 소화관의 다양한 조건들 하에서 생존하였다.
항세균 펩타이드는 광범위한 항균활성을 가진 저분자 단백질들로서 가장 유망한 새 항균제 개발 후보들이다. 다내제성 세균들을 처리하기 위해 양이온성이면서도 글라이신이 풍부한 강력한 항균제인 분자량 1kDa 이하의 펩타이드를 전통 한국발효식품인 김치로부터 분리된 Bacillus amyloliquefaciens CBSYD1로부터 정제하였다. 16S rRNA 염기서열 분석 결과 CBSYD1 균주는 Bacillus amyloliquefaciens subsp. Plantarum FZB42(T)와 99.79%의 유사도를 보였다. 에드만 분해법을 사용하여 분석한 결과 YD1의 아미노산 서열은 Ala-Pro-Lys-Gly-Val-Gln-Gly-Pro-Asn-Gly이었다. 몇 개의 생물정보 서버들을 이용하여 검색한 결과 YD1의 아미노산 서열이 독특하다고 판단되었다. YD1은 그람 음성 및 그람 양성 세균들에 대하여 항균활성을 보였다. Escherichia coli KCTC1923 (E. coli), methicillin-resistant Staphylococcus aureus B15 (MRSA) 및 vancomycin-resistant enterococci (VRE) 에 대한 YD1의 최소억제농도는 8 - 64 µg/mL의 범위 내에 있었으며, 이는 시판 중인 기준 항생제들보다 더 강력한 항균활성을 갖고 있음을 보여준다. YD1의 항균 기전은 YD1이 세균 세포 내로 투과해 들어간 후 DNA와의 상호작용을 통해 결국 세균 세포를 사멸시키는 것이라고 판단되었다. YD1의 아미노산 서열 중 세포투과성 모티프로서 가장 가능성이 높은 부분은 Gly-Pro-Asn-Gly이다.
RAW 264.7를 YD1으로 처리한 결과 Nrf-2의 전사 및 번역 활성이 높아졌으며, 이에 상응하여 heme oxygenase-1 (HO-1)의 수준이 높아졌다. 또한 YD1 처리군은 산화 스트레스군에 비해 항산화 효소들의 수준이 높게 나타났다. YD1은 RAW 264.7 세포 내에서 일산화탄소(NO) 와 활성산소종(ROS)의 생성을 감소시킴으로써 높은 항산화 활성을 보여주었다.
본 연구는 YD1은 천연 항균/항산화제가, 해당 균주는 프로바이오틱 후보가 될 수 있음을 시사한다. YD1은 임상 적용이 가능한 유망한 항균제가 될 수 있을 것이다. 또한 본 연구는 Bacillus amyloliquefaciens CBSYD1와 같은 프로바이오틱(유익균)으로부터 유래된 펩타이드 YD1이 천연 식품 제조 공정에서 산화반응을 억제하는 용도로 사용될 수 있음도 시사한다.
- Alternative Title
- Bacillus amyloliquefaciens에서 생산된 펩타이드 YD1의 항균 및 항산화 작용기작연구
- Alternative Author(s)
- Md. Saifur Rahman
- Affiliation
- Department of Pharmacy, Graduate School of Chosun University
- Department
- 일반대학원 약학과
- Advisor
- Jin Cheol Yoo
- Awarded Date
- 2017-08
- Table Of Contents
- Table of Contents
List of Tables--vi
List of Figures--vii
Abstract--x
Abstract (Korean)--xiii
Abbreviations--xvi
1. CHAPTER ONE: INTRODUCTION--01
1.1. Bioactive peptide--02
1.2. Bioactive peptides as pharmaceutical ingredients--04
1.3. Peptide and peptide class--04
1.3.1. Milk peptides--06
1.3.2. Ribosomal peptides--06
1.3.3. Nonribosomal peptides--07
1.3.4. Peptones--07
1.4. Production of peptides--08
1.4.1. Enzymatic hydrolysis--08
1.4.2. Microbial fermentation--11
1.5. Fermented food of Korea--12
1.5.1. Category one--13
1.5.2. Category two--13
1.5.3. Category three--14
1.6. Kimchi fermentation and its microorganisms--15
1.7. Peptide families--21
1.7.1. Opioid peptides--21
1.7.2. Immunomodulating peptides--21
1.7.3. Mineral-binding peptides--24
1.7.4. Antithrombotic peptides chain--24
1.7.5. Antimicrobial peptides--24
1.7.6. Antioxidative peptides--26
AIMS OF THE STUDY--27
2. CHAPTER TWO: THE STRAIN CBSYD1 AND PROBIOTIC--28
2.1. INTRODUCTION--29
2.2. MATERIALS AND METHODS--33
2.2.1. Materials--33
2.2.2. Bacterial strain isolation and identification--33
2.2.3. Tolerance towards bile salt--34
2.2.4. Bile Salt plate assay--35
2.2.5. Gastrointestinal tract condition tolerance of Bacillus CBSYD1--35
2.2.6. Bacteriocin activity assay--36
2.2.7. Media optimization--36
2.2.8. Bacteriocin production and protein precipitation--37
2.3. RESULTS AND DISCUSSION--39
2.3.1. Strain isolation and identification--39
2.3.2. Tolerance towards bile salt--39
2.3.3. Bile salt hydrolase--43
2.3.4. Gastrointestinal tract condition tolerance of Bacillus CBSYD1--43
2.3.5. Media optimization--44
2.3.6. Antibacterial activity of Cd.YD1--48
2.4. CONCLUSION--50
3. CHAPTER THREE: ANTIMICROBIAL MECHANISM STUDY--51
3.1. INTRODUCTION--52
3.2. MATERIALS AND METHODS--58
3.2.1. Culture media for YD1 production--58
3.2.2. Antimicrobial activity--58
3.2.3. Purification of YD1--59
3.2.4. Electrophoresis and In-situ analysis--59
3.2.5. Amino acid sequencing and computational analysis--59
3.2.6. Stability of YD1--60
3.2.7. Cytotoxicity--60
3.2.8. Synergism or antagonism of YD1 with antibiotics--60
3.2.9. Time-kill interaction between an antibiotic and YD1--61
3.2.10. Lysis of gram-negative spheroplasts--62
3.2.11. DNA binding assay--62
3.2.12. Transmission electron microscopy (TEM)--63
3.3. RESULTS AND DISCUSSION--64
3.3.1. Media of culture--64
3.3.2. Production, purification, and antimicrobial activity of YD1--65
3.3.3. Effects of YD1 in synergism and E. coli cell--71
3.3.4. Amino acid sequencing, computational analysis, and antimicrobial study--73
3.4. CONCLUSION--83
4. CHAPTER FOUR: ANTIOXIDANT MECHANISM STUDY--84
4.1. INTRODUCTION--85
4.1.1. Environmental stress and factors--86
4.1.2. ROS and its complication in humans--87
4.1.3. Role of exogenous antioxidants in oxidative stress--89
4.1.4. Antioxidant peptides from fermented food kimchi--91
4.2. MATERIALS AND METHODS--93
4.2.1. Radical-scavenging activity assays--93
4.2.1.1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay--93
4.2.1.2. 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging assay--93
4.2.1.3. Ferric reducing antioxidant power (FRAP) assay--94
4.2.1.4. Cupric-reducing antioxidant capacity (CUPRAC) assay--94
4.2.1.5. Oxygen radical absorbance capacity (ORAC) assay--95
4.2.2. Cell viability--95
4.2.3. Measurement of cellular NO generation--95
4.2.4. Measurement of intracellular ROS generation--96
4.2.5. Preparation of cell lysates and western blot--96
4.2.6. Gene expression and reverse transcription PCR (RT-PCR) analysis--97
4.2.7. Statistics--97
4.3. RESULTS AND DISCUSSION--100
4.3.1. Purified YD1 as an antioxidant--100
4.3.2. Antioxidant activity and mechanism study--101
4.3.2.1. Inhibitory effects of YD1 on cellular NO and ROS generation in RAW 264.7 cells--103
4.3.2.2. Effects of YD1 on gene expression of antioxidants and Phase II antioxidant enzymes--105
4.4. CONCLUSION--115
5. FUTURE DIRECTIONS--116
6. REFERENCES--118
Appendix-1--137
Appendix-2--140
- Degree
- Doctor
- Publisher
- Department of Pharmacy, Graduate School of Chosun University
- Citation
- 라흐만 사이퍼. (2017). Anti-microbial and anti-oxidant mechanism study of a novel peptide YD1 from Bacillus amyloliquefaciens.
- Type
- Dissertation
- URI
- https://oak.chosun.ac.kr/handle/2020.oak/13243
http://chosun.dcollection.net/common/orgView/200000266255
- Appears in Collections:
- General Graduate School > 4. Theses(Ph.D)
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