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Antioxidant and Antibacterial Activities of Citrus Press-Cake Dried by High Speed Drying and Far-infrared Radiation Drying

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Author(s)
Mahinda Senevirathne
Issued Date
2009
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000004640
Abstract
감귤류는 천연 플라보노이드의 원천으로 전세계적으로 가장 많이 생산되는
과실 중 하나이다. 감귤 껍질은 과실보다 많은 양의 플라보노이드가 함유되어
있다. 감귤쥬스 공정 중에 많은 양의 감귤 부산물(감귤박)이 만들어지고
있으며, 이러한 감귤박 부산물에도 많은 양의 플라보노이드가 함유되어 있을
것이다. 그러나 감귤쥬즈 공정 후 생기는 감귤박은 산도가 아주 낮기 때문에
감귤 이외의 나무에는 비료로 사용하기 조차 힘들 뿐만 아니라, 수분함량이
90%이상으로 보관상의 어려움도 있어 전량 폐기되고 있는 실정이다. 가치는
있지만 폐자원인 감귤박을 효율적으로 이용하기 위해서는, 감귤박이 부패하기
전에 급속히 건조시키는 방안이 필요하다. 그러나, 현재 많이 사용되고 있는
건조방법은 비용이 비싸거나 건조하는데 시간이 오래 걸려 대량공정에 있어서
경제적으로 비생산적이다. 따라서, 이 연구에서는 건조 공정 효율을 높이기
위하여, 초고속건조(HSD), 원적외선 건조(FIR)공법을 이용하였다. 이 두
방법으로 건조한 감귤 부산물의 기능성을 검토하기 위하여
동결건조(FD)공법으로 제조한 시료와 함께 항산화 및 항균 활성을 측정하였다.
첫 번째 파트에서는 초고속 및 원적외선 건조를 한 감귤 부산물 추출물의
메탄올 추출물에 대한 항산화 활성을 검증하였다. 이에 일반적으로 시료
건조에 가장 이상적인 방법으로 많이 사용하는 동결 건조를 통해 건조한 감귤
부산물 추출물과 비교 검증하였다. 총폴리페놀 함량 및 총플라보노이드
함량은 발색법으로 측정하였다. HPLC 분석을 통하여 감귤 부산물 추출물에는
주로 flavone 과 polymethoxylated flavone 계열의 플라보노이가 주로
함유되어 있는 것을 확인 하였다. Hesperidin 은 감귤박의 주요 구성
플라보노이드 성분으로, 이 연구에서 HPLC 분석을 통하여 자유기
라디칼(ABTS∙+) 소거율을 측정한 결과 flavone 계열의 hesperidin 이 주로
소거활성 능력을 나타내었고, 다른 종류의 플라보노이드 물질들도 우수한
소거활성을 나타낸 것을 확인 할 수 있었다. 따라서, 이러한 결과들은 감귤
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부산물내에 존재하는 폴리페놀 및 플라보노이드 물질들에 의해 우수한 항산화
활성 보여주고 있다.
두 번째는, 초고속 건조 및 원적외선 건조를 한 감귤 부산물의
효소추출물을 제조하여 라디컬 소거 활성 및 DNA damage 보호효과 통하여
항산화 활성을 측정하였다. 총 6 개의 효소 추출물(AMG, Celluclast,
Pectinex, Termamyl, Ultraflo 및 Viscozyme) 중에서 가장 우수한 항산화
활성을 보였던 AMG 효소 추출물은 우수한 라디칼 소거 활성, 지질과산화 생성
억제 활성 및 DNA 보호 효과를 나타내었다. 이러한 결과들은, 세포
실험에서도 감귤박 AMG 추출물에 의해 세포 생존률을 증가시켰고, 세포내
ROS 량도 감소시켰다. 뿐만 아니라 세포 수준에서 우수한 지질과산화
억제활성을 나타내기도 하였다. 이러한 결과는 AMG 효소 추출물의
항산화제로서의 가능성을 보여 주고 있다.
마지막으로, 감귤 부산물의 초고속건조 및 원적외선 건조물의 메탄올
추출물에 대한 항균 활성을 어병 세균과 식품 오염 미생물에 대하여
검증하였다. 그 결과 감귤 부산물의 원적외선 건조 추출물에서 대부분의
미생물에 대해 우수한 항균 활성을 나타내었을 뿐만 아니라 그람양성균의
생육도 억제 시킨 것을 확인 할 수 있었다. 이러한 결과는 감귤 부산물 유래
물질을 식품 보관에 사용시 천연 방부제로 사용이 가능 할 것으로 사료되며,
더 나아가서는 새로운 항생제 소재로 도출이 가능하리라 사료된다.
그러나 식품 안전성 확보를 위하여 향후 식품 산업공정에 대한 연구가 더
수행되어야 할 것이다.
결론적으로 초고속건조 공법은 감귤박을 건조하는데 시간을 감소시키고
하나의 공정과정으로 대량을 건조 시킬 수 있는 효율적이고 효과적인 건조
방법으로 간주 된다.
Citrus is an important fruit with the largest production in the world and potentially it
represents a rich source of natural flavonoids. Citrus peel shows higher amount of
flavonoids than that in other parts of the fruit. The half of the fruit weight removed as
waste. Therefore, citrus juice processing plants release large amount of press-cake and
they may contain high amount of flavonoids as these press-cake mainly represent by the
peel. To use this valuable product effectively, it is necessary to dry them quickly before
they are subjected to spoilage. Though, there are several drying methods available at
present, most of them are expensive and time consuming methods. Therefore, to
transform this huge amount of agricultural product into dried from some efficient method
is needed. Hence, in order to increase the efficacy in the drying process, Okadora Korea
system was introduced in this study as the high speed drying (HSD) system. Existing
drying methods such as far-infrared radiation drying and freeze drying were used as
counterpart to the HSD method. Thereafter, the antioxidant activities and antibacterial
activities of citrus press-cake (CPC) dried by HSD, far-infrared radiation dried (FIR) and
freeze dried (FD) were investigated.
The antioxidant activities of methanolic extracts from CPC dried by HSD and FIR were
evaluated for their antioxidant potential by various assays. Also, flavonoid content was
determined by using HPLC, and they showed high amount of flavonone and
polymethoxylated flavones. Hesperidin was found as the major compound available in
CPC. Further, the extracts from CPC were evaluated by HPLC coupled on-line to an
ABTS+ radical scavenging detection system to determine and identify the antioxidant
compounds at same time and results confirmed that hesperidin was the key compound in
viii
CPC. The extracts from CPC showed strong antioxidant activities in all the assays tested.
Hence, the results obtained in this study suggest that the total flavonoid and other
polyphenolics in the CPC extracts could be good radical scavengers.
Then, the antioxidant activities of enzymatic digests from CPC were evaluated by
different radical scavenging assays and comet assay. The enzymatic digests were
prepared from the CPC by using six enzymes (AMG, Celluclast, Pectinex, Termamyl,
Ultraflo and Viscozyme). As the AMG digest from CPC showed higher DPPH radical
scavenging activity and total phenolic content at the preliminary investigations, it was
selected for further antioxidant experiments. The AMG digest from CPC showed higher
activities in the antioxidant assays tested. Further, digests showed strong lipid
peroxidation inhibitory activities and remarkable inhibitory activities against H2O2-
induced DNA damage.
Further, protective effect of the methanolic extracts from CPC against oxidative stressinduced
damage in Vero cells was evaluated by different cell based assays. The results
showed that the extracts from CPC could increase the cell viability and reduce the
intracellular ROS level. Further, the extracts decreased the malondialdehyde levels in the
oxidative stress-induced Vero cells and the fluorescence intensity in the comet tail of the
cells pretreated with the extracts by enhancing the inhibitory effect in DNA damage.
Based on the results, we can conclude that the extracts from CPC have potential ROS
scavenging effects to inhibit, retard or prevent biological molecular damage of ROS in
live cells.
The antibacterial activities of methanolic extracts from CPC against fish pathogenic and
food born disease causing bacteria were evaluated in the final part of this study. Due to
ix
the evolving resistance of microorganisms to existing antibiotics, there is an increasing
need for new antibiotics not only in human but also in veterinary medicine. The extracts
from CPC showed highest antibacterial activities in the assays such as disk diffusion,
minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
assays. Also, scanning electron microscopic (SEM) results showed that the gram (+)
bacteria was inhibited by the extracts. Therefore, the extracts from CPC have the
potential as natural antibacterial agent for food preservation and medicinal use. However,
it should be further researched for use in the food industries to improve food safety by the
control or elimination of food born pathogenic bacteria in the tested samples. In
conclusion, high speed drying method is an effective and efficient method to transform
wet CPC into dried form considering its short drying time, the amount it transform at asingle turn, and the antioxidant and antibacterial activities
Affiliation
제주대학교 대학원
Department
대학원 식품공학과
Advisor
김수현
Awarded Date
2009. 2
Table Of Contents
BACKGROUND = 1
Citrus flavonoids and distribution = 1
Antioxidant and antibacterial activities of citrus flavonoids = 6
Formation of radicals involved in cellular oxidation process = 8
Structure-activity relationship of falvonoids = 10
PART Ⅰ: Effective Drying of Citrus Press-Cake by High Speed Drying and Far-infrared Radiation Drying and Their Antioxidant Activities = 12
ABSTRACT = 13
INTRODUCTION = 14
MATERIALS AND METHODS = 16
Materials = 16
Principle of high speed drying process = 16
Principle of far-infrared radiation drying = 17
Principle of freeze drying process = 19
High speed drying, far-infrared radiation drying or freeze drying of CPC = 19
Extraction of bioactive compounds from CPC = 20
Total phenolic content assay = 23
Total flavonoid content assay = 23
HPLC determination of flavonoid constituents in CPC = 23
DPPH radical scavenging assay = 24
Superoxide radical scavenging assay = 24
Hydroxyl radical scavenging assay = 25
Alkyl radical scavenging assay = 25
Hydrogen peroxide scavenging assay = 26
Metal chelating ability = 26
Lipid peroxidation inhibition by ferric thiocyanate (FTC) assay = 26
HPLC coupled on-line to ABTS^(+) assay for the rapid identification of antioxidants = 27
Statistical analysis = 28
RESULTS AND DISCUSSION = 29
Part Ⅱ :Antioxidant Properties of Enzymatic Digests from Citrus Press-Cake Dried by High Speed Drying and Far-infrared Radiation Drying and Their Protective Effect on H2O2- induced DNA Damage = 52
ABSTRACT = 53
INTRODUCTION = 54
MATERIALS AND METHODS = 57
Materials = 57
High speed drying or far-infrared radiation drying of CPC = 57
Preparation of enzymatic digests = 58
Total polyphenolic content assay = 58
Total flavonoid content assay = 58
DPPH radical scavenging assay = 59
Superoxide radical scavenging assay = 59
Hydroxyl radical scavenging assay = 60
Alkyl radical scavenging assay = 60
Hydrogen peroxide scavenging assay = 61
Metal chelating ability = 61
Lipid peroxidation inhibition by ferric thiocyanate (FTC) method = 61
Determination of the DNA damage reduction (Comet assay) by CPC = 62
Statistical analysis = 63
RESULTS AND DISCUSSION = 64
PART Ⅲ: Protective Effect of Citrus Press-Cake Dried by High Speed Drying and Far-infrared Radiation Drying Against Oxidative Stress-induced Damage in Vero Cells = 80
ABSTRACT = 81
INTRODUCTION = 82
MATERIALS AND METHODS = 84
Materials = 84
High speed drying or far-infrared radiation drying of CPC = 84
Preparation of methanolic extracts from CPC = 85
Cell culture = 85
Cell viability assay = 85
Intracellular ROS determination by DCFH-DA = 86
Lipid peroxidation inhibitory assay = 86
Investigation on the reduction of DNA damage (Comet assay) = 87
Nuclear staining with Hoechest 33342 = 88
Statistical analysis = 89
RESULTS AND DISCUSSION = 90
PART Ⅳ: Antibacterial Activity of Citrus Press-Cake Dried by High Speed Drying and Far-infrared Radiation Drying Against Food-born and Pathogenic Bacteria = 110
ABSTRACT = 111
INTRODUCTION = 112
MATERIAL AND METHODS = 115
Microorganisms and culture = 115
High speed drying or far-infrared radiation drying of CPC = 115
Extraction of bioactive compounds from CPC = 116
Antimicrobial assay (disk diffusion assay) = 116
Determination of minimum inhibitory concentration (MIC) = 117
Minimum bactericidal concentration (MBC) assay = 120
Scanning electron microscope (SEM) observation = 120
Statistical analysis = 121
RESULTS AND DISCUSSION = 122
REFERENCES = 134
ACKNOWLEDGEMENT = 154
Degree
Doctor
Publisher
제주대학교 대학원
Citation
Mahinda Senevirathne. (2009). Antioxidant and Antibacterial Activities of Citrus Press-Cake Dried by High Speed Drying and Far-infrared Radiation Drying
Type
Dissertation
Appears in Collections:
General Graduate School > Food science and Engineering
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