Ehime University Institutional Repository

竹下, 浩子

藤田, 昌子

宇都宮, 早智

Japanese

高等学校・家庭基礎における消費者教育の展開 : 食品ロスの授業を通して

八木, 赳憲

当真, 要

森田, 展樹

石掛, 桂士

阿立, 真崇

山下, 陽一

上野, 秀人

長崎, 信行

竹粉および竹粉堆肥被覆による雑草抑制効果

高橋, 基泰

Takahashi, Motoyasu

近世英国農村社会経済慣行史論

Ehime University (愛媛大学)博士(学術)thesi

Manita, Soontornjanagit

（第３号様式）(Form No. 3) 
学 位 論 文 要 旨 
Dissertation Abstract 
氏名： 
Name 
Manita Soontornjanagit 
学位論文題目： 
Title of Dissertation 
Studies on the occurrence of aflatoxin M1 in powdered milk and ochratoxins 
in coffee products, which were commercially available in Thailand 
(タイ国内で市販されていた粉ミルクのアフラトキシンM1及びコーヒー
製品のオクラトキシン類の汚染に関する研究) 
学位論文要旨： 
Dissertation Abstract 
 Occurrence of mycotoxins, which are secondary toxic metabolites produced by fungi, in foods 
and feeds are health risks to humans and animals. Thailand is located in tropical regions where is an area 
suitable for growth of fungi. Possibility contaminated with mycotoxins in the Thai farm products is high. 
However, there is a few data on mycotoxins in commercial foods in Thailand. So that, to make clear the 
risks of mycotoxins in Thai commercial foods, immunoaffinity column-HPLC methods for aflatoxin M1 
(AFM1) in powdered milk and ochratoxin A (OTA) and B (OTB) in coffee products were developed and 
commercially available 79 powdered milks, 30 roasted and 38 instant coffees in Thailand, and 32 
Vietnamese roasted coffees, which were for reference, were collected, and were analyzed. 
 The results of AFM1 in 79 Thai powdered milks were that 12 samples (15%) were contaminated 
with 0.005 to 0.135 ng/mL of AFM1 (the positive average; 0.024 ng/mL and the overall average; 0.004 
ng/mL) in reconstituted powdered milk. Two samples (0.066 and 0.135 ng/mL) exceeded the limit of E.U. 
(0.05 ng/mL), but none exceeded the limit of Codex (0.5 ng/mL). The liver cancer risk of AFM1 in these 
powdered milks was sufficiently low. However, powdered milks made in Thailand contained more AFM1 
than imported powdered milks. 
In total, the 59 (59%) of the 100 commercial coffee samples from Vietnam and Thailand were 
contaminated with OTA or B, and the OTA values in these coffees were lower than the regulatory limit of 
E.U. (5 µg/kg in roasted, 10 µg/kg in instant coffee). Arabica coffees are cultivated in the mountains at 
high altitude in the north Thailand as Royal project. It was good news that only 4 (13.3%) of 30 samples in 
Thai Arabica roasted coffee were contaminated with OTA with an average of 0.66 μg/kg (the overall 
average; 0.17 μg/kg). The concentration of OTA was lower than previous reports in other countries. 
73.7% of Thai instant and 81.3% of Vietnamese roasted coffee were contaminated with OTA. The overall 
averages were 2.19 μg/kg in Thai instant and 0.75 μg/kg in Vietnamese roasted coffee. The concentration 
of OTA in these coffees was a relatively high. However, as the results of risk evaluation, the risk of OTA 
in commercial Thai and Vietnamese coffees was acceptably low. This report was the first on OTA and B 
in Thai instant coffee. 
From my studies, the risks of AFM1 in commercial powdered milk and OTA in coffee product were 
low in Thailand. Also, OTA in Vietnamese Robusta coffee product was acceptably low. In order to reduce 
AFM1 in powdered milks made in Thailand, it is necessary to lower regulation for Thai dairy cows to 
stricter regulation level, and then to enhance the education of the farmers to enforce cultivation and 
preservation management of the crops for cow feeds at the same time.  


English

Studies on the occurrence of aflatoxin M_1 in powdered milk and ochratoxins in coffee products, which were commercially available in Thailand

愛媛大学Ehime University博士(理学)thesi

YAYU, INDRIATI ARIFIN

Medical Geology in Artisanal and Small-scale Gold Mining Area Polluted by Mercury, Northern Sulawesi, Indonesia

Ehime University (愛媛大学)博士(工学)thesi

FIRMAN, HUSAIN

     
 
氏   名 (Name) Firman Husain 
論 文 名：  
(Dissertation Title)  DEVELOPMENT OF A SEAWALL OF DOUBLE WATER CHAMBER TYPE FOR 
EFFECTIVE WAVE POWER EXTRACTION AND REFLECTED WAVE 
DISSIPATION 
 
 
 
 
 
 
 
 
 
 
 
In the present study, a new type of seawall called a water chamber type is introduced. The water chamber 
model proposed here has two water chambers partitioned by two vertical curtain walls with different draft 
depths. This seawall is proposed for realizing the following two purposes: The first is to reduce reflected 
waves and also wave overtopping amount. And the second is to extract incoming wave energy for the 
utilization of electric power. Existence of the curtain wall triggers a pumping mode wave resonance in the 
chamber.  
For expanding the function of the new seawall model and increasing the B/C performance, the newly 
proposed water chamber model is also examined as a wave extraction device. In this study, both the single 
and double water chamber types of seawall are examined as a wave energy extraction device, in addition to 
the function of a reflection wave dissipater. A special feature of this study is to apply a horizontal water 
turbine in the water chamber instead of an air turbine in the air chamber as used in the almost all previous 
studies for extracting the wave energy. 
In order to realize the objectives described above, the thesis consists of seven chapters, including the first 
introductory chapter and followed by the conclusion of Chapter 7. In Chapter 1, the introduction related to 
this research was described; history of seawall, types of seawall and material construction include on content. 
The framework of this research was also discussed in this chapter. In Chapter 2, a theoretical analysis for 
solving the water wave boundary value problem around the coastal structure was described. Especially, the 
source distribution method is introduced to solve the water wave boundary value problem. In order to analyze 
the wave dissipation effect around the low reflective seawall, the damping wave model was introduced and 
developed. In Chapter 3, a theoretical procedure to find out an effective cross section of water chamber 
seawall was clarified. Theoretical considerations regarding the pumping or piston mode wave resonance in 
the water chamber was discussed precisely. In order to derive the rational design method of the single water 
chamber seawall, the governing parameter for describing dissipation effects of reflected waves was presented. 
In Chapter 4, effectiveness of the single water chamber seawall with guide vanes was mainly examined. The 
major interest is the ability of wave energy extraction rates as well as the dissipation function of the reflected 
waves from the seawall. In Chapter 5, in order to increase the ability of wave power extraction and 
dissipation of reflected waves, the double water chamber model comprised of two curtain walls was newly 
introduced. Especially, focusing on the wave amplifications of the piston mode resonance in the two 
chambers, various cross sections of the seawall were tested. Intending the wave power extraction device of 
OWC type, the rational design method for effective cross section was examined extensively. In Chapter 6, 
firstly, in order to improve the effectiveness of the single water chamber model, an extension model to it with 
an addition of a new water chamber was proposed. By using various locations of the guide vanes mainly in 
the second water chamber, ability of wave energy extraction was examined extensively. The resultant 
dissipation effect of reflected waves was also discussed. In order to take much more advantage of the double 
water chamber structure of the seawall, a new allocation of the guide vane was further examined. As a 
preliminary study, the wave amplifications in the two chambers were examined for various cross sections of 
the seawall model. By adopting the optimum cross section of the double water chamber model, ability of 
wave energy extraction and dissipation of wave reflection were clarified.  
Based on overall examinations and discussions of this study, the major conclusions can be summarized as 
follows: 
1) The dissipation of reflected waves by a single water chamber model comprised of a front curtain wall and 
a rear vertical wall is strongly dependent on the two dimensionless parameters, i.e., the ratio of a wave length 
to a breadth of the water chamber and the one of a wave length to a draft depth of the curtain wall. The 
maximum dissipation takes place under the condition of piston or pumping mode wave resonance in the 
water chamber. A new length parameter expressing the water chamber is proposed for the rational design of 
the water chamber.  
2) From the examination of the two different models of a single water chamber type with and without gap 
spacing around the guide vanes, it can be confirmed that the gap spacing is effective to activate rotations of 
the turbine. It is also seen that the wave reflection is comparatively large when the energy extraction device is 
mounted in the water chamber, especially for higher wave height conditions.     
3) Wave energy extraction rate of the single water chamber model is about 18% at maximum. The rate 
becomes lower for higher wave height conditions. It may be caused by higher hydrodynamic resistance in the 
guide vane. The possible wave power in prototype scale is about 25kW for wave height H=2m and 65kW for 
H=4m.  
4) A double-water-chamber type of seawall, in which a horizontal plate is attached to the lower end of a 
second vertical barrier, is more effective to reduce wave reflections and also wave amplifications in the two 
chambers as compared to the model without a horizontal plate.                                                                                             
5) Effective range of wave frequency for the dissipation of reflected waves becomes narrow with increasing 
the bottom channel height under the horizontal plate. However, wave height amplifications in the water 
chambers, especially in the second water chamber, magnified significantly. It may contribute the higher 
efficiency of wave power extraction with the use of OWC type device.  
6) The new parameter for estimating the performance of the second water chamber of a 
double-water-chamber type with a horizontal plate was proposed. It can be used for the rational design of the 
new double-water-chamber type seawall.  
7) According to the examinations of four different arrangements of guide vanes in the double-water-chamber 
model, which are extensions of the single water chamber model described above, the maximum efficiency of 
the extraction of wave energy is about 22%. For higher wave condition, the efficiency decreases as compared 
to the case of lower waves, that is about 12%. The higher fluid resistance around the guide vanes for the 
higher wave condition might be responsible for the reduction in efficiency. Although the efficiency of the 
proposed seawall is not enough, the structure plays a second role as a breakwater sufficiently, typically wave 
reflection Cr being about 0.3 in average for a wide range of wave period.  
8) In order to take much more advantage of the double water chamber structure of the seawall, a new 
allocation of the guide vane was further examined. As a preliminary study, the wave amplifications in the two 
chambers were examined for various cross sections of the seawall model. It was confirmed that the double 
water chamber model with an overlapping part of the first vertical wall on the guide vane is optimum for 
wave amplifications in both the chambers.  
9) The maximum efficiency of the wave energy conversion for the double water chamber model with an 
overlapping part of the first vertical wall on the guide vane is about 30%. For a higher wave height condition, 
the efficiency of wave energy conversion is a little bit decreased and the maximum wave power extraction 
rate is about 23%. Both the rates observed here are about two times as large as that of the single water 
chamber model. This optimal condition is closely related to the optimal wave amplification in the water 
chamber described above. It was suggested that the gap spacing below the first curtain wall and the 
overlapping structure of the first wall on the guide vane are important to increase the efficiency and wave 
amplifications in the water chambers.    
10) The possible extraction of wave power in prototype scale for the overlapping model is about 50 kW for a 
wave height H = 2 m and 120 kW for H = 4 m in average. 


DEVELOPMENT OF A SEAWALL OF DOUBLE WATER CHAMBER TYPE FOR EFFECTIVE WAVE POWER EXTRACTION AND REFLECTED WAVE DISSIPATION

Ehime University (愛媛大学)博士(医学)thesi

八木, 専

YAGI, Sen

（第 12 号様式） 
 
 
学 位 論 文 の 要 約 
（ 研 究 成 果 の ま と め ） 
 
 
 
            氏   名 八木 専 
 
 
            学位論文名 炎症性腸疾患モデルマウスを用いた炭酸脱水酵素 I エピトープの 
同定とその腸炎抑制効果 
 
 
    学位論文の要約 
[背景と目的] 
炎症性腸疾患は原因不明の慢性炎症性疾患である。申請者らは炎症性腸疾患の病態に主要な
腸内細菌抗原である炭酸脱水酵素 I(Carbonic anhydrase I:CA I)が関与していることを同定
し、同抗原でパルスした制御性樹状細胞（Reg-DC）を用いた細胞療法と経口免疫寛容療法が、
炎症性腸疾患モデルマウスにおける腸炎抑制に有用であることを報告した。本研究では、同抗
原による免疫治療の開発を想定して、CA I のアミノ酸配列の中で免疫寛容を誘導するエピトー
プ領域を同定し、同 CA I ペプチドによる腸炎抑制効果をみることを目的として研究した。 
 
[材料と方法] 
1. CA I 蛋白の配列の中で、MHC class Ⅱと強結合しうる、つまり二次構造、親水性、抗原性、
翻訳後修飾がないペプチド配列を網羅的に検索した。 
2. 8～12 週齢の BALB/c マウスの脾臓から 3×105個の CD4+CD25- T 細胞を分離し、8～12 週齢
の SCID マウスに一度腹腔内投与し腸炎を誘発した。 
3. 腸炎モデルマウスの腸間膜リンパ節(MLN)のリンパ球と候補ペプチドを共培養し、培養上清
中の interleukin（IL）-17 濃度を enzyme-linked immunosorbent assay(ELISA)法で測定し
た。 
4. 8～12 週齢の BALB/c マウスの骨髄細胞を IL-10、transforming growth factor（TGF）-β1、
顆粒球マクロファージコロニー刺激因子で 8 日間培養後、lipopolysaccharide で 24 時間培
養して Reg-DC を作成した。 
5. 同定した候補ペプチドでパルスした 1×106個の Reg-DC を腸炎誘発と同時にマウスに投与し
た。コントロールとしてリン酸緩衝液（PBS）、scramble ペプチドでパルスした Reg-DC を
用いた。 
6. 腸炎誘発後 7 日毎に体重測定を行い、4 週目に腸管長および結腸の組織所見を検討した。 
7. 腸炎誘発後 4 週目に MLN および腸管の転写因子、炎症性サイトカインの mRNA 量を、real-ti
me RT-PCR 法で測定した。MLN 細胞（PMA、ionomycine 刺激下）、結腸切片を 72 時間培養し
た上清のサイトカイン濃度を測定した。 
8. 腸炎誘発後 7 日目に MLN と腸管を採取し、Foxp3+制御性 T 細胞及び CD103+樹状細胞について
フローサイトメトリーで解析を行った。 
 
 [結果と考察] 
1. CA I アミノ酸配列のうち MHC classⅡに強結合する候補ペプチド配列として KPLSISYNPAT
AKEIVNVGHSFHVIFD(AA46-72)、KEIVNVGHSFHVIFDD(AA58-73)、KVLDALNSVKTKGKRAPFTNFD(AA1
60-181)、EGEPAVPVLSNHRPPQPLKGRTVRASF(AA235–261)の 4 つを同定した。 
2. 3 つのペプチドで CA I 蛋白全長と同様の IL-17 産生パターンを示した。 
3. 候補ペプチドのうち CA I58-73 をパルスした Reg-DC (Reg-DCCA Ⅰ58-73)だけが腸炎モデルマウ
スに投与したところコントロールと比較して、体重減少、腸管長の短縮はみられず、腸管
組織において炎症像(炎症細胞浸潤、腸管壁の肥厚、杯細胞、粘液の減少)が改善していた。 
4. Reg-DCCA Ⅰ58-73群では、MLN の Foxp3、Il10、Tgfb mRNA 発現が増加、retinoic acid-related
 orphan receptor gamma t (Rorγt) mRNA 発現は低下し、IL-6、tumor necrosis factor(T
NF)-α、interferon-γ産生は低下していた（p<0.05）。 
5. Reg-DCCA Ⅰ58-73群では、腸管の Tgfb mRNA 発現は増加、RorγT mRNA 発現は低下し、IL-17、T
NF-α産生は低下していた（p<0.05）。 
6. Reg-DCCA Ⅰ58-73群は PBS 群、Scramble 群と比較し、MLN 及び腸管の Foxp3+制御性 T 細胞、CD1
03+樹状細胞が増加していた（p<0.05）。 
CA I 蛋白の配列から MHC class Ⅱに強結合するペプチドを同定した。その中で AA58-73 をパ
ルスした Reg-DC では腸炎を改善した。腸炎改善機序として Foxp3+CD4+CD25+制御性 T 細胞、CD10
3+樹状細胞の誘導及び Th17 細胞誘導の抑制が考えられた。 
 
[結論] 
CA Iアミノ酸配列の中で、炎症性腸疾患モデルマウスの腸炎を抑制できるペプチド領域を同
定した。同ペプチドでパルスしたReg-DCが抗原特異的に免疫を抑制し、腸炎を改善することを
明らかにした。本研究で明らかとしたCA Iペプチドにより、炎症性腸疾患に対する免疫治療を
構築しうる可能性が示唆された。なお、この学位論文の内容は、以下の原著論文に既に採択済
である。 
 
主論文：Sen Yagi, Masanori Abe, Masakatsu Yamashita, Kenichirou Mori, Hirofumi Yamanishi, E
iji Arimitsu, Yasunori Yamamoto, Eiji Takeshita, Yoshio Ikeda, and Yoichi Hiasa. Carbonic anhyd
rate I epitope peptide improves inflammation in a murine model of inflammatory bowel disease. 
Inflammatory Bowel Diseases 2016 年 採択済 


Carbonic Anhydrate I Epitope Peptide Improves Inflammation in a Murine Model of Inflammatory Bowel Disease

Ehime University (愛媛大学)博士(医学)doctoral thesi

木下, 徹

KINOSHITA, Tetsu

（第３号様式）     
 
 
学 位 論 文 要 旨 
 
 
 
            氏   名 
 
木 下   徹 
 
            論 文 名 
 
ユビキノール(還元型コエンザイム Q10)の長期摂取による 
地域住民の QOL 改善効果 
 
 
 
 
 
学位論文要旨     
     
【背景】 
ユビキノール(還元型コエンザイム Q10)はアンチエイジング素材としてサプリメント等に用
いられている成分である。本研究では、高齢化が進行した地域の住民がユビキノールを長期摂
取することによる、Quality of Life  (QOL=生活の質)の改善または維持効果について検証した。 
【方法】 
愛媛県上島町在住の 124 名の成人(男性 36 名、女性 88 名、22-86 歳)から本研究参加の同意
を得た。参加者は 100-120mg のユビキノールを含むソフトカプセルまたは顆粒を、1 日 1 回、6
か月(n=22)または 12 か月(n=102)継続して摂取した。ユビキノールの摂取を開始する前および
摂取した後(6 または 12 か月後)で、血中のユビキノール濃度を測定し、QOL を SF-36 により評
価した。 
【結果】 
ユビキノールの継続摂取により、血中ユビキノールの平均値は有意に上昇した(1.05→4.25 
µg/ml, P<0.01)。また、女性の参加者において、SF-36 のサブスケールのうち日常役割機能(身
体)(+3.1、P=0.04)、活力(+3.0、P<0.01)、社会生活機能(+3.2、P<0.01)、心の健康(+2.3、
P=0.02)、精神系サマリー(+1.7、P=0.03)など、主に精神系の QOL スコアが有意に上昇した。
一方、男性参加者においては、いずれのサブスケールにおいても有意な変化は見られなかった。
血中ユビキノール濃度の変化量と SF-36 スコアの変化量の間には、男女ともに有意な相関は見
られなかった。ユビキノール摂取前(ベースライン)における血清ユビキノール濃度を低・中・
高の 3 分位に層別して分析したところ、女性においては、ベースラインでのユビキノール濃度
が低～中レベルの参加者で、活力や心の健康などの精神系 QOL スコアが有意に上昇していた。
その一方で、ベースラインでのユビキノール濃度が高レベルの女性参加者においては、SF-36
のいずれの項目でも有意な変化は見られなかった。男性参加者については、いずれの層でも
SF-36 スコアの有意な変化は見られなかった。 
【考察】 
ベースラインでの血中ユビキノール濃度が低～中程度の女性に有意な SF-36 スコアの改善が
見られたのは、ユビキノールによるミトコンドリアでの ATP 産生活性化によるものと考えられ
る。一方、男性で有意な SF-36 スコアの改善が見られなかったのは、ベースラインでの血中ユ
ビキノール濃度が女性よりも有意に高かったことおよび SF-36 スコアも女性に比べ高かったこ
とが原因と考えられる。血中ユビキノール濃度の変化量と SF-36 スコアの変化量に有意な相関
が認めれなかったが、これは血中ユビキノール濃度が上がるほど SF-36 スコアも上がるわけで
はないことを表している。従って、血中ユビキノール濃度には最適閾値があり、それ以上に上
昇しても血中のユビキノールは QOL の変化には影響しないのではないかということが考えられ
る。本研究の大きな特長は、過去の先行研究では病者を対象として Hospital-based で行われ
ていたコエンザイム Q10 に関する研究を、地域住民を対象に Community-based で実施し、市販
レベルの用量(100mg/日程度)でも女性への精神系 QOL に有効な可能性があることを示したこと
である。高齢化が急速に進む我が国では、高齢者の健康維持・増進のための政策・取組みが求
められており、本研究のような地域住民を対象とするアンチエイジング素材を活用した臨床研
究は、今後よりその価値を増すものと考えられる。 
【結論】 
本研究の結果、ユビキノールの継続摂取による女性の精神系 QOL 改善の効果が示唆された。
特に、血中ユビキノール濃度が低～中程度の女性に効果が期待できることが示された。 
 
 
 
キーワード（３～５） 
 
 
 
ユビキノール 
Quality of Life (QOL) 
臨床研究 
ATP 産生作用 
 
 
 
 


The Effects of Long-Term Ubiquinol Intake on Improving the Quality of Life of Community Residents

佐藤, 創

SATOH, Hajime

（第７号様式） 
 
学位論文審査結果の要旨 
 
   氏     名       佐藤  創  
 
 
 
 
 
   審 査 委 員 
 
 
 
 
 
 
 
 
 
                                      
 
                                      
 
                                      
 
                                      
 
                                      
 
 
論  文  名： 
外科大侵襲手術術後凝固異常における可溶性フィブリンモノマー(SF)の臨床的有用性 
 
 
審査結果の要旨： 
【研究内容のまとめ】 
背景：可溶性フィブリンモノマー(Soluble Fibrin；SF)は、鋭敏な凝固指標として臨床的
有用性が報告され、深部静脈血栓症の診断や DIC の診断において注目されている。しか
し、外科大侵襲手術術後におけるその臨床的意義については未だ不明であり、申請者ら
は、当該科における外科大侵襲手術症例を対象に、周術期における SF と従来の凝固線溶
系マーカーの、DIC 移行予測因子としての意義を検討した。 
方法：申請者らは SIRS モデルとして 44 例の外科大侵襲手術を対象に、後方視的検討を
行った。術後経過中 DIC に至らなかった 7 例を除く 37 例が、術後 SIRS 関連凝固異常
（SIRS associated Coagulopathy；SAC）にとどまった 16 例と DIC にまで至った 21
例の 2 群に割り付けられた。検討方法として、対象症例に対し術前・術後 1・2・3・5・
7・10 日に採血を行い、採血項目としては SF(ラテックス免疫比濁法)・DIC マーカーで
ある血小板・FDP・PT とし、SIRS 項目(体温・心拍数・呼吸数・白血球)を検討し、急
性期 DIC 診断基準に基つき DIC スコアを算出した。 
結果：術後全ての患者が術後 1 日目に SAC に至り、DIC 移行群は術後 2 日目に DIC へ
移行した。多変量解析の結果、術後 1 日目の SF と FDP の 2 因子が DIC 移行群の危険因
子として他の因子に比較し有意差を認めた(オッズ比 14.4、7.8)。β値を用いた予測式の
感度は 71%、特異度は 94%であり、臨床的に認容性があるものと思われた。これらの結
果から、DIC 移行の危険因子は術後 1 日目の SF と FDP であり、とくに SF が最も強い
予測因子である可能性が示され、 
SF を測定することにより、手術の侵襲度を把握し、術後の凝固異常を早期に予測するこ
とが可能であり、これにより凝固異常にも配慮したよりきめ細かい周術期管理が可能に
なる可能性がある。 
 
審査委員は、本論文に対し、以下の質問を行い、申請者は的確に応答した。 
１．本研究で、両群に肝硬変の有無、肝炎の有無あるいはそれら合併症に差を認めたか。 
２. 炎症性サイトカインの測定は行ったか。 
３．SAC と DIC の２つのグループの差は何が原因か。 
４. D-ダイマーと比較して SF の有用性は。 
５. 大侵襲後 DIC と敗血症性 DIC の差はあるか。 
 
以上より、審査委員は，申請者が本論文関連領域に対して学位授与に値する十分な見識
と能力を有することを全員一致で確認し，本論文が学位授与に値すると判定した。 
  
（第８号様式） 
 
最終試験の結果の要旨 
 
   氏     名      佐藤  創 
 
 
 
 
 
   審 査 委 員 
 
 
 
 
 
 
 
 
 
  主 査                                  印 
 
  副 査                                  印 
 
  副 査                                  印 
 
  副 査                                  印 
 
  副 査                                  印 
 
 
      実施年月日                                       平成 27 年 2 月 8 日 
      試験方法（該当のものを○で囲むこと。）              口 頭     筆 答 
      試験結果の要旨 
 
申請者は、愛媛大学大学院医学系研究科に在学中であり、所定の単位を修得している。
平成 28 年 2 月 8 日に開催された公開審査会において、申請者は、提出論文内容の発表を
すべて英語で行った。その後の質疑応答は日本語で行い、 提出論文の内容及び関連領域
に関した別紙に示す 様々な質問に対し明確に応答し、学位授与に値する十分な見識と能
力を有すると評価し、最終試験に合格と判定した。 


Clinical significance of soluble fibrin in coagulopathy caused by highly invasive surgery

Ehime University (愛媛大学)博士(農学)thesi

Waraporn, PRASERT

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1 
学位論文全文に代わる要約 
Extended Summary in Lieu of Dissertation 
 
氏名： 
Name 
 
Waraporn PRASERT 
学位論文題目： 
Title of Dissertation 
 
A Study of Food Nano-Emulsification Using Low-Energy Method in Aqueous 
Phase/Polyoxyethylene Sorbitan Fatty Acid Ester/Vegetable Oil Systems 
(ポリオキシエチレンソルビタン脂肪酸エステル水溶液/食用油系におけ
る省エネルギーなナノエマルション調製に関する研究) 
 
学位論文要約： 
Dissertation Summary 
 
Nano-emulsions are emulsions with droplet sizes from 20 nm up to 200-500 nm, are thermodynamically 
unstable, and appear transparent or translucent to the naked eye. Usually, nano-emulsions are highly stable to 
gravitational separation because the relatively small particle size means that Brownian motion effects dominate 
gravitational forces. Low-energy emulsification method uses energy from the chemical potential of the 
components. It is suggested that remarkable changes in the curvature of the surfactant assembly plays an 
important role in low-energy nano-emulsification methods. However, the studies of this emulsification methods 
using food ingredients are limited.  
This work tries to use food ingredients in the study of nano-emulsification using low-energy method in 
aqueous phase/polyoxyethylene sorbitan fatty acid ester (Tween®)/vegetable oil systems. Water or 20% sucrose 
solution was used as an aqueous phase, four types of polyoxyethylene sorbitan fatty acid esters i.e. Tween20, 
Tween40, Tween60 and Tween80 were used as surfactants, and vegetable oil was used as oil phase. The fatty acid 
content of vegetable oil was C16:0 7.78%, C18:0 3.13%, C18:1 45.3%, C18:2 34.21%, C18:3 6.47%, and others 
3.11%. 
The triangular phase diagram (Fig. 1) was used to study phase behavior of surfactant existing at 
equilibrium with desired properties. The phases in the diagrams were determined using visual methods, crossed 
plate polarizers, polarized light microscopy (Olympus, BX51, Japan), consistency index of viscosity evaluated by 
viscometers (DV-III Ultra; Brookfield, USA , TV-20 and TV-30; Toki Sangyo, Japan), Small Angle X-ray 
Scattering (SAXS) techniques using a Nano-viewer SAXS instrument (Rigaku Co., Tokyo, Japan), and the 
conductivity measurement using a conductivity meter (Eutech Instruments, Fisher Scientific, Singapore).  
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Each arrow in the diagram represented an emulsification course. The letters A-H are codes for 
emulsification courses that starting from A-H point at ratio of aqueous phase to surfactant are 0.1:0.9, 0.2:0.8, 
0.3:0.7, 0.4:0.6, 0.5:0.5, 0.6:0.4, 0.7:0.3, and 0.8:0.2, respectively. Surfactant and aqueous phase were mixed at 
the desired ratio by a stirrer at a rotational speed of 300 rpm, followed by dropwise addition of vegetable oil. 
Water was then added dropwise, until the ratio of aqueous phase, surfactant, and vegetable oil of prepared 
emulsion equal to 0.8:0.1:0.1at the point shown as the star in the phase diagram. The droplet size of the prepared 
emulsions was measured using a laser diffraction particle-size analyzer (SALD-7100; Shimadzu, Kyoto, Japan). By 
overlaying the emulsification course onto each phase diagram, phase behavior along the emulsification process 
could be predicted. It is clear that the phase diagram or phase behavior along the emulsification process correlates 
with the droplets size of emulsions which prepared by different pathway. 
Surfactant
Aqueous Phase Oil Phase
0.5
0.5
0.5
0.40.30.20.1 0.80.70.6 0.9
0.1
0.1
0.2
0.2
0.3
0.3
0.4
0.4
0.6
0.6
0.7
0.7
0.8
0.8
0.9
0.9
A
B
C
D
E
F
G
H
 
Fig. 1 Phase diagram and emulsification courses using a low-energy method. 
The effect of sucrose on phase behavior of aqueous phase/Tween®/vegetable oil systems and food 
nano-emulsification using low-energy methods were studied. The results show as Fig. 2. In the 
water/Tween20/vegetable oil system, there were two phase types; surfactant phase coexisting with vegetable oil 
(S + O), and micellar phase coexisting with vegetable oil (Wm + O). In the sucrose solution/Tween20/vegetable 
oil system, there was minimal expansion in the boundary of S + O towards a higher aqueous content instead of 
Wm+O region in the water/Tween20/vegetable oil system and the re-crystalized sucrose (Suc) could be seen. This 
expansion of the S+O phase might be due to the increasing hydrophobicity of Tween20.  
In binary compositions of Tween40 and water system showed a lamellar liquid crystalline phase (La). 
On the other hand, in a sucrose-Tween40 binary system, a liquid crystalline phase (Lc) was formed at high 
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sucrose solution content (higher than 50%). A sponge phase (L3) was formed at the ratio of sucrose solution to 
Tween40 was 0.3:0.7 and 0.4:0.6 (C- and D- point). In ternary composition, water/Tween40/vegetables oil 
system showed coexisting of cubic phase and vegetable oil (I1 + O), and a small area of L3 phase. In the sucrose 
system, the boundary of the S+O phase did not change; however, the L3 region expanded to lower vegetable oil 
content at the same Tween40 concentration and to lower Tween40 content at the same sucrose solution content. 
The expansion of an L3 phase has been attributed to the decrease in the cloud point of the surfactant by sucrose. It 
is known that the effective HLB of the surfactant shifts toward the hydrophobic side in the presence of sugar 
(Ikeda et al., 2013; Wakisaka et al., 2014). In addition, re-crystallization of sucrose (Suc) and region of three 
coexisting phases, Wm + O + Lc, could be seen in the diagram of sucrose solution/Tween40/vegetables oil system. 
In Tween60 systems, solid Tween60 (Solid T60) existed with other phases in both phase diagrams of 
water and sucrose systems. Adding sucrose caused re-crystallization of sucrose and all Lc phases existing in 
water/Tween60/vegetable oil system changed to La phase in sucrose solution/Tween60/vegetable oil system. In 
sucrose system, La phase could have be seen for all area in phase diagram in term of sole La, La + I1, La + Wm + O, 
La + O, and La + O + Suc phases. 
In Tween80 systems, the phases of binary compositions of water and Tween80 were a hexagonal 
crystalline phase (H1) at D- (0.4:0.6) and E-point (0.5:0.5), and coexisting phases of H1 and L3 (H1+L3) at C-point 
(0.3:0.7). Adding sucrose transformed H1 into La phase at D-point and an L3 phase appeared at F-point (0.6:0.4). 
For the ternary compositions system, the cubic phase (I1) region in the sucrose solution/Tween80/vegetable oil 
system was smaller than that in the water/Tween80/vegetable oil system. Moreover, I1 and I1 + O phases 
transformed to Lc + Wm + O phases, Wm + O region expanded towards a lower aqueous content, and some part of 
Wm + O and I1 phase changed into an L3 phase leading to the expansion of the L3 region (L3 or L3 + O) in the 
sucrose solution/Tween80/vegetable oil system.  
The smallest nano-emulsion droplet sizes using Tween20 as surfactant for both the water and sucrose 
systems were produced by emulsification courses near the inversion point, the boundary between S + O and  
Wm + O, which was located at a ratio of aqueous phase to Tween20 of 3.5:6.5. The results of Tween40 and 
Tween80 systems indicated that small droplet sizes could be prepared in both water and sucrose systems when 
emulsification course passed through an L3 phase. The big droplet sizes could have been a result of passing 
through the H1 and/or I1 phase, a highly viscous phase, during emulsification process. On the other hand, the 
low-energy emulsification methods using Tween60 as surfactant were unsuccessful both in water and sucrose 
system at 25 °C. It was thought that presented of Solid T60 was a reason of this problem.  
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Fig. 2 Phase diagram of the aqueous phase/Tween®/vegetable oil systems at 25˚C, where Tween® are Tween20 (T20), 
Tween40 (T40), Tween60 (T60), and Tween80 (T80). Water (W) or 20% sucrose solution (S) was used as the aqueous phase. 
Phase types are denoted as micellar (Wm), sponge phase (L3), hexagonal phase (H1), lamellae phase (La), surfactant phase (S), 
cubic phase (I1), liquid crystal phase (Lc), vegetable oil (O) and solid Tween60 (Solid T60). 
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Therefore, the next part will study the effect of temperature on phase behavior of the surfactants to solve 
this problem. The effect of temperature (25, 40, 50, 60, and 70 °C) on food nano-emulsification using low-energy 
method in water/Tween60/vegetable oil systems was studied. The result is shown as Fig.3. 
 
 
Fig.3 Phase diagram of the water/Tween60/vegetable oil systems at 25, 40, 50, 60, and 70 ˚C. 
The results of the Tween60 system can be concluded that solid Tween60 at 25 °C disappeared by 
increased temperature from 25 to 40 °C due to an increase in solubility of surfactant. Increasing temperature from 
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25 °C to 40, 50, 60, and 70 °C caused phase transformation of La to H1 or L3 or Lc phase, H1 to L3, L3 to S + O or 
Wm phase, and Lc and I1 phase to Wm phase as shown in Fig. 3.  
Solid T60 existing at 25 °C is a disadvantage to nano-emulsification using the low-energy method 
resulting in unsuccessful emulsification, while nano-emulsification was successful at higher temperature. At 
40 °C, the emulsification courses that started from H1 phase and passed through I1 phase for D-course resulted in 
the formation of very big (1,424.89 ± 82.79 nm) droplet sizes of emulsion. For C-course, droplet size of emulsion 
was smallest (179.56 nm), while the standard deviations of droplet distribution (SDd) which was used as 
representative of polydispersity index (PDI) in this study was high (130.33). It is considered that the 
emulsification C-course started at L3 phase promoted small droplet size formation and then, passed through I1 and 
Lc + Wm + O phase that led to high SDd. Droplet size of emulsions prepared at 50 ºC and 60 ºC were not different 
compared within the same emulsification course. Both phase diagrams of these temperatures are same. Smallest 
emulsions were prepared passing through D- and E-course for both temperature started with L3 phase. However, 
emulsions prepared at 60 ºC showed lower SDd than that prepared at 50 ºC. Increasing prepared temperature to 
70 ºC resulted in bigger droplet size than those at 50 ºC and 60 °C. At this temperature (70 ºC), an L3 phase had 
already transformed to Wm phase and viscosity of the system was very low.  
To extend the study of the effect of temperature, other Tween®; Tween20, 40, and 80 were selected, 
since the hydrophilic head group of these surfactants are same as Tween60 while the structures of hydrophobic 
tail are different with each other. The effect of temperature (25, 40, and 60 ºC) on nano-emulsification and the 
phase types along the emulsification course (C- and D-course) in water/Tween20, 40, or 80/vegetable oil system 
were studied. Moreover, emulsions prepared using low-energy emulsification method were compared with the 
emulsions prepared using high-energy emulsification method. 
The results of low-energy emulsification methods showed that temperature did not show obviously 
difference effect on phase behavior in Tween20 system. The boundary between Wm+ O and S + O at 40 ºC and 
60 °C were similar with that at 25 ºC in this system. Increasing temperature resulted in minimized the properties 
difference between Wm+ O and S + O phases. Increasing temperature caused increased in droplet size of prepared 
emulsion in Tween20 system. At 60 °C, very big droplet size emulsion (more than 1,000 nm) could be produced, 
consequently leading to phase separation. However, the phenomenon of phase behavior of Tween20 system 
should be more studied in the future. 
In Tween40 and Tween80 system, increasing temperature resulted in phase transformation from La to H1 
or L3, H1 to L3, L3 and I1 to Wm or S phase. The changes in phase behavior along the emulsification process 
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resulted in differences in droplet size and homogeneity of prepared emulsions of Tween40 and Tween80 systems. 
It is clear that L3 structure gives an advantage in the formation of small droplet sized emulsions with low SDd, 
while H1 and I1 structures do not favor the formation of small emulsion droplets. 
The emulsion droplet size for all emulsions prepared by the low-energy method was smaller than that 
emulsions prepared by high-energy method. Very large size of droplets, bigger than 10,000 nm, was obtained 
using the high-energy method. These results pointed that emulsification using low-energy method had higher 
ability to form small droplet sized emulsions than using high-energy method in water/Tweens®/vegetable oil 
systems.  
The emulsion stability was studied in prepared emulsions using low-energy emulsification method of 
Tween20, 40, 60, and 80 systems. The results showed that prepared emulsions which had composition of 
water:Tween®:vegetable oil equal to 0.8:0.1:0.1exhibited good stability without increase in the average of droplet 
size over 4 months of storage period. However the fluctuation of droplet size could be seen in the samples whose 
droplet size bigger than 500 nm, especially the prepared emulsion using B- and D-course at 40 °C in Tween60 
system, and using D-course at 25 °C in Tween80 and Tween40 systems. All of these emulsification courses 
involved with H1 and/or I1 phases. Moreover, emulsion separation (creaming) was visually observed in the 
samples whose droplet sizes bigger than 500 nm. This could be attributed to the creaming velocity of oil droplets 
which increases with an increase in the droplet radius, hence emulsions containing larger droplets will cream 
more rapidly than those containing smaller ones (Yang et al., 2012). 
It can be noted that H1 and I1 phases led to very big droplet size and heterogeneity of emulsions, resulted 
in fuctuation of droplet size and creaming during storage.  
The surfactant concentration of all samples in this study was fixed at 10%, which might be high content 
for emulsion stabilization. In order to study the stability of emulsions at lower surfactant concentrations, emulsion 
prepared by C- and D-course in all Tween® systems at 25, 40 and 60 °C were diluted to 1.0% and 0.5% by pure 
water and stored at 25 °C for 4 weeks. The results showed that most emulsion samples showed high stability 
through storage time (4 weeks), especially nano-emulsions with droplet sizes smaller than 500 nm. The average 
droplet sizes did not vary significantly during storage time. Surfactant concentration of 0.5 and 1.0% did not 
significantly affect droplet size of all samples. 
The representative emulsions in sucrose solution were the prepared emulsion using D-course for 
Tween20 system, and the prepared emulsion using C-course for Tween40 and Tween80 systems. Each emulsion 
was selected from the smallest droplet size emulsion in each sucrose solution/Tween®/vegetable oil system. The 
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8 
results were as same as in water system that all samples had high stability without changing their droplet size and 
surfactant concentration of 0.5 and 1.0% did not significantly affect droplet size of all samples through storage 
time. These small droplet emulsions significantly improved emulsion stabilities by against creaming and 
flocculation due to the substantially reduced rates of gravitational separation and enhanced Brownian motion of 
oil droplets (Solans et al., 2005). Moreover, sucrose molecules increased viscosity of continuous phase compared 
with water, then decreased the opportunities of droplets collisions which resulted in enhance of stability. 
It is known that Ostwald ripening is one of the most common mechanisms for emulsion destabilization. 
The solubility of oil in water is an important parameter on the Ostwald ripening rate in the Lifshitz-Slezov and 
Wagner (LSW) theory. Ostwald ripening is prevented by long chain triglycerides of the oil phase. The oil phase 
in the present study was vegetable oil consisting of long chain triglycerides which have low solubility in water, 
resulted in high stability of emulsion in all system. In addition, it is assumed that all Tweens® had high ability to 
completely adsorb at the surface of oil droplets. Low-energy emulsification facilitated surfactant adsorption and 
orientation along the interface between oil and water due to the formation of suitable aggregate structures in the 
water, surfactant and oil mixtures. 
In overall results indicate that food nano-emulsification using low-energy method is useful and 
successively lead to high emulsion stability. Therefore, it is important to select conditions such as stating 
composition, preparation pathway, and temperature that involve with L3 phase and avoid H1 and I1 phases during 
the emulsification process. 
References 
Ikeda, S., Miyanoshita, and M., Gohtani S. Effects of sugars on the formation of nanometer-sized droplets of 
vegetable oil by an isothermal low-energy emulsification method. Journal of Food Science, 78(7): 
E1017–E1021. (2013) 
Solans, C., Izquierdo, P., Nolla, J., Azemar, N., Garcia-Celma, and M.J. Nano-emulsions. Current Opinion in 
Colloid and Interface Science,10:102-110. (2005) 
Wakisaka, S., Nakanishi, M., and Gohtani, S. Phase behavior and formation of O/W nano-emulsion in vegetable 
oil/mixture of polyglycerol polyricinoleate and polyglycerin fatty acid ester/water systems. Journal of 
Oleo Science, 63(3), 229–237. (2014) 
Yang,Y., Marshall-Breton, C., Leser, M.E., Sher, A.A., and McClements, D.J. Fabrication of ultrafine edible 
emulsions: Comparison of high-energy and low-energy homogenization methods. Food hydrocolloids, 
29:398-406. (2012) 


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