book of proceedings

BOOK OF PROCEEDINGS

Tunisian-Japanese Symposium on Society, Science & Technology 1155--1188 N Noovveem mbbeerr 22001133 H Hootteell EEllm moouurraaddii –– YYaassm miinnee H Haam mm maam meett,, TTuunniissiiaa

Proceedings TJASSST 2013 November 15th-18th, 2013. Hammamet, Tunisia

Session I: Life Sciences, Food & Agriculture

FOREWORD The Tunisia-Japan Symposium on Science, Society and Technology (TJASSST 2013) is held from 15-18 November 2013, Hammamet, Tunisia. The objective of this symposium is to contribute to a sustainable development and to promote international scientific exchanges between Tunisia and Japan through the strengthening of a multidisciplinary research network. It will focus on the sustainable development issues through science and technology in North-African and Mediterranean countries, as well as on the social impacts resulting from such activities. The

symposium is organized by the Centre of Biotechnology of Borj-Cédria (CBBC) under the auspices of the Ministry of Higher Education and Scientific Research in Tunisia. The Alliance for Research on North Africa (ARENA), the North African and Mediterranean Center for Research and Education (CANMRE) and the University of Tsukuba are responsible for the Japanese side.

The success of our conference largely relies on the generous support of our sponsors, but more importantly, this success will fully be dependent on you, our guests. The strong participation of delegates to all sessions will warrant the richness of exchanges between young and established researchers. On behalf of the organizing committee, I wish you a fruitful and a pleasant meeting and stay in Hammamet. Prof Chedly Abdelly Director General of CBBC

i



Tunisia-Japan Symposium on Science, Society and Technology (TJASSST 2013) November 15th-18th, 2013 Hammamet, Tunisia

COMMITTEES

Organizing Committee Tunisia

Japan Co-Chairmen

Prof . Chedly Abdelly (CBBC)

Prof. Mitsutoshi Nakajima(U. Tsukuba) Prof. Hiroko Isoda (U. Tsukuba)

Secretary Prof.Ridha Mhamdi (CBBC)

Prof. Kenichi Kashiwagi (U. Tsukuba)

Members Prof. Ridha M hamdi (CBBC)

Prof. Katsuhiro Akimoto (U. Tsukuba)

Prof. Riadh Ksouri (CBBC)

Prof. Hideomi Koinuma (U. Tsukuba)

Dr. Mounawer Badri (CBBC),

Prof. Takahiro Morio (U. Tsukuba)

Dr. Ahmed Debez (CBBC)

Prof. Mitsuteru Irie (U. Tsukuba)

Dr. Rim Nefissi (CBBC)

Prof. Junkyu Han (U. Tsukuba)

Dr. Samiha Bouzayene (CBBC)

Prof. Yoshikazu Suzuki (U. Tsukuba)

Dr. Inès Cherif (CBBC),

Prof. Kosuke Matsubara (U. Tsukuba)

Dr. Hana Sammoud (CBBC)

Prof. Islam Muhammad (U. Tsukuba)

Dr. Tarek Slatni (FST, CBBC)

Prof. Marcos A. Neves (U. Tsukuba)

Dr. Mohamed Gandour (CBBC)

Prof. Maki Iwasaki (U. Tsukuba)

Prof. Néji Besbes (CNRSM)

Prof. Atsushi Kawachi (U. Tsukuba)

Dr. Mohamed Kefi (CERTE)

Prof. Hajime Kamiyama (U. Tsukuba)

Dr. Fatma Hachani (CERTE)

Prof. Tariq Shezad (U. Tsukuba)

Dr. Mohamed Mehdi Bessem (CRTEn)

Dr. Myra O. Villareal (U. Tsukuba)

Dr. Talel Sahmim (Technopark)

Dr. Marino S. Morikawa - Sakura (U. Tsukuba) Dr . Wang Zheng (U. Tsukuba) Ms. Tamaki W. Kitagawa (U. Tsukuba) Mr . Akihiko Yahata (U. Tsukuba)

ii



Scientific Committees Session Coordinators Tunisia

Japan 1- Life Sciences, Food & Agriculture

Prof. Ridha. Mhamdi (CBBC)

Prof. Tatsuhito Fujimura (U. Tsukuba)

Prof. Riadh Ksouri (CBBC)

Prof. Toru Matsui (U. of the Ryukyus)

2- Environment Prof. Sami Sayadi (CBS)

Prof. Maki Tsujimura (U. Tsukuba)

Prof. Mohamed Ksibi (ENIS)

Prof. Mitsuteru Irie (U. Tsukuba)

Prof. Ahmed Ghrabi (CERTE)

3- Energy & Materials Prof. Adel Mnif (CNRSM)

Prof. Katsuhiro Akimoto (U. Tsukuba)

Prof. AmenAllah Guizani (CRTEn)

Prof. Yoshikazu Suzuki (U. Tsukuba)

4- Mathematics & ICT Prof. Habib Ouerdiane (FST)

Prof. Nobuaki Obata (Tohoku U.) Prof. Keisuke Kameyama (U. Tsukuba)

5- Management & Innovation Prof. Lassaad Mezghani (IHEC)

Prof. Kenichi Kashiwagi (U. Tsukuba)

6- Humanities & Social Sciences Prof. Mabrouk Mannai (FLM)

Prof. Etsuko Aoyagi (U. Tsukuba)

7- SATREPS Prof. Sami Sayadi (CBS)

Prof. Hiroko Isoda (U. Tsukuba)

8- Posters Prof. Moncef Harrabi (INAT)

Prof. Takahiro Morio (U. Tsukuba)

iii



Supporting institutions Japan International Cooperation Agency (JICA) Japan Science and Technology Agency (JST) Alliance for Research on North Africa (ARENA) North African and Mediterranean Centre for Research and Education (CANMRE) Centre de Recherches et des Technologies de l’Energie (CRTEn) Centre National de Recherche en Sciences des Matériaux (CNRSM) Centre de Recherche et des Technologies des Eaux (CERTE) Institut des Sciences et Technologies de l'Environnement (ISSTE) Société de Gestion de la Technopole de Borj Cedria Pôle Industriel et Technologique de Gabès (Politech) Centre de Biotechnologie de Sfax (CBS) Université Ez-Zitouna Université de Tunis I Université de Tunis El Manar Université de Carthage Université de Manouba Université de Jendouba Université de Sousse Université de Monastir Université de Kairouan Université de Sfax Université de Gafsa Université de Gabes Université Virtuelle Institut des Régions Arides (IRA) Institution de la Recherche et de l'Enseignement Supérieur Agricoles (IRESA) Association Tunisienne de Biotechnologie (ATBiotech) Association Tunisienne des Ressources Génétiques (ATRG) F-LAMBDA Company Bouattour Equipements et Services (BES)

Sponsors Japan Society for the Promotion of Science (JSPS) Ministry of Higher Education, Scientific Research (MHESR) – Tunisia

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Table of Contents Session I: Life Sciences, Food & Agriculture

Page

Relationship between physico-chemical characteristics and spaghettis making quality for selected durum wheat varieties Daaloul Bouacha Olfa, Amamou Sameh

1

LFA 1

LFA 2

Physicochemical, sensorial and rheological characteristics of a soy milk yogurt Essid Ines, Njoumi Sondes, Gligum Héla, Bel Hadj Safouan

5

Study of the pharmacokinetics of carbamazepine and its clinical efficacy in a Tunisian bipolar patients Chahra Chbili, Souhail Bannour, Saad Saguem, Bechir Ben Hadj Ali

9

LFA 3

LFA 4

Study of different extraction methods of phenolic compounds in orange peel M’hiri N, Ioannou I., Mihoubi Boudhrioua N and Ghoul M

13

LFA 5

Study of the antibacterial activity of two functionalized dressings El Ghoul Yassine, Salah Fatma, Roudesli Sadok

17

Growth and protein profile changes in Brassica juncea L. leaves exposed to cadmium Manel Taamalli, Angelo D’Alessandro, Tahar Ghnaya, Federica Gevi, Anna Maria Timperio,Chedly Abdelly, Lello Zolla

21

LFA 6

LFA 7

Phenolic profile and antioxidant capacity of ethyl acetate extract of fenugreek seeds O. Belguith-Hadriche, M. Bouaziz, A. El Feki, F. Makni-Ayedi

25 29

LFA 8

Higher baseline tumor necrosis factor [TNF]-α and early postoperative interleukin [IL]-10 level are associated with postoperative sepsis in cardiac surgery: A preliminary study M. Ben Azaiz, R. Arbi, C. Romthani, W Brahem, W. Ghothban, I. Labbane, Z Hajjej, S. Shnik, M Ferjani, E. Ghazouani Study of three types of a carbonated hydroxyapatite biomaterial implantation in vivo in rats of "Wistar" strain Salha Boulila, Kais Mnafgui, Hassane OudaDesse, Hafed El Feki & Abdelfattah El Feki

32

LFA 9

Color stability of anthocyanins from three Tunisian cultivars of strawberry (Camarosa, Selva and Tulda) in a jam model Hidri Dhekra, Riahi Hamadi and Debbabi Hajer

36

LFA 10

Anti-inflammatory effects of Ruta chalepensis L. extracts on LPS-stimulated RAW 264.7 cells Mohamed Kacem, Gaëlle Simon, Stéphane Cérantola, Laurent Misery, Abdelfattah Elfeki and Nicolas Lebonvallet

40

LFA 11

LFA 12

Evaluation of the susceptibility of five olive cultivars to Pseudomonas savastanoi pv savastanoi Samira Krid HadjTaieb, Imen Mougou, Mohamed Ali Triki and Ali Rhouma

44

LFA 13

Essential oil characteristics of Pistacia terebinthus L. fruits growing wild in Tunisia Ines Maalej, Chokri Jribi and Mohamed Makni

48

Mechanism of the melanogenesis stimulatory effect of Cymbopogon shoenanthus in B16 murine melanoma cells Mai Maeda, Myra O. Villareal, Junkyu Han and Hiroko Isoda

54

LFA 14

Inhibitory Effect of Caffeic acid and Caffeic Acid Phenetyl Ester on Oxidative Stress in Human Epidermal Melanocyte Sakura Eri B. Maezono, Myra O. Villareal, Junkyu Han , and Hiroko Isoda

58

LFA 15

Response variation to water deficit of parental genotypes of recombinant inbred lines populations of the annual legume Medicago truncatula Saoussen Mahfoudh, Kamel Hessini, Chedly Abdelly, Mounawer Badri

63

LFA 16

Fumigation resistance in two moth pests infesting stored dates in Tunisia Jouda Mediouni Ben Jemâa and Emna Eyet Limam

70

LFA 17

v



Selection of efficient and tolerant rhizobial strains to enhance production of chickpea (Cicer arietinum) and Faba bean (Faba vicia minor) grain legumes under water deficit. Rakia Mhamdi, Ridha Mhamdi and Haythem Mhadhbi

74

LFA 18

Molecular mechanism analysis of insulin resistance improvement effect of Cyanidin-3-glucoside in 3T3-L1 adipocyte Toshiya Matsukawa, Tetsuya Inaguma, Junkyu Han, Hiroko Isoda

78

LFA 19

LFA 20

Molecular phylogeny and evolution of alcohol dehydrogenase (ADH) genes in palms species Imen Rekik, Amine Elleuch, Noureddine Drira

82

LFA 21

Chemical and Biological characterization of some by-products Nasri Saida, Ben Salem Hichem

87 90

LFA 22

Identification of 6-Octadecynoic acid from Marrubium vulgare L. as a PPARγ agonist Anna Ohtera, Yusaku Miyamae, Naomi Nakai, Atsushi Kawauchi, Kiyokazu Kawada, Junkyu Han, Hiroko Isoda, Mohamed Neffati, Kazuhiro Maejima, Toru Akita, Naoki Mori, Kazuhiro Irie, Taiho Kambe, Seiji Masuda & Masaya Nagao

LFA 23

Uptake and transport of iron in N2-fixing common bean nodules Slatni Tarek, Vigani Gianpiero, Dell’Orto Marta, Zocchi Graziano & Abdelly Chedly

94

State-of-the-art and future perspectives of functional compounds from olive, argan, date fruits and their by-products in North Africa Zheng Wang, Marcos A. Neves, Mitsutoshi Nakajima & Mohamed Zahar

98

LFA 24

Fluorescence fingerprint Technology for Food Safety Junichi Sugiyama, Kaori Fujita, Masatoshi Yoshimura, Mizuki Tsuta, Mario Shibata and Mito Kokawa

102

LFA 25 LFA 26

Research Topics at Food Engineering Division in National Food Research Institute, Japan Hiroshi Nabetani

108

Scaling-up Production Technology Foreseeing Novel Applications for Antioxidant Food Materials Neves M. A., Kobayashi I., Nakajima M.

111

LFA 27

116

LFA 28

Antidiabetic effects of isoflavones, present in Japanese traditional soy foods, in cultured myocytes and type 2 diabetic model mice Sun Hee Cheong, Keisuke Furuhashi, Katsuki Ito, Masato Nagaoka, Myoung Jin Son, Takayuki Yonezawa, Yutaka Miura & Kazumi Yagasaki

LFA 29

In-vitro model for axon pruning: a phosphatidylserine independent pathway Insaf Bahrini and Rikinari Hanayama

120

Specific induction of apoptosis by 1, 8-cineole in two human colon cancer cells. Soichiro Murata, Risa Shiragami, Chihiro Kosugi, Tohru Tezuka, Masato Yamazaki, Atsushi Hirano, Yukino Yoshimura, Kiyohiko Shuto, Nobuhiro Ohkohchi, Keiji Koda

124

LFA 30

Argan oil inhibits melanin biosynthesis in B16 murine melanoma cells by activating MITF in B16 murine melanoma cells1 M. O. Villareal, S. Kume, T. Bourhim, F. Z. Bakhtaoui, K. Kashiwagi, J. Han, C. Gadhi, H. Isoda

128

LFA 31

132

LFA 32

Next generation sequencing (NGS) for simultaneous mapping and isolation of genes by MutMap+ R. Fekih, H. Takagi, M. Tamiru, A. Abe, S. Natsume, H. Yaegashi, S. Sharma, S. Sharma, H. Kanzaki, H. Matsumura, H. Saitoh, C. Mitsuoka, H. Utsushi, A. Uemura, E. Kanzaki, S. Kosugi, K. Yoshida, L. Cano, S. Kamoun, R. Terauchi Cell cycle checkpoint engineering: novel construction method of gene-amplified CHO cell line for therapeutic antibody production Tomomi Tsutsui, Kyoung Ho Lee, Rina Matsuyama, Masayoshi Onitsuka & Takeshi Omasa

137

LFA 33

Hyperspectral Scattering Imaging and Parallel Factor Analysis for the Spectral Decomposition of Multi-layered Biological Materials M. Tsuta, K. Fujita, M. Shibata, M. Yoshimura, M. Kokawa and J. Sugiyama

140

LFA 34

LFA 35

Agroinoculation: Simple tool for complex gene function analysis Anis Ben-Amar, Ahmed Mliki

143

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Valorization of Bio-resources in Semi-Arid and Arid Land -Olives, Halophytes and Aromatic plantsJunkyu Han, Mahmoud Ben Othman, Feten Zar Kalai, Imen Samet, and Hiroko Isoda

147

LFA 36

LFA 37

Halophilic bacteria isolated from Tunisia and Okinawa T. Matsui, T. Uezu, L. El Bassi, N. Nugara, H. Abdennaceur, and H. Isoda

151

Studies on traditional edible plants with anti-oxidative and anti-inflammatory activities, which are cultivated in Fukui, Japan Masakazu Takahashi, Kyoichi Kobayashi, and Hajime Ohigashi

155

LFA 38

Prospects for biological protection of olive trees in Tunisia with entomopathogenic bacteria against main insect pests Imen Blibech, Mohieddine Ksantini, Ikbal Chaieb and Sami Aifa

159

LFA 39

Inhibitory activities of nisin and potassium sorbate on aerobic spore-forming Bacteria isolated from raw milk Chedia Aouadhi, Slah Mejri and Abderrazak Maaroufi

164

LFA 40

Characterization of virgin olive oils from seven selected oleasters (Olea Europea L.) according to its sterols and triterpenic dialcohols compositions: comparison with others Tunisian cultivars Bechir Baccouri, Hedia Manai, Douja Daoud and Mokhtar Zarrouk

167

LFA 41

Genetic diversity of Pyrenophora teres f. teres in Tunisia as revealed by pathotype and AFLP markers Aida Bouajila, Néjia Zoghlami, Michael Baum, Kumarse Nazari & Abdelwahed Ghorbel

170

LFA 42

LFA 43

The importance of the prophylactic pest management tools to control Tuta absoluta in Tunisia Asma Cherif, Rihab Ben Jbara, Ameni Aissa and Kaouthar Grissa Lebdi

174

Bioinformatics analysis of a putative salt stress tolerance gene Vv-α-gal/SIP from grapevine (Vitis vinfera L) Samia Daldoul and Ahmed Mliki

178

LFA 44

LFA 45

Salt-induced early changes in ABA and antioxidant enzyme activities of barley plants Dorsaf Allal, Marta Pinto Marijuan, Maria Manuela Chavez and Chedly Abdelly

182 186

LFA 46

Lipid peroxidation and oxidative stress in rat erythrocytes induced by two halo-acetates and the protective effect of date palm fruit extract Amira El Arem, Fatma Ghrairi, Amira Tahouri, Mouna Zekri, Emna Behija Saafi, and Lotfi Achour

LFA 47

Evolution of olives antioxidants of three olive table varieties during their treatment Hayet Fourati and Mohamed Bouaziz

190

LFA 48

Secondary metabolites and antioxidant activity of Cistus monspeliensis leaves methanolic extract Hanene Ghazghazi, Chedia Aouadhi, Asma Bettaib, Abderrazak Maaroufi and Hasnaoui Brahim

194

Study of the phenomenon of diapauses according to two protocols applied to the carob moth (Ectomyelois ceratoniae Zeller) under laboratory conditions Hached Wiem, Lebdi Grissa Kaouther Impact of the bacteria inoculation on Hordeum vulgare response to salt stress

197

LFA 49

LFA 50

201

Haifa Hammami, Wisal Metoui, Ian Dodd and C. Abdelly Molecular research on the genetic diversity of Tunisian Aegilops geniculata Roth populations and Triticum durum Desf Based on RAPD markers A. Mahjoub, Khaled Mguis, Mustapha Rouaissi, Nadia Ben Brahim, and Mohamed El Gazzah

205

LFA 51

LFA 52

Genotypic and symbiotic diversity of rhizobia nodulating Lathyrus sativus in Tunisia Mnasri Bacem, Donia Khedher, Haifa Boukriba & Ridha Mhamdi

210

Effect of salt stress on growth, osmotic and hydraulic conductance of wild barley (Hordeum maritimum) Mejri Maroua, Hessini Kamel, Ferchichi Selma and Abdelly Chedly

214

LFA 53

LFA 54

Biochemical composition of Tunisian olive oils and variations induced by growing area Imen Oueslati, Nesrine Jridi, Guido Flamini and Mokhtar Zarrouk

218

vii


LFA 55


Effects of sodium arsenate exposure on liver fatty acid profiles W. Kharroubi, M. Dhibi, Z. Haouas, I. Chreif, F. Neffati, M. Hammami, R. Sakly

viii

222



LFA 1

Relationship between physico-chemical characteristics and spaghetti making quality for selected durum wheat varieties Daaloul Bouacha Olfaa, Amamou Sameha, Sfayhi Dorrab, Rezgui Salaha and Nouaigui Sadokc (a)

National Agronomic Institute of Tunisia, (b) National Agronomic Research Institute of Tunisia, (c) High School of Food Industries of Tunisia; [email protected]

Abstract Four durum wheat varieties were examined for their test weight (TW), thousand kernel weight (TKW), protein content (P), yellow berry (YB), Ash content, gluten content (Gc), gluten index (Gi), SDS-sedimentation and color. Semolina extracted from these cultivars was characterized for SDS-sedimentation (SDSS) volumes, gluten content and gluten index and for color. Spaghettis made of these semolina samples were examined for color, cooking time (CT), cooking losses (CL) and water absorption (Wa). Results showed that strong relationships were found between physico-chemical and technological quality parameters and spaghetti cooking quality. SDSS and CL were negatively correlated (r = - 0.86). Semolina Gc and the yellow fraction of the color were positively correlated (r=0.90). P was negatively correlated with CT, CL and Wa. While a strong positive correlation was significant between P and SDSS (r = - 0.73). These correlations could explain the differences observed in the spaghettis cooking quality between landraces and high yielding cultivars. Landraces seemed to outperform high yielding cultivars for physico-chemical and technological quality parameters and appeared to be more adapted to the spaghetti manufacturing process giving better cooking quality spaghettis. Gi and SDSS could be considered predictive parameters for pasta quality evaluation.

Keywords : Durum wheat, Quality, Spaghetti, Gluten strength 1. Introduction Durum wheat is the main raw material for processing into pasta products. The durum grains milling produces a coarse particle called semolina, ideal for making pasta (Sissons, 2008). The quality for the miller is represented by high extraction rate expressed by the properties of the wheat kernel that is milled into semolina. Pasta quality is defined by color characteristics and cooking quality. Superior pasta quality should have a bright yellow color (D‘Egidio et al., 1990) and should maintain its texture after cooking and not become a thick, sticky mass (Sissons, 2008). In general the specificity of a durum wheat cultivar for pasta products is determined by its grains protein composition (MacRitchie, 1992). However, it has been observed that the same protein content can be present in pasta samples with contrasting rheological and cooking properties, indicating that other gluten characteristics are most important in transformation process (Kosmolak et al., 1980; Gupta et al., 1994). This investigation aimed the evaluation of the relationship between physico-chemical and technological characteristics of durum wheat grains and spaghetti making quality amongst four Tunisian durum wheat cultivars.

2. Material and Methods Four Tunisian durum wheat cultivars were used in this study. They are composed by two traditional cultivars (Chili and Biskri) and two high yielding ones (Karim and Khiar). These cultivars are known to have contrasting qualities (Daaloul Bouacha et al., 2009; Zaibet et al., 2007). Semolina samples extracted from these durum wheat cultivars

1



were used in spaghetti processing, using a laboratory pasta machine (La Monferrina, model Dolly, Italy). After extrusion, spaghetti was dried using a drier chamber (La Monferrina, Model EC 25, Italy).

2.1. Durum wheat grains and semolina quality evaluation : Test weight (TW) (NT 51.61., 1993), thousand kernel weight (TKW) (NF V03-702., 1981), yellow berry (Garrido-Lestache et al., 2005) Protein content (P) (NT 51.54, 1991) and ash content (NT, 1990) were determined for whole grains. Gluten content, gluten index (ICC, 1986), Sodium-Dodecyl-Sulfate sedimentation (SDSS) (Carter et al., 1999) and yellow index (using Minolta Chroma Meter colorimeter) were assessed on whole grain flours and semolina samples.

2.2. Pasta quality evaluation : Pasta was characterized by yellow index determination on dried spaghetti, cooking time (CT) according to NF ISO 73.04 (1989), cooking losses percentages according to Aalami et al. (2007) and water absorption indicating the swelling capacity of cooked spaghetti.

3. Results and Discussion 3.1. Quality characterization of grains, semolina and spaghettis of the four cultivars : ANOVA analysis carried out on the grains, semolina and spaghetti from the four studied cultivars ‗characteristics showed that genotypic effect controlled almost all grain quality characteristics, except for yellow berry. These results are in accordance with many previous studies (Lerner et al., 2006; Li et al., 2013); which stipulated that qualitative parameters such as protein and gluten content were controlled by genotypes, and showed also a significant effect of interaction between genotypes and environments. In addition, genotypes effect was significant for SDSS, yellow index for semolina and for cooking losses percentages and yellow index for spaghetti. Overall means comparison amongst cultivars for all studied parameters showed that cultivar Chili presented high protein content (17.65%) and gluten content (43.89 %) levels; followed by Biskri with means respectively of 17.33% and 41.10%, then Karim with means respectively of 15.35% and 37.82 % and Khiar with means of 14.93% and 32%, respectively (Table 1). Table 1. Overall means amongst cultivars of test weight (TW), thousand kernel weight (TKW), ash content (ash), Sodium-Dodecyl-Sulfate sedimentation (SDSS), protein content (P), gluten content (G), gluten index (Gi) and yellow index (b*) assessed on grains. Grains (cultivars)

TW (Kg/hl)

TKW (g)

Ash (%)

SDSS (ml)

P (%)

G (%)

Gi

b*

Chili

79.76ab

53.01b

1.50b

14.23a

17.65a

43.89 a

49.26 b

13.10 a

Biskri

81.58a

55.98a

1.85a

12.08b

17.33a

41.10 b

53.42 b

12.54 b

Karim

80.42ab

50.26c

1.73a

5.33d

15.35b

37.82 c

48.35 b

12.41 b

Khiar

78.66b

44.88d

1.72a

9.95c

14.93b

32.00 d

64.39 a

13.25 a

Means with the same letters do not differ significantly (p0.05) to those of cultivars Biskri (5.16 ml) and Khiar (5.95 ml); therefore it was significantly higher than that of cultivar Karim (3.7 ml). However, yellow index showed similar values for cultivars Chili and Khiar, significantly higher than those observed for cultivars Biskri and Karim. Gluten index varied from 40.95 to 65.35 showing an overlapping ranking amongst cultivars.

2



Table 2. Overall means amongst cultivars of Sodium Dodecyl Sulfate-sedimentation (SDSS), gluten content (G), gluten index (Gi) and yellow index (b*) assessed on semolina. Semolina (cultivars)

SDSS (ml)

b*

G (%)

Gi

Chili

5.80 a

19.01 a

38.35 a

40.95 b

Biskri

5.16 a

17.11 b

36.35 a

65.35 a

Karim

3.7 b

17.87 b

35.74 a

51.20 ab

Khiar

5.95 a

19.61 a

33.75 a

57.31 a

Means with the same letters do not differ significantly (p 800 nm were removed (Fig. 2c)). Fig.2 Data pre-processing

2.3 Chemometrics The quantification models were developed using partial least squares (PLS) regression with leave-one-out cross validation to the FF data of the calibration samples. The performance of PLS models depends on the number of latent variables (LVs) used. The optimum number of LVs was determined by minimizing the root-mean-square error of the prediction of cross-validation. The calibration model was applied to the validation dataset to evaluate the accuracy of the model. The fitting of the calibration model to the calibration and validation datasets was finally evaluated by the coefficient of determination (R2), standard error of calibration (SEC), and standard error of prediction (SEP) .

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Proceedings TJASSST 2013 November 15th – 18th, 2013 Hammamet, Tunisia


3. Results and Discussion: 3.1 Detection of Mycotoxin in Wheat Major mycotoxin in wheat is deoxynivarenol(DON). There are other mycotoxin , nivarenol(NIV) and zeararenon(ZEA). They appear in major crops such as wheat, corn and other cereal grains. They reduce yield and quality. They caused symptoms such as vomiting, diarrhea and headaches upon human and animal ingestion. Such damages are reported around the world. The wheat samples for this experiment were artificially contaminated with fuzarium graminearum in the field. Four levels of contaminated wheat were harvested. They were

Fig.3 FF of Contaminated Wheat Flours

ground into flour by the milling machine (Cyclone Sample Mill, UDY Corp., USA). Fig.3 shows FF of each flour. Low, medium, medium-high and high in Fig.3 mean level of contamination. Some fluorescence peaks can be found in Fig.3, however, little difference among the 4 levels. To predict quantitative contamination

Fig.4 PLS Regression

level, PLS regression was applied. Actual contamination level was measured by HPLCUV. Fig.4 shows schematic diagram of PLS regression. Fig.5 is the prediction of DON concentration in contaminated wheat flour. Both calibration and

validation

correlations

datasets show

between

actual

significant values

and

Fig.5 Prediction of DON

predicted values. It is well known that not only DON but also other mycotoxins like NIV, ZEA were also contaminated at the same time.

However, degree of contamination is different from DON.

FF also reflects on these

contaminations. There could be created the model to predict for NIV and ZEA from the same FF. Fig. 6 shows the results of NIV and ZEA prediction. Both results have good correlations. Especially, the remarkable point is sensitivity to predict NIV. The order is almost ppb levels. Because it is too little, the conventional chemical analysis cannot detects it. So actual value of NIV and ZEA was measured by LC/MS/MS.

104



As a result, it is clear that the FF can predict DON, NIV, ZEA at the same time.

3.2 Prediction of Aerobic Bacteria Population on Beef surface 60 lean beef pieces, consisting of two lots (15 pieces / lot) each of Australian and Japanese cattle, were purchased from a local meat store (Ibaraki, Japan) and they were cut into 45 x 45 x 8 mm pieces at the store.

Fig.6 Prediction of NIV and ZEA

Samples were stored aerobically by putting them into sterilized plastic Petri dishes with lids. Each lot (15 pieces) of lean beef samples were stored in an incubator at 15 °C and analyzed after 0, 12, 24, 36 and 48 hours of storage. For analysis, three samples were used for both fluorescence fingerprint measurement and microbial determination. The sample was placed between a quartz plate with 0.5 mm thick and an acrylic plate with1 mm thick (Fig. 7(a)) and mounted in the sample holder in the spectrophotometer. A Fluorescence spectrophotometer (FHitachi

High-Technology

Corp.)

(b)

(a) Quartz plate

mounted with a front-surface sample holder

Meat sample

was used to measure FF. FFs were measured in

Acrylic plate

the range of 200 ~ 900 nm for both excitation and emission wavelengths with 10 nm intervals.

40 mm

7000,

No. 1

No. 2

No. 3

No. 4

45 mm 45 mm

Four locations (Fig. 7(b), cross marks, No.1 4) were measured for one sample at room temperature. A total of 240 FFs (4 lots x 5 different time of storage x 3 samples x 4

Fig.7 Sample preparation for FF and APC measurement (Cross mark No.1 ~ 4: FF measurement position, Shaded area: swab both surfaces of meat and quartz plate for APC measurement)

positions) were collected. After FF measurements, 40 mm squared areas on both quartz plate and beef sample were wiped with a sterile swab (Fig.7(b), shaded area). To ensure adequate sampling, the sample was swabbed in a horizontal pattern and again in a vertical pattern, while being rotated between the index finger and thumb in a back and forth motion. Serial dilutions of the swab sample were prepared with the phosphate buffer solution in which the swab was immersed, then aerobic plate count (APC [CFU/cm2], CFU: Colony forming unit) were determined by incubating 1 ml of appropriate dilution on PetrifilmTM Aerobic count plates (Sumitomo 3M Ltd.) for 48 hr at 35 °C . A total of 60 APCs (4 lots x 5 different time of storage x 3 samples) were determined through the entire experiment. Fig. 8 shows the time variation of aerobic plate count determined in Australian beef sample (cross and triangle symbols) and Japanese beef (circle and square symbols). Both initial aerobic plate count and growth rate varied among the lots.

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PLS regression was applied to FF to develop a model for the prediction of aerobic plate count (APC) . Fig. 9 shows the result of PLS regression. In this case for the beef meat, prediction model for the aerobic plate count was made with seven latent variables (LV), which gave best result with highest correlation and lowest SEC. From the result for validation set (Fig. 9(a)), good correlation (R2 = 0.819) and small SEP (SEP = 0.752 log [CFU/cm2]) was obtained and the accuracy of the model was verified. 8.0

Calibration（LV 7）

9

Y = 0.889 * X + 0.472 2 R = 0.889 RMSEC = 0.548

8

2

])

6.0

7

5.0 Log( Predicted APC[CFU/cm

log ( APC [CFU/cm2] )

7.0

4.0 3.0 2.0 Japanese 1 Australian 1

1.0

Japanese 2 Australian 2

0.0 0

12

24 Elapsed time [hr]

36

48

6 5 4 3 2 1

Fig.8 Time variation of the aerobic plate count on the surface of beef meat (Japanese 1 & 2: samples of Japanese cattle, Australian 1 & 2: samples of Australian cattle)

1

2

3

4

5

6

7

8

9

2

Log( APC[CFU/cm ] )

Validation (LV 7)

9

Y = 0.810 * X + 1.078 2 R = 0.819 RMSEP = 0.752

2

])

8 7

Log( Predicted APC[CFU/cm

The distribution of the regression coefficient of this model is shown in Fig. 10. The wavelength conditions with high regression coefficient value are considered to contribute largely to the model. High regression coefficient values are observed in the fluorophores related regions (A - D). It seems

6 5 4 3 2

that each peak was caused by the following intrinsic 1

fluorophores (wavelength condition of excitation and

1

(B) NAD(P)H (Ex 320 nm / Em 460 nm), (C) Porphyrins (Ex 430 nm / Em 600 nm), and (D) Flavins (Ex 460 nm / Em 520 nm). As a result, the regression model was build depending on the information of these fluorophores.

Excitation wavelength [nm]

(Em) 330 & 660 nm),

4

5

6

7

PLS Regression Coef. 0.02 0.015 0.01

700

0.005

600

0

D

500

C

B

400

300

400

-0.01

A

A

200 200

-0.005

500

600

700

800

-0.015 900

-0.02

Emission wavelength [nm]

Fig.10 Distribution of the regression coefficient of PLS model

References:

Fujita K., Tsuta M., Kokawa M., Sugiyama J.(2010). J.Food and Bioprocess Technology, 3(6), 922-927 Lakowicz JR. (1990) Principles of Fluorescence Spectroscopy, 3rd edition, Springer-Verlag, New York,

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9

Fig.9 PLS regression for APC on beef

800

300

8

2

900

(A) tryptophan (Excitation (Ex) 290 nm / Emission

3

Log( APC[CFU/cm ] )

emission maximum in FF is shown in parentheses) (Prased, 2003):

2



Kokawa M., Fujita K., Sugiyama J., Tsuta M., Shibata M., Araki T, Nabetani H. (2011) Biosci Biotechnol Biochem. 75(11), 2112-2118 Kokawa M., Fujita K., Sugiyama J., Tsuta M., Shibata M., Araki T, Nabetani H. (2011) J Cereal Sci. 55, 15-21 Kokawa M., Sugiyama J., Tsuta M., Yoshimura M., Fujita K., Shibata M., Araki T., Nabetani H. (2012) Food Bioprocess Technol. 5(5), Oto N., Oshita S., Makino Y., Kawagoe Y., Sugiyama J., Yoshimura M.(2010) Meat Sci. 93(3), 579-585

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LFA26

Research Topics at Food Engineering Division in National Food Research Institute, Japan Hiroshi Nabetania (a) Food Engineering Division, National Food Research Institute, NARO 2-1-12 Kan-nondai, Tsukuba, Ibaraki 305-8642, Japan

Abstract At Food Engineering Division in National Food Research Institute (NFRI), based on a food engineering approach, new food technologies are being studied as unit operations by analyzing the processes, improving the system, and incorporating cutting-edge technologies such as nanotechnologies and information technologies (IT). Some successful technologies are using our research and have already contributed to our daily life through safe and high quality foods. In this paper, some research topics in our research division will be introduced.

Keywords: Hydrostatic Pressure Processing, Ultra-fine Grinding, Fluorescence Fingerprint, Aqua-gas, Membrane Separation Technology, Biodiesel

1. Development of Hydrostatic Pressure (HHP) Processing for High Quality Foods Foods processed by high pressure are different in the quality from those by heat. High pressure enables minimally processed high quality foods without cooked odor and with retained fresh color, flavor and taste. Microorganisms such as bacteria can be inactivated by high pressure. Therefore, high-pressure process is expected to enable pasteurization of food. In addition, liquid surrounding foodstuff can be impregnated with a help of high pressure. Taking advantage of these specific properties in high pressure treatment, some foods have already been commercialized. Starch can be gelatinized by heat, and it can also be gelatinized by HHP. Behavior of the pressure gelatinization is dependent on the botanical origins of starch. For example, corn starch gelatinizes at less high pressure than potato starch. However, the pressure gelatinization has not yet been elucidated sufficiently. In NFRI, by using calorimetry, X-ray diffractometry, microscopy, and viscometry, various starches are currently under investigation1). In addition, protein can be denatured by HHP. Denaturation of egg protein is studied toward effective utilization of HHP-processing for novel egg products. Middle-heat treatment was applied in combination with HHP processing to prevent recovery of pressure-injured Listeria monocytogenes in milk2).

2. Development of Ultra-fine Grinding Method for Food Materials by Use of Jet Mill Rice grains are usually consumed as cooked whole grains in Japan but the consumption is decreasing year by year. In order to expand the consumption not only as whole grains but also as rice flour, we are doing a study about ultra-grinding of rice3-5). At first, we tried to make the ultra-fine rice flour, using hammer mill and jet mill under dry grinding condition. In the jet mill, the compressed air is released from a nozzle. Sample is crashed into a ceramic board at high velocity along the air flow. The coarse flours are crashed repeatedly and the fine flours are collected by a cyclone classifier. Flour obtained by the jet mill showed finer and sharp size distribution and the minimum mean size was about 3m. However, we could not obtain the flour of less than submicron mean size. The flours from 120 m to 50 m mean size indicated the similar viscosity. This means that the pasting property is not influenced in the range even though the mean size is changed. As a result, we found that the viscosity was drastically decreased in less than 5 m mean sizes. It is considered that the drastic change relate to the crash of the starch granule.

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3. Detecting and Imaging Technology for Food Components by Use of Fluorescence Fingerprint A non-destructive imaging method using the Fluorescence Fingerprint (FF) was developed by Tsuta et al. at NFRI6). The FF, also known as the Excitation-Emission Matrix (EEM), is a set of fluorescence spectra acquired at consecutive excitation wavelengths, creating a three-dimensional diagram. Its pattern is unique for every constituent, rather like a fingerprint. Compared to conventional fluorescence spectroscopy, its large amount of information enables fine distinctions between constituents which differ only slightly in fluorescence characteristics. We have already applied FF to visualization of the distribution of gluten and starch in bread dough, determination of the geographic origin of mangoes, rapid estimation of aerobic bacteria population on beef surface etc.

4. Utilization of Aqua-gas (superheated steam with hot water droplets) for Food Processing Superheated steam (SHS) has been applied for food processing because of its various advantages including efficient heat transfer by latent heat and radiation, as well as low oxygen environment. High drying capacity is one of the advantages of SHS when it is used for drying. However, it causes some difficulties when SHS is applied for blanching of vegetables and for cooking. To prevent the drying of food materials during SHS heating, a system using SHS and a spray of hot water micro droplets (WMD) was developed and named Aqua-gas7). The Aqua-gas system prevented water absorption and dissolution of solid content of the potato, which is usually caused by the hot water or saturated steam heating. In addition, WMD prevented the drying of potato caused by SHS heating. It was also found that the heat transferability of SHS was enhanced by the presence of WMD. Because of its efficient heat transferability, Aqua-gas system enable surface pasteurization of fresh vegetables with slight quality changes with short time heating. Quality and shelf life of potato salad made with the potato heated with Aqua-gas system and fresh vegetables pasteurized with Aqua-gas system were improved compared with the potato salad cooked by a conventional method.

5. Utilization of Membrane Separation Technology in Food Industry Membrane separation technologies such as microfiltration, ultrafiltration and reverse osmosis have many advantages over other separation technologies because they require less energy and no heat treatment. Their application to food industries has been developed successfully in Japan. Recently nano-filtration technology which is a new category of membrane technology placed between reverse osmosis and ultrafiltration is attracting a great deal of attention. In the Food Engineering Division, we are trying to apply membrane separation technology to food processing, and some novel processes such as membrane process for highly concentrated fruit juice8), simple refining process for edible oil9, 10), and purification and concentration process for functional polysaccharides from botanical resources11) and dipeptides12) from animal resources have been developed.

6. Development of Non-catalytic Alcoholysis Process for Production of Biodiesel Fuel from Used Frying Oil Biodiesel fuel is a replacement for diesel as a fuel produced from biomass resources. It is usually defined as a fatty acid methyl ester derived from vegetable oil or animal fat. Production of biodiesel fuel by use of conventional method (alkaline catalyst method) requires deacidification process prior to the reaction and refining process to remove the catalyst after the reaction. These processes increase total cost required for production of biodiesel fuel. In order to solve the problem, we recently proposed a method called superheated methanol vapor 13, 14) method . In a process with this method, superheated methanol vapor is continuously bubbled into the oil in the reactor vessel and reacted with triglycerides to form fatty acid methyl ester and glycerol. The fatty acid methyl ester and glycerol formed flows out of the reactor together with unreacted methanol vapor

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and is collected using a condenser. Reaction using the superheated methanol vapor method can be conducted at atmospheric pressure. The method does not require refining process after the reaction because no catalyst is used in this method and fatty acid methyl ester can be separated from glycerol simply by sedimentation. The method does not require deacidification process prior to the reaction because not only triglyceride but also free fatty acid can be converted into fatty acid methyl ester by use of the method15). Therefore, both initial and running costs required for biodiesel production are thought to be reduced by applying the method. Now, we are trying to apply the method to free fatty acid from oil refining process and used frying oil which do not compete with edible use.

7. Conclusions Some research topics at the Food Engineering Division in NFRI have been introduced in this paper. We hope some of them will lead to collaborative search subjects between Tunisia and Japan.

References 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15)

Yamamoto, K. et al., Food, 3, 57(2009). Koseki, S. et al., Food Microbiol., 25, 288(2008). Hossen, M. S. et al., Jpn J. Food Eng., 12, 29(2011). Hossen, M. S. et al., Cereal Chemistry, 88, 6(2011). Sotome, I. et al., Jpn. J. Food Eng., 10,95(2009). Tsuta, M. et al, Transactions of the Asabe, 50, 2127(2007). Sotome, I. et al., , LWT Food Sci. Technol., 42, 1035(2009). Nabetani, H., Membrane, 21、102(1996) (in Japanese). Subramanian, R. et al., Food Res. Int., 31, 587-593(1998). Miyagi, A. et al., Membrane, 29, 26-33(2004) (in Japanese). Kamada, T, et al., Food Sci. Technol., 8, 172(2002). Yanai, N. et al., Membrane, 29, 17(2004) (in Japanese). Yamazaki, R. et al., Japan Journal of Food Engineering, 8, 11(2007). Joelianingsih et al., Renewable Energy, 33, 1629 (2008). Joelianingsih et al., Journal of Chemical Engineering of Japan, 40, 780 (2007).

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LFA 27

Scaling-up Production Technology Foreseeing Novel Applications for Antioxidant Food Materials Neves, M. A.,a Kobayashi, I.,b Nakajima, M.a (a) ARENA, Faculty of Life and Environmental Sciences, University of Tsukuba, Japan; (b) Food Engineering Division, National Food Research Institute, NARO, Tsukuba, Japan. Tel: +(81)29-853-4703; E-mail: [email protected]

Abstract The health benefits associated with functional lipids such as carotenoids, polyphenols, -3 fatty acids among other natural antioxidants have been studied for many decades. However, most of these bioactive compounds not only are water-insoluble but also possess low solubility saturation in oil at room temperature. The low solubility of the functional lipids impairs their bioavailability and limits their use in food formulations. The formulation of functional lipids into emulsions or micro- / nanoparticles is expected to improve their bioavailability. On this context, the authors have investigated the formulation and characterization of monodisperse Oil-in-Water (O/W) emulsions loaded with lipophilic bioactive compounds such as ß-carotene or -oryzanol, -3 PUFAs, or polyphenols using microchannel (MC) emulsification. Moreover, O/W nanodispersions containing such bioactive compounds have been formulated using high-pressure homogenization, investigating their physicochemical stability as well. The authors also developed a large MC emulsification device including a newly designed asymmetric MC array chip to realize the mass production of uniformly sized droplets on a liter per hour scale, so that satisfying the minimum droplet productivity needed for industrial-scale production. The overall goals of this research are to develop novel bioactives delivery systems such as food micro- /nanodispersions with enhanced bioavailability and controlled release of bioactive compounds, and to evaluate their functional properties.

Keywords: Lipid, emulsion, bioactive, antioxidant, scale-up 1. Introduction The application of compounds such as PUFAs into food products is restricted by their susceptibility to oxidative degradation, a major concern to food manufacturers since it leads to the development of undesirable flavors (rancidity) and potentially toxic reaction products (Ribeiro, et al., 2009). Such intermediary compounds are unstable and cause oxidation of pigments, flavors, and vitamins. Other compounds, such as ketones, aldehydes, alcohols, hydrocarbons, acids, and epoxides, formed during the oxidation of unsaturated fatty acids, produce off-flavors and can interact with fish proteins to produce offcolors (Thanonkaew, et al., 2006). If lipids containing highly unsaturated fatty acids are incorporated into processed foods, they would likely be in the form of emulsions stabilized by small-molecule emulsifiers or proteins (Donnelly, et al., 1998). Oil-in-water (O/W) emulsions may be an effective method for supplementing formulations with -3 PUFA (Neves, et al., 2008a; Neves, et al., 2012). According to previous literature, emulsions containing lipophilic antioxidants dissolved in finely dispersed oil droplets have been reported to increase their absorption in vitro (Ribeiro, et al., 2006), as well as in vivo (Garaiova, et al., 2007). For preparing such O/W emulsions, the lipophilic bioactives are dissolved in an oil phase and subsequently emulsified with an

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aqueous phase containing an emulsifier to stabilize the oil droplets. Such substances in the form of fine droplets have a better water dispersibility compared to those in bulk form (Neves, et al., 2008b). Among the various emulsification processes currently in use, traditional emulsification devices (e.g., colloid mills and high-pressure homogenizers) apply shear, extension, and impact to the systems, resulting in the production of polydisperse emulsions. On the other hand, less energy-intensive emulsification techniques have been under development, such as MC emulsification, a unique and very mild process based on spontaneous droplet formation (Kawakatsu, et al. (1997). Most recently, straight-through MC, which is an array of channels vertically microfabricated on a silicon chip, generally producing monodisperse droplets with average droplets diameter lower than 30 m and considerable throughput capacity, has been under development for mass producing uniformly sized droplets (Kobayashi, et al., 2012). On this concern, the authors have investigated the formulation of O/W emulsions loaded with bioactive molecules, such as -carotene, prepared using straight-through microchannel (MC) arrays.. We also evaluated the formulation of size-controlled O/W emulsions loaded with -3 PUFAs using MC emulsification. In this case, the effect of various levels of disperse phase flux and continuous phase flow velocity on the droplet formation was investigated. Moreover, the effect of lipid structuring on the oxidative stability of fish O/W nanoemulsions prepared by high-pressure homogenization was also evaluated. Most recently, scaling up of MC arrays has been under development, foreseeing potential applications in cosmetics or chemical industries as well.

2. Material and Methods 2.1 Encapsulation of bioactive compounds using microchannel emulsification Refined soybean oil incorporating the lipophilic molecule (3.2 g/L of -carotene) was dispersed in a continuous water phase containing sucrose monolaurate. The disperse phase was pressurized through the asymmetric straight-through MC, and dispersed into the continuous phase producing an O/W emulsion. Upon formulation, the average droplet size (dav) and coefficient of variation (CV) were measured, in order to evaluate the efficiency of emulsification process and emulsion stability upon storage.

2.2 Effect of lipid structuring on the oxidative stability of fish O/W nanoemulsions Aiming to retard lipid oxidation in O/W nanoemulsions, we evaluated the effectiveness of structuring PUFA-rich fish oil with tripalmitin, a long-chain high MP (66°C) saturated triglyceride, which was mixed with Menhaden fish oil (weight ratio 1:1). The continuous phase contained 1 wt% decaglycerol monolaurate (ML750) in Milli-Q water. Pre-mixtures containing oil and water phases (ratio 1:9 w/w) were prepared using a rotor-stator (5,000 rpm, 5 min), and homogenized using a high-pressure homogenizer (150 MPa, 1 cycle) at room temperature or at 70°C. Nanoemulsions were stored either at 5 or 30 °C, meanwhile lipid hydroperoxides (LOOH) were monitored using ferric thiocyanate (McClements and Decker, 2000).

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3. Results and Discussion 3.1 Encapsulation of bioactive compounds using microchannel emulsification The emulsification process conducted using the MC array enabled the production of -carotene-loaded monodispersed O/W emulsions, with a dav of 27.6 m and CV 2.3% (Figure 1).

(a)

(b)

dav = 27.6 m CV = 2.3 %

30 m

50 m

Fig. 1 Micrographs of -carotene-loaded O/W emulsion obtained using straight-through MC emulsification. (a) Droplets generation; (b) Monodisperse oil droplets.

3.2 Effect of lipid structuring on the oxidative stability of fish O/W nanoemulsions The results indicated that both fish oil-based emulsions as well emulsions structured with tripalmitin had monomodal size distributions (results shown in Fig. 2, left-side) with average droplet size 130 nm. The microstructure of nanoemulsions containing structured lipids was observed under polarized light, revealing lipid crystals enclosed in the oil phase, which may formed as effect of by the cooling rate, directly related to lipid crystallization (Batte, et al., 2007). By structuring fish oil with tripalmitin, we were able to effectively retard lipid oxidation, with an actual difference from 190 to 55 mol/kg.

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Fig. 2 Left-side: Size distribution of fish O/W submicron emulsions freshly prepared using Nanomizer (operating pressure: 150 MPa; 1 cycle). Right-side: Oxidative stability of fish O/W nanoemulsions. Tripalmitin-based emulsion was used as control. All samples were stored at 5C (Neves et al., 2009).

4. Conclusion This paper outlined recent developments towards the development of highly monodisperse O/W emulsions loaded with bioactive compounds with precisely controlled droplet sizes and unique interfacial properties, by using MC emulsification which is a promising technique to produce lipid-based monodisperse emulsions. This feature might have a great potential for increasing the shelf life of food emulsions, as well as to stabilize O/W emulsions loaded with bioactive lipophilic molecules, which generally are very sensitive to oxidation and heat. MC emulsification is also a practically robust process, since the droplet size and its distribution of the resultant emulsions are basically insensitive to the flow of the dispersed and continuous phases. Optimization of processing conditions, choice of emulsifier, and other ingredients are the most important variables to achieve the desirable droplet size as well as suitable stability for each application. The fish oil-based nanoemulsions had monomodal size distributions with average diameter of 130 nm. The lipid oxidation was effectively retarded by structuring fish oil with a high melting point lipid.

Acknowledgements This work was partially supported by JST-JICA Science and Technology Research Partnership for Sustainable Development Program (SATREPS), Japan.

References Batte, H. D., Wright, A. J., Rush, J. W., Idziak, S, H, J. and Marangoni, A. G. (2007). Food Biophys., 2, 29. Donnelly, J.L., Decker, E.A. and McClements, D.J. (1998). J. Food Sci., 63, 997-1000. Garaiova, I., Guschina, I. A., Plummer, S. F., Tang, J., Wang, D. and Plummer, N. T. (2007). Nutr. J. 6, 1-9.

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Nakashima, T., Shimizu, M. and Kukizaki, M. (1991). Key Eng. Mater, 61/62, 513. Kawakatsu, T., Kikuchi, Y. and Nakajima, M. (1997). J. Am. Oil Chem. Soc., 74, 317-321. Kobayashi, I., Wada, Y., Neves, M. A., Uemura, K. and Nakajima, M. (2012). Green Proc. Synth., 1, 353. Neves, M. A., Fujiu, K., Ribeiro, H. S., Kobayashi, I. et al., (2008a). Ind. Eng. Chem. Res., 47, 6405-6411. Neves, M. A., Ribeiro, H. S., Kobayashi, I. and Nakajima, M. (2008b). Food Biophysics, 3, 126-131. Neves, M. A., Kobayashi, I. and Nakajima, M. (2009). J. Biosci. Bioeng., 108, S1, S137. Neves, M. A., Nakajima, M. and Kobayashi, I. (2012). J. Food Drug Anal. 20, S1, 184-188. Neves, M. A., Kobayashi, I., Ribeiro, H. S. et al. (2013). Bionanotechnology. Wiley & Sons Inc. p. 605. McClements, D. J. and Decker, E. A. J. Food Sci., 65, 1270-1282 (2000). Ribeiro, H. S., Guerrero, J. M. M., Briviba, K. et al. (2006). J. Agric. Food Chem., 54, 9366-9369 Ribeiro H. S., Schuchmann H. P. et al. (2009). Encapsulation of carotenoids. Springer, New York, p. 211. Thanonkaew, A., Benjakul, S., Visessanguan, W. and Decker, E.A. (2006). Food Chem., 95, 591–599.

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LFA 28

Antidiabetic effects of isoflavones, present in Japanese traditional soy foods, in cultured myocytes and type 2 diabetic model mice Sun Hee Cheonga,b, Keisuke Furuhashia, Katsuki Itoa, Masato Nagaokaa, Myoung Jin Sona Takayuki Yonezawac, Yutaka Miuraa & Kazumi Yagasakia,c (a) Department of Applied Biological Chemistry, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan; (b) Department of Biotechnology, Konkuk University, Chungju 380-701, Republic of Korea; (c) Graduate School of Medicine, The University of Tokyo, Bunkyo-Ku, Tokyo 113-8654, Japan E-mail: [email protected]

Abstract In this study, we investigated the in vitro effect of daidzein, an isoflavone in soy foods, and its metabolite equol on glucose uptake, AMP-activated protein kinase (AMPK) phosphorylation, and glucose transporter 4 (GLUT4) translocation to plasma membrane in L6 myocytes, and their in vivo antihyperglycemic effects in obese-diabetic model mice. Glucose uptake, AMP-activated protein kinase (AMPK) activation and glucose transporter 4 (GLUT4) translocation to plasma membrane were studied in cultured L6 myocytes by glucose uptake assay, Western blotting analyses. In addition, we tried to visually confirm GLUT4 translocation by immunocytochemical method using a GLUT4 cDNA-coding vector. Antidiabetic effects of daidzein and equol were studied in type 2 diabetic model mice. Daidzein and equol are found to promote glucose uptake, AMPK phosphorylation and GLUT4 translocation in cultured L6 myocytes under a condition of insulin absence. They suppressed the increases in blood glucose levels in either db/db, ob/ob or KK-Ay mice. Dietary supplemented daidzein also suppressed the increases in urinary glucose excretion in KK-Ay mice. Moreover, equol treatment improved the impaired glucose tolerance in ob/ob mice, and was demonstrated to improve expression of hepatic gluconeogenesis-related genes in terms of glucose metabolism. In conclusion, one of the mechanisms by which daidzein and equol suppress blood glucose levels is promotion of glucose uptake by muscle cells through GLUT4 translocation via AMPK signaling pathway.

Keywords: AMPK, Daidzein, Equol, GLUT4, Myocytes, Type 2 diabetes 1. Introduction: The total number of patients with type 2 diabetes (T2D) is increasing globally by population growth, aging, urbanization, and increasing physical inactivity and prevalence of obesity [1]. The skeletal muscles, which account for the majority of insulin-mediated glucose uptake in the post-prandial state, play an important role in maintaining glucose homeostasis. In skeletal muscles, insulin increases glucose uptake through a signaling that leads to activation of PI3K and Akt, resulting in increased translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Another GLUT4 translocation promoter is AMP-activated protein kinase (AMPK). If a certain compound could promote AMPK signaling in the absence of insulin, then the compound is considered to have a potential to overcome insulin resistance. We have recently reported concerning molecular mechanisms that genistein, an isoflavone present in soy bean-derived foods,

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possesses a potential to suppress rises in the blood glucose levels and to improve glucose intolerance inT2D model KK-Ay mice [2]. The present study attempted to elucidate the effects of daidzein, another isoflavone present in soy bean-derived foods, and its metabolite equol on glucose metabolism through the study on molecular mechanisms for glucose uptake using cultured L6 myocytes in vitro. To determine the effects of daidzein and equol on T2D in vivo, we examined their effects on blood glucose levels and glucose intolerance using db/db, KK-Ay and ob/ob mice.

2. Materials and Methods: L6 myoblasts were cultured and differentiated to myotubes [3], and then subjected to glucose uptake assay as described previously [4] (Figure 1). Phosphorylation (= activation) of AMPK and translocation of GLUT4 to plasma membrane in L6 myotubes were verified by Western blotting analyses [5].

In addition, we tried to visually confirm GLUT4 translocation in L6 myoblasts by immunocytochemical method using HaLoTag-glut4 expression vector that was constructed in our laboratory and transfected into L6 myoblasts [6]. Antidiabetic effects of daidzein and equol were studied in type 2 diabetic model KK-Ay, db/db and ob/ob mice. Blood glucose levels [4], intraperitoneal glucose tolerance test (IPGTT) [4] and urinary glucose excretion in KK-Ay mice [2] were measured as previously described. Hepatic gene expression of enzymes related to glucose metabolism was examined as described previously [7].

3. Results: Daidzein and equol were found to promote glucose uptake, AMPK phosphorylation and GLUT4 translocation by Western blotting analyses in L6 myotubes under a condition of insulin absence. Promotion by daidzein and equol of glucose uptake as well as GLUT4 translocation to plasma membrane by

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immunocytochemistry was also demonstrated in L6 myoblasts transfected with a HaLoTag-glut4 expression vector. Daidzein suppressed the rises in blood glucose levels in db/db and KK-Ay mice and urinary glucose excretion in KK-Ay mice, while daidzein promoted AMPK phosphorylation in gastrocnemius muscles of db/db mice. Daidzein significantly suppressed HOMA-IR, an in vivo index of insulin resistance, in db/db mice. Equol suppressed the increase in blood glucose levels and improved glucose intolerance in ob/ob mice. Equol also tended to decrease HOMA-IR in ob/ob mice. Furthermore, equol treatment was demonstrated to improve expression of hepatic gluconeogenesis-related genes in terms of glucose metabolism.

4. Discussion: One of the mechanisms by which daidzein and equol suppress blood glucose levels is promotion of glucose uptake by muscle through GLUT4 translocation via AMPK signaling pathway like other phytochemicals [8]. Effective dose of equol seems to be lower than that of daidzein at both in vitro and in vivo levels.

5. Conclusion: Daidzen and equol are demonstrated to have antidiabetic potential through not only GLUT4 translocation via AMPK signaling pathway but also normalization of gene expression of enzymes related to glucose metabolism. From these findings, daidzein [9] and equol [10] are preventive and in some case reductive against T2D.

Acknowledgements: This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) and in part by a grant from the Takano Life Science Research Foundation, Japan.

References: 1. Wild, S., Roglic, G., Green, A., Sicree, R. and H. King (2004) Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care. 27, 1047-1053. 2. Ha, B.G., Nagaoka, M., Yonezawa, T., Tanabe, R., Woo, J.T., Kato, H., Chung U.I., and K. Yagasaki (2012) Regulatory mechanism for the stimulatory action of genistein on glucose uptake in vitro and in vivo. J. Nutr. Biochem. 23, 501-509. 3. Yagasaki, K., Morisaki, N., Kitahara, Y., Miura, A. and R. Funabiki (2003) Involvement of protein kinase C activation in L-leucine-induced stimulation of protein synthesis in l6 myotubes. Cytotechnology. 43, 97-103. 4. Kawano, A., Nakamura, H., Hata, S., Minakawa, M., Miura, Y. and K. Yagasaki (2009) Hypoglycemic effect of aspalathin, a rooibos tea component from Aspalathus linearis, in type 2 diabetic model db/db mice. Phytomedicine. 16, 437-443.

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5. Minakawa, M., Kawano, A., Miura, Y. and K. Yagasaki (2011) Hypoglycemic effect of resveratrol in type 2 diabetic model db/db mice and its actions in cultured L6 myotubes and RIN-5F pancreatic β-cells. J. Clin. Biochem. Nutr. 48, 237-244. 6. Minakawa, M., Miura, Y. and K. Yagasaki (2012). Piceatannol, a resveratrol derivative, promotes glucose uptake through glucose transporter 4 translocation to plasma membrane in L6 myocytes and suppresses blood glucose levels in type 2 diabetic model db/db mice. Biochem. Biophys. Res. Commun. 422, 469-475. 7. Son M.J., Minakawa, M., Miura, Y. and K. Yagasaki (2013) Aspalathin improves hyperglycemia and glucose intolerance in obese diabetic ob/ob mice. Eur. J. Nutr. 52, 1607-1619. 8. K. Yagasaki (2013) Anti-diabetic phytochemicals that promote GLUT4 translocation via AMPK signaling in muscle cells. Nutr. Aging, in press. (doi: 10.3233/NUA-130032) 9. Cheong, S.H., Furuhashi, K., Ito, K., Nagaoka, M., Yonezawa, T., Miura, Y. and K. Yagasaki (2013) Daidzein promotes glucose uptake through glucose transporter 4 translocation to plasma membrane in L6 myocytes and improves glucose homeostasis in type 2 diabetic model mice. J. Nutr. Biochem. in press. 10. Cheong, S.H., Furuhashi, K., Ito, K., Nagaoka, M., Yonezawa, T., Miura, Y. and K. Yagasaki (2013) Antihyperglycemic effect of equol, a daidzein derivative, in cultured L6 myocytes and ob/ob mice. Mol. Nutr. Food Res. in press. (doi: 10.1002/mnfr.201300272).

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LFA 29

In-vitro model for axon pruning: a phosphatidylserine independent pathway Insaf Bahrinia and Rikinari Hanayamaa (a) Laboratory of Immune Network, WPI-Immunology Frontier Research Center, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Tel: +81-6-6879-4936; Fax: +81-6-6879-4935; E-mail: [email protected]

Abstract Selective elimination of neural processes, known as axon pruning is a common phenomenon which occurs during both development and pathological conditions such as injury and neurodegenerative diseases. Previous studies demonstrate that the engulfing action of glia cells is essential in this process. However, the molecular mechanisms underlying axon pruning are largely unknown. Recent findings raised the possibility that axon pruning and apoptotic cell engulfment may share common molecular machinery for their removal. Phagocytes engulf apoptotic cells after recognition of PS expressed on their surface. We aimed to examine whether PS exposure on degenerating axons triggers their engulfment by glia cells. We showed that Microglia cells promote the removal of axons undergoing degeneration via a pathway distinct from that of PS recognition. Preliminary results showed that microglial phagocytic activity might be mediated by exosomes.

Keywords: PC12 cells, microglia, NGF, Phosphatidylserine, axon pruning, phagocytosis, exosomes. 1. Introduction The rat pheochromocytoma PC12 cell line has been a valuable model for studying the neurotrophic and differentiating effects of nerve growth factor (NGF, reviewed in Levi and Alemá, 1991). Exposure of PC12 cells to NGF results in their conversion from immature adrenosympathetic precursor-like cells to cells that resemble mature sympathetic neurons (Greene and Tischler, 1982). Dead, dying, or aged cells display eatme signals, such as phosphatidylserine (PS) on their surface (Fadok et al., 1998). Viable but stressed cells can reversibly expose PS (Tyurina et al., 2007). Exposed PS is bound by extracellular adaptor proteins, including milk-fat globule EGF factor-8 (MFG-E8, also known as lactadherin) via a C2 domain and via a RGD domain to the vitronectin receptor on phagocytes, thereby activating phagocytosis (Hanayama et al., 2002). A point mutation in the RGD motif of MFG-E8 (D89E) completely masks the PS on apoptotic cells. Recent findings raised the possibility that axon pruning and apoptotic cell engulfment may share common molecular machinery for their removal (Hosmane et al., 2012).

2. Experimental methods PC12 cells were maintained in DMEM medium containing 10% FCS, 10% heat-inactivated horse serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Neuronal PC12 cells were cultured on glass slides precoated with Polyethyleneimine (PEI, Sigma). Axonal differentiation was induced by treatment with NGF (recombinant rat β-NGF, R&D Systems) at a concentration of 100ng/ml for 10 d in serum-free DMEM. Fresh medium and NGF were added every 2 d. NGF withdrawal was realized by washing cells 3 times with complete medium. For apoptosis induction, medium was replaced with serum-free medium without NGF. Apoptosis inhibitor QVD (Calbio) was added to degeneration medium at 2 μM as control. Cells were fixed using 1% paraformaldehyde (PFA) and mounted with coverslips for microscope observation.

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2.1. Annexin staining PS exposure was detected by Annexin V binding. PC12 cells were cultured in 35 mm glass bottom culture dishes pre-coated overnight with 2 μg/cm2 rat tail collagen type I (Roche). Cells were washed 3 times with complete medium. Then, 5 µl of FITC-Annexin V (BioLegend) in 500 µl of complete medium without phonol was added into each coverslip to make the staining solution. Cells were observed directly using multi-area time lapse confocal microscopy. 2.2. Phagocytosis assay Mouse microglial cell line MG6, mouse embryonic fibroblast cell line NIH-3T3 and mutant NIH-3T3 (NIH-3T3-TIM4) were used for phagocytosis assays. PC12 cells were cultured in presence of NGF for 10 d. After NGF withdrawal, PC12 cells were incubated with either MG6 cells at a density of 5.104 cell/cm2 or NIH-3T3 or NIH-3T3-TIM4 at 1.104 cell/cm2 in presence (20 µg/ml) or in absence of D89E protein. 2.3. Mechanisms of axon pruning In order to investigate the mechanisms by which MG6 cells promote axonal pruning, PC12 cells were incubated in presence of MG6 conditioned medium overnight. Exosome secretion from neuronal PC12 cells was induced by depolarization by adding 25 mM KCl as described by Fauré et al. (2006). Exosomes were purified from the culture medium by ultracentrifugation (100000 g for 2 h) and labeled with PKH26 red fluorescent dye (Sigma) according to manufacturer’s instructions. Purified exosomes were characterized by immunoblotting using anti-flotillin-2 antibody. Labeled exosomes were added to MG6 culture and incubated overnight. The next day, MG6 cells were trypsinized and added to PC12 cells.

3. Results and Discussion 3.1. Model for the role of glia in axon pruning When PC12 cells are exposed to NGF, a progressive increase in neurite length was observed. The cell process longer than the cell body diameter was considered as neurite. Maximum induction occurred between 7 and 10 days and corresponds to about 80% of neurite-bearing cells. In absence of NGF and in presence of serum, cells showed positive staining with Annexin V (Fig. 1). Apoptosis inhibitor QVD added to PC12 culture didn’t have any effect on the de-differentiation in the absence of NGF (data not shown). As shown in Figure 2, axonal degeneration was markedly enhanced by the presence of MG6 cells. Addition of MFG-E8/D89E that has a very high affinity for PS didn’t prevent axonal degeneration in presence of MG6 cells. Moreover, the incubation with NIH-3T3-TIM4 cells, which have a high capacity to phagocytose apoptotic cells, didn’t have any effect on axons (Fig. 2). 30 min

2 h 30 min

4 h 30 min

Fig.1. FITC Annexin-V staining of PC12 cells at 2h interval using live imaging confocal microscopy (10).

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A

B

Fig.2. Morphology of neuronal PC12 cells after NGF withdrawal and co-culture with MG6 cells, NIH-3T3 cells (3T3) and NIH-3T3-TIM4 cells (Tim4). Average neurite length (A) was estimated from 10 neurite-bearing cells in four different areas. Percentage of neurite-bearing cells (B) and average neurite length were quantified from two independent experiments. Control and w/o NGF correspond resp. to cultures in presence and in absence of NGF.

Mechanisms of axon pruning The incubation of PC12 cells in presence of MG6 cells conditioned medium didn’t have any effect on neurite length and number of neurite-bearing cells. Live imaging of phagocytosis indicated that MG6 cells migrate to axons undergoing degeneration and promote their engulfment and/or retraction by engulfing axonal debris (data not shown). Our preliminary results demonstrated that exosomes secretion from PC12 cells was induced by depolarisation and that purified exosomes were engulfed by MG6 cells after overnight incubation (Fig. 3). C

KCl

Flotillin

Fig.3. Purification of exosomes from PC12 cell cultures. Top panel: Exosome secretion is stimulated by depolarization. C and KCl correspond respectively to exosomes purified from PC12 cells in absence of KCl and 30 min after addition of 25 mM KCl. Bottom panel: MG6 cells engulfing exosomes (labeled with red) observed by confocal microscopy (60).

4. Conclusion Here, we have demonstrated that PC12 cells can be used as a good model to study the mechanisms of axon pruning. The presence of MG6 in culture accelerated axon retraction and/or degeneration in absence of NGF. Live imaging results confirmed that glia cells engulf degenerating axons (Watts et al., 2004). However axon undergoing degeneration expressed phosphatodylserine, this mechanism is distinct from cell

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death. MFG-E8 was recently reported to have an important role in phagocytic activity of microglial cells in response to apoptotic cells (Liu et al., 2013). Blocking PS with MFG-E8 didn’t prevent axon removal by MG6 cells. We also showed that the action of MG6 is not mediated by a soluble factor secreted by MG6 cells. Interestingly, exosomes-engulfed MG6 had an enhanced phagocytic activity, suggesting a role for exosomes in phagocytic property of microglial cells. Exosomes are microvesicles of endosomal origin secreted by cells including neurons (Fauré et al., 2006). Exosomes have been implicated in cell-to-cell communication (Valadi et al., 2007). Taken together, this study provide the framework to further investivate the mechanisms underlying axon-glia intereactions during axon puning.

References Fadok, V.A., Bratton, D.L., Konowal, A., Freed, P.W., Westcott, J.Y. and P.M. Henson (1998) Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF. J. Clin. Invest. 101, 890-898. Fauré, J., Lachenal, G., Court, M., Hirrlinger, J., Chatellard-Causse, C., Blot, B., Grange, J., Schoehn, G., Goldberg, Y., Boyer, V., Kirchhoff, F., Rapaso, G., Garin, J. and R. Sadoul (2006) Exosomes are released by cultured cortical neurones. Mol. Cell. Neurosci. 31, 642-648. Greene, L.A. and A.S. Tischler (1982) PC12 pheochromocytoma cultures in neurobiological research. Adv. cell. Neurobiol. 3, 373-414. Hanayama, R., Tanaka, M., Miwa, K., Shinohara, A., Iwamatsu, A. and S. Nagata (2002) Identification of a factor that links apoptotic cells to phagocytes. Nature 417(6885), 182-187. Hosmane, S., Tegenge, M.A., Rajbhandari, L., Uapinyoying, P., Kumar, N.G., Thakor, N. and A. Venkatesan

(2012) Toll/interleukin-1 receptor domain-containing adapter inducing interferon-β

mediates microglial phagocytosis of degenerating axons. J. Neurosci. 32(22), 7745-7757. Levi, A. and S. Alemá (1991) The mechanism of action of nerve growth factor. Annu. Rev. Pharmacol. Toxicol. 31, 205-222. Liu, Y., Yang, X., Guo, Ch., Nie, P., Liu, Y. and J. Ma (2013) Essential role of MFG-E8 for phagocytic properties of microglia cells. Plos One 8(2), 1-8. Tyurina, Y.Y., Basova, L.V., Konduru, N.V., Tyurin, V.A., Potapovich, A.I., Cai, P., Bayir, H., Stoyanovsky, D., Pitt, B.R., Shvedova, A.A., Fadeel, B. and V.E. Kagan (2007) Nitrosative stress inhibits the aminophospholipid translocase resulting in phosphatidylserine externalization and macrophage engulfment: implications for the resolution of inflammation. J Biol Chem. 282, 84988509. Valadi, H., Ekström, K., Bossios, A., Sjöstrand, M., Lee, J.J. and J.O. Lötvall, J.O. (2007) Exosomemediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 9, 654-659. Watts, R.J., Schuldiner, O., Perrino, J., Larsen, C. and L. Luo (2004) Glia engulf degenerating axons during developmental axon pruning. Curr. Biol. 14, 678-684.

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LFA 30

Specific induction of apoptosis by 1, 8-cineole in two human colon cancer cells Soichiro Murata1, 2, Risa Shiragami1, Chihiro Kosugi1, Tohru Tezuka1, Masato Yamazaki1, Atsushi Hirano1, Yukino Yoshimura1, Kiyohiko Shuto1, Nobuhiro Ohkohchi2, Keiji Koda1 1. Department of Surgery, Teikyo University Chiba Medical Center, 3426-3 Anesaki, Ichihara, Chiba, 299-0111, Japan. 2. Department of Surgery, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan. E-mail: [email protected]

Abstract: Aims of the work: Several essential oils possess pharmacological effects. Among the various constituents of essential oils, 1, 8-cineole has been shown to possess pharmacological effects such as anti-bacterial and anti-inflammatory effects. The effect of 1, 8-cineole on human colorectal cancer cells, however, has not reported previously. The aim of this work is to evaluate the anti-tumor effect of 1, 8-cineole on human colorectal cancer cells in in vitro and in vivo. In this study, we have investigated the anti-proliferative effect of 1, 8- cineole on human colon cancer cell lines HCT116 and RKO by WST-8 and BrdU assays. The cytotoxicity of 1, 8-cineole was investigated by LDH activity and TUNEL staining. The mechanism of apoptosis by 1, 8-cineole was determined by western blot analyses. In in vivo study, RKO cells were injected to the SCID mice and the effect of 1, 8-cineole was investigated. Specific induction of apoptosis, not necrosis, was observed in human colon cancer cell lines HCT116 and RKO by 1, 8-cineole. 1, 8-cineole treatment was associated with inactivation of survivin and Akt and activation of p38. These molecules induced cleaved PARP and caspase 3, finally causing apoptosis. In xenotransplanted SCID mice, the 1, 8cineole group significantly inhibited tumor progression compared to the control group. These results indicated 1, 8-cineole suppressed human colorectal cancer proliferation by inducing apoptosis. Based on these studies 1, 8-cineole would be an effective strategy to treat colorectal cancer.

Keywords : 1, 8-cineole, colorectal cancer, apoptosis, HCT116, RKO 1. Introduction : The use of plant-derived natural products for medicinal benefits has played an important role almost all over the world. In cancer therapy, the focus is on strategies that suppress tumor growth through cell cycle disruption [1] and activate the apoptotic program in the cell [2]. Several essential oils from plants possess medical benefits. Among the various constituents of essential oils, 1, 8-cineole has been shown to possess pharmacological effects. The content of 1, 8-cineole in the essential oils varies in the different Eucalyptus species, from 25 to 90% [3, 4]. 1, 8-cineole has been used as a percutaneous penetration enhancer [5], antiinflammatory [6, 7] or antihypertensive [8] agent. 1, 8-cineole was reported to induce apoptosis in leukemia cell lines [9]. In this study, we found that 1, 8-cineole exerts antitumor activity on human colon cancer cell lines HCT116 and RKO. We investigated whether 1, 8-cineole induces apoptosis in two human colorectal cancer cell lines and in a xenograft model.

2. Material and Methods: 2.1 Reagents. 1, 8-cineole (eucalyptol) was obtained from Sigma (St. Louis, MO, USA). For cell culture experiment, 1, 8-cineole was dissolved in ethanol and then in water at a concentration of 0.1 mg/mL. In an in vitro experiment, 5-50mM of 1, 8-cineole was added to the medium, and cell viability assay and western blot experiments were performed 24 h later. 2.2 Cells. The human CRC cell lines HCT116 and RKO were used. Stock cultures were grown in highglucose DMEM containing 10% FBS and 1% antibiotics. The cells were grown in growth medium at 37°C in a 95% air, 5% CO2-humidified incubator. 2.3 Cell viability assay. To measure the cytotoxicity of 1, 8-cineole against these cancer cells, 3×103 cells were plated per well onto 96-well plates. Following overnight culture, 1, 8-cineole and oxaliplatin were added at specified concentrations. After 24 h of incubation, cell viability was measured by the WST-8 to formazan using a Cell Counting kit-8 (Dojindo Laboratories, Kumamoto, Japan). 2.4 Cell proliferation assay. To measure the cell proliferation activity of 1, 8-cineole against HCT116 and RKO cancer cells, 3×103 cells were plated per well onto 96-well plates. Following overnight culture, 1, 8cineole was added and after 24 h of incubation, cell proliferation was measured with a BrdU assay kit (Roche Diagnostics, Penzberg, Germany). 2.5 Lactate Dehydrogenase (LDH) Activity Assay. In order to evaluate the activity of the cytoplasmic enzyme lactate dehydrogenase (LDH) released from the cytosol when cells were damaged under stress,

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HCT 116 and RKO cells were seeded (1×105 cells/mL) on 96-well plates. The LDH activity was determined using a commercial kit (Takara Bio, Tokyo, Japan). 2.6 Apoptosis assay. The In situ Cell Death Detection kit (Roche diagnostics, Basel, Switzerland) was used for the demonstration of apoptotic cell death of cell culture. 3×10 4 cells were plated per well and were incubated with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction mixture according to the manufacturer’s recommendations. 2.7 Western blot analysis. For western blot analysis, total protein extracts of HCT116 cells were obtained 24 h after 1, 8-cineole treatment, and separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. All antibodies were purchased from Cell Signaling Technology, Beverly, MA, USA. 2.8 Animals. Seven-week-old male Severe Combined Immunodeficiency (SCID) mice (Clea, Tokyo, Japan), weighing 24-28 g, were utilized. The mice were separated into two groups as follows: control group, mice without any treatment (n=10); and 1, 8-cineole group, mice with 1, 8-cineole treatment (n=9). 2×106 cells of RKO were injected subcutaneously into the right flank of each mouse with a 27-gauge needle. Tumors were detected by palpation and measured periodically with calipers. Seven days after tumor injection, 50 mg/kg of 1, 8-cineole was injected subcutaneously every 3 days. Twenty one days after inoculation, mice were sacrificed and tumors were removed for examination. 2.9 Statistical analysis. All data are expressed as the mean ± SD of samples. Comparisons between various points were made using one-way ANOVA. Comparisons between the two groups were made using MannWhitney U-test. Significant data were examined by the Bonferroni–Dunn multiple comparisons post-hoc test. In all cases, a P-value 90% were assumed to belong to the same pathotype. For AFLP analysis, the total population and within the subpopulations (Hs) thus: DST = Ht – Hs (Nei, 1973). Population differentiation (Gst) was calculated from gene diversity between subpopulations (DST) and gene diversity in the total population (ht) as given by Gst = DST/ht. All the above calculations were undertaken by POPGENE 1.32 (Yeh et al., 1995).

3. Results and Discussion Genetic diversity A total of 401 discernible and polymorphic AFLP bands were generated with six primer combinations selected across the 78 isolates of 17 Tunisian populations of P. teres f. teres. Primers varied in their ability to detect variation at both within and between populations. The within populations HS varied from 0.127 for E11XM50 primer to 0.194 for E11XM47 primer. Evaluation of the genetic diversity of P. teres isolates from Rihane and local landraces revealed high genotypic diversity based on AFLP markers. Genotypic diversity of all populations ranged from 0.75 to 0.88 (with average of 0.80). Primers varied in their ability to detect variation at both within and between populations. Most of the gene diversity was found within populations (60%) with only 36% of gene diversity accounting to be among P.teres populations. The overall genetic differentiation (Gst) across all the populations was moderate (0.398) with moderate Nm value of