Bioaccumulation of Heavy Metals by Non-living Rhodococcus Erythropolis B4.

Transcription

1 2014 5th International Conference on Food Engineering and Biotechnology IPCBEE vol.65 (2014) (2014) IACSIT Press, Singapore DOI: /IPCBEE V65. 2 Bioaccumulation of Heavy Metals by Non-living Rhodococcus Erythropolis B4. A. Djefal-Kerrar 1+, K. Abdoun-Ouallouche 1, L. Khadraoui 2, A. Belounis 2. 1 Division of Nuclear Applications, Nuclear Research Centre of Algiers. 02, Bd Frantz Fanon, Algiers, Algeria. 2 Faculty of Science, M'Hamed Bougara University of Boumerdes, Algeria. Abstract. The sorption of lead and mercury ions from aqueous solutions by dead biomass of Rhodococcus erythropolis B4 was investigated in the batch mode. The influence of initial ph, initial concentration of ions and contact time were studied. The metal concentration was analyzed by Atomic Absorption Spectrophotometry (AAS). Analyses by The Fourier Transform Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray (EDX) were performed to show the interactions between cells and metals ions. Maximum sorption capacities of lead and mercury were found to be 75 mg.g -1 and 300mg.g -1 respectively. The Langmuir and Freundlich models were applied to the experimental data and the Langmuir model was found to be in better correlation with experimental data. Competitive biosorption experiments were performed with Pb 2+ together with Hg 2+. Keywords: waste-water treatment; Biosorption; Rhodoccocus erythropolis; Modelling; Adsorption; Heavy metals. 1. Introduction The pollution of the environment with toxic heavy metals is spreading through the world along with industrial progress [1], [2]. The toxicity produced by lowest concentrations of heavy metals ions in industrial wastewaters is a subject of public concerns, since these ions can reach food chain and persist in nature [3]. According to the water standards used in most countries, levels of heavy metals ions in waste waters must be controlled and reduced to permissible limits [4]. Several methods are available for removing heavy metals, such as chemical precipitation, membrane filtration and ion exchange [2]-[5]. Since these traditional methods are often ineffective and/or very expensive when used for removal of heavy metals at very low concentrations, using of microorganisms offers a potential alternative[3], [4]. Biological process for removal of metal ions from liquids can be done by accumulation by viable microorganisms or by adsorption onto dead microorganism s surface [6]. Biosorption by dead biomass is relatively rapid and can be reversible. It involves physicochemical interactions between the metal and functional groups as ketones, aldehydes, carboxyls present on the microorganism s surface [2]-[7]. Among the group of bacteria, we can distinguish Gram positive and Gram negative. Cell wall of Gram negative bacteria which contains peptidoglycan are somewhat thinner and also not heavily cross-linked like the Gram positive ones which contains moreover teichoic acids, thus Gram positive bacteria have more potential binding sites for metal ions and are better biosorbent [8], [9]. The Rhodococcus genus, an aerobic gram positive, non motile, mycolate containing and belonging to the class of actinomycetes has a considerable importance in biotechnology applied to the environment, because of its high metabolic diversity and his wide range of enzymatic capacities[1]-[10]. + Corresponding author. Tel: ; Fax: address : kerasdjef@yahoo.fr 7

2 q e (mg.g -1 ) The objective of this work was the study the adsorption capacity of dead biomass of Rhodococcus erythropolis B4 strain for lead and mercury, as a function of initial ph, initial metal ions concentrations and contact time. 2. Material and Methods 2.1. Microorganism and growth conditions Rhodococcus erythropolis B4 strain was grown at 30 C under agitation of 150 rpm, in a liquid medium containing 10 g l -1 glucose, 5g.l -1 peptone, 3g.l -1 yeast extract as well as 3 g. l -1 malt extract. Biomass was harvested after 24 h of incubation Metal solutions Lead and mercury solutions were obtained by dissolving an accurate quantity of lead acetate (CH 3 COO) 2 Pb, 3H 2 O, and mercury chloride (HgCl 2 ) in deionized water to obtain stock solutions of 1g l Batch biosorption experiment Factors affecting lead and mercury adsorption rate and biosorption capacity by Rhodococcus erythropolis B4 were examined in a batch system. Kinetics of biosorption, isotherms of adsorption and influence of ph on the biosorption capacity of biomass were studied. The concentrations of non adsorbed metal ions by Rhodococcus erythropolis B4 biomass were determined by means of AAS. Analysis by FTIR, SEM and EDX were performed to show interactions between cells and metals ions. 3. Results and Discussion 3.1. Effect of ph The results presented in figure 1 show an adsorption increase with increasing ph values for both metals. At very acidic ph (2 and 3) we observe a low yield of biosorption, about 3% and 5% for Pb and 7% and 17% for Hg. It s only at ph 4 that we observe a significant biosorption increase of about 78% for Pb with a capacity of mg g -1 and 85% for Hg with a capacity of mg g -1 respectively. This allows us to say that the biosorption of metals by dead biomass of Rhodococcus erythropolis is strongly dependent on ph. 160 Pb Hg ph Fig. 1: Effect of ph on the biosorption capacity of Pb and Hg by dead biomass of Rhodococcus erythropolis (agitation speed: 150 rpm, weight of biomass: 0.01g, initial concentration of Pb and Hg: 10 mg l -1, room temperature, contact time: 5h) The Gram-positive cell walls are composed of linear polymers of peptidoglycan covalently tied together around the cell membrane [11]. The peptidoglycan forms a carboxyl and hydroxyl rich giant macromolecule [11]. The presence of these acid groups, especially the carboxylic ones, confers to the surface a very phdependent charge [12] Effect of contact time initial metals concentration 8

3 q e (mg.g -1 ) qe (mg.g -1 ) The results presented in figure 2 show that more than 52 % of Hg and 92 % of Pb are absorbed only after 15 min of contact. The maximal elimination takes place after 5 hours of contact for Pb (97%) with a capacity of mg g -1 and for Hg (93%) with a capacity of mg g -1. In Fig. 3, it is shown that the increase of initial metal concentrations results in an increase of the biosorption capacity for both Pb and Hg. This behavior is explained by the fact that for high metals concentrations, there are a high number of ions in solution, implying thus a high adsorption capacity. Dursun (2006) [4] reported that the initial metal concentration provides a driving force to overcome mass transfer resistances between the biosorbent and biosorption medium. A stable biosorption capacity is observed beyond 75 mg l -1 concentration for Pb and 300 mg l -1 for Hg. This stability is due to the insufficient availability of sites for sorption in comparison to the number of molecules to be adsorbed. The phase of saturation is thus attained [13], [14] Pb Hg Pb Hg contact time (min) C 0 (mg.l -1 ) Fig. 2: Effect of contact time on the biosorption capacity of lead and mercury from dead biomass of Rhodococcus erythropolis (agitation speed: 150 rpm, biomass weight: 0.01g, initial concentration of Pb and Hg: 10 mg l -1, ph: 5, room temperature). Fig. 3: Effect of initial concentration of metallic solutions on the biosorption capacity of Pb and Hg by dead biomass of Rhodococcus erythropolis (agitation speed: 150 rpm, weight of biomass: 0.01g, ph: 5, room temperature, Contact time: 24) SEM and EDX analysis SEM micrographs and EDX spectra obtained before and after contact of the dead biomass of Rhodococcus erythropolis with Pb and Hg are presented in figures 5 and 6. a b c Fig. 4: Micrographs of the dead biomass of Rhodococcus erythropolis observed by SEM before (a) and after contact with the metal ions Pb (b) and Hg (c) (x G 12000). The coccobacillar form of Rhodococcus erythropolis cells is well seen by SEM observations in Fig. 4. We distinguish a clear and distinct cluster of morphologically uniform cells (fig. 5a). These micrographs do not clearly reveal the presence of new particles on the cell surface after contact (Fig. 5 b and c). However, EDX analysis confirms the adsorption of metal ions. The characteristic peaks of Pb and Hg appear distinctly 9

4 and confirm that the biomass has well fixed the metal ions (Fig.6). a b c Fig. 5: Dead biomass of Rhodococcus erythropolis analyzed by EDX before (a) and after contact with Pb metal ions (b) and Hg metal ions (c) FT-IR analysis The FT-IR spectra of Rhodococcus erythropolis before and after contact with Pb and Hg are presented in Fig. 7. a b 1403,6 1448,8 1033,9 1077,9 1316,1 637,8 Absorbance 3708,8 3737,1 1241,1 2854,1 Absorbance 1241,1 637,8 1403,6 3575,9 2924,7 1733,5 1549,5 2924,7 1448,8 1033,9 1549,5 3293,8 1660,9 1627,8 1637,5 1643,7 1077,9 1637,5 3293,8 1660,9 0,80 Biomasse morte + Pb Biomasse morte 0,75 Biomasse morte + Hg Biomasse morte 0,75 0,70 0,70 0,65 0,65 0,60 0,60 0,55 0,55 0,50 0,50 0,45 0,45 0,40 0,40 0,35 0,35 0,30 0,30 0,25 0,25 0,20 0,20 0,15 0,15 0,10 0,10 0,05-0,00 0, Nombre d'onde (cm-1) Nombre d'onde (cm-1) Fig. 6: FT-IR spectrum of dead biomass of Rhodococcus erythropolis before and after contact with Pb (a) and Hg ions (b). The model of the IR biomass spectrum shows a distinct and a strong absorption at 3575 and 3293 cm -1 indicative of the existence of OH groups and NH groups [15]. The absorption peak at 2924 cm -1 can be assigned to a C H group and the absorption peak at 1733 cm -1 is indicative of the C=O group. The absorption peak at 1077 cm -1 can be assigned to a C OH group [16] and the peak at 637 cm -1 represents the group C SH 2. According to the obtained results, no difference is observed between the Rhodococcus erythropolis spectra before and after contact with the metal ions except that the absorbance of the peaks in the Pb and Hg loaded biomass is slightly lower than in the native one which indicate that there is a metal binding process taking place on that surface of the biomass Modeling of adsorption The equilibrium sorption isotherms are one of the most important data to understand the mechanism of the biosorption (Fig. 8 a and b). Results (Table 1) indicate that the Langmuir model describes well the data of lead and mercury equilibrium adsorption by dead biomass of Rhodococcus erythropolis. Maximum loading capacities obtained are 95,23 mg.g -1 for lead and 147, 05 mg.g -1 for mercury.these values approximate the experimental ones which are 98, 43 mg.g -1 for Pb and 134, 07 mg.g -1 for Hg. Lead and mercury adsorption occurs on a homogeneous surface by monolayer sorption with interactions between adsorbed molecules. 4. Conclusion It is demonstrated that dead biomass of Rhodococcus erythropolis B4 is a potential candidate for biosorption of lead and mercury from aqueous solutions. The process is ph, initial metal concentration and 10

5 Ce/qe (g.l -1 ) Logqe contact time dependant. Sorption of Pb and Hg on this strain is found to follow a monolayer type of adsorption. The experimental data fit well to the Langmuir isotherm model. 2,5 2,0 Hg Pb 2,2 2,0 Hg Pb 1,5 1,8 1,0 0,5 0, Ce(mg.l -1 ) 1,6 1,4 1,2 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 2,2 2,4 2,6 Log Ce Fig. 7a: Linear transformation of the Langmuir biosorption of lead ( ) and mercury ( ) by dead biomass of Rhodococcus erythropolis B4 Fig. 7b: Linear transformation of the Freundlich biosorption of lead ( ) and mercury ( ) by dead biomass of Rhodococcus erythropolis B4 Table 1: Adsorption constants obtained from Langmuir and Freundlich models of dead biomass of Rhodococcus erythropolis B4. Adsorbat Langmuir model Freundlich model q max (mg. g -1 ) b (l. mg -1 ) R 2 K F (mg.g -1 ) n R 2 Pb 2+ 95,23 0,085 0,94 4,75 5,37 0,60 Hg ,05 0,040 0,98 3,38 2,56 0,69 5. References [1] H. Aurelio, A. Jaureguibeitia, M. Begona Prieto, R. F. Concepcion, J. L. Serra and MJ, Llama, Biological treatment of phenolic industrial wastewaters by Rhodococcus erythropolis UPV-1. Enzyme and Microbial Technology 31(2002) [2] B. Bueno, M.Torem, F.Molina and L.Mesquita, Biosorption of lead (II) and copper (II) by R.opacus: Equilibrium and Kinetic studies. Minerals Engineering 21 (2008) [3] C. J. E. Basurco, R. J.deCarvalho, M. L. Torem, Evaluation of equilibrium, kinetic and thermodynamic parameters for biosorption of nickel(ii) ions onto bacteria strain, Rhdococcus opacus. Minerals Engineering. 22 (2009) [4] A. Y. Dursun, G. Uslu, O. Tepe, Y. Cuci, H. I. Ekiz, A comparative investigation on the bioaccumulation of heavy meatl ions by growing Rhizopus Arrhizus and Aspergillus niger. Minerals Engineering 15 (2003) [5] I. Kiran, T. Akar, S. Tunali, Biosrption of Pb (II) and Cu (II) from aqueous solutions by pretraited biomass of Neurospora crassa. Process Biochemistry 40 (2005) [6] B. Preetha, T. Viruthagiri, Bioaccumulation of chromium(vi), copper(ii) and nickel(ii) ions by growing Rhizopus arrhizus, Biochemical engineering journal 34 (2007) [7] Y. Goksungur, S. Uren, U. Guvenç, Biosorption of cadmium and lead by ethanol treated waste baker s yeast biomass. Bioresource technology 96 (2005) [8] N. Das, R. Vimala and P. Karthika, Biosorption of heavy metals. An overview. Indian journal of biotechnology. 7 (2008) [9] K. Chojnacka, Biosorption and bioaccumulation the prospects for practical applications Environment International 36 (2010) [10] L. Martinkova, B. Uhnakovaun, M. Patekun, J. Nesveraun and V. Krenun, Biodegradation potential of the genus Rhodococcus. Center of Biocatalysis and Biotransformation, Institute of Microbiology, Academy of Sciences of 11

6 the Czech Republic. 35(1) (2009) [11] S. Kelly, K. Kemner, J. Fein, D. Fowle, M. Boyanov, B. Bunker, and N. Yee, X-ray absorption one structure determination of ph-dependent U-bacterial cell wall interactions. Geochemica et Cosmochimica Acta, 66(22) (2002) [12] A. Plette, M. Benedetti, and W. Van Riemsdjik, Competitive binding of protons, calcium, cadmium and zinc to isolated cell walls of a Gram-positive soil bacterium. Environmental Science and Technology, 30 (1996) [13] T. Akar, S. Tunali, I. Kiran, Botrytis cinerea as a new fungal biosorbant for removal of Pb(II) from aqueous solutions, Biochem. Eng. J. 25 (3) (2005) [14] M. Isik, Biosorption of Ni (II) from aqueous solutions by living and non-living ureolytic mixed culture. Colloids and surface B : Biointerfaces 62 (2008) [15] H. Li, Y. Lin, W. Guan, J. Chang, L. Xu, J. Guo, G. Wei, Biosorption of Zn (II) by live and dead cells of Streptomyces ciscaucasicus strain CCNWHX Journal of Hazardous Materials 179 (2010) [16] J. Pan, G. Xiaopeng, L. Ruixia, and T. Hongxiao, Characteristic features of Bacillus cereus cell surfaces with biosorption of Pb (II) ions by AFM and FT-IR. Colloids and Surfaces B: Biointerfaces. 52(1) (2006)

7 2014 5th International Conference on Food Engineering and Biotechnology IPCBEE vol.65 (2014) (2014) IACSIT Press, Singapore DOI: /IPCBEE V65. 3 Treatment Comparison Efficiency of Microbial Amended Agro-waste Biochar Constructed Wetlands for Reactive Black Textile Dye Beenish Saba 1,2, Madeeha Jabeen 2, Tariq Mahmood 2 and Irfan Aziz 3 1 Department of Civil and Environmental Engineering and Geodetic Science, The Ohio State University Columbus Ohio, USA 2 Department of Environmental Sciences 3 Department of Agronomy, PMAS Arid Agriculture University Rawalpindi Abstract. Textile effluents are chief industrial polluters because of color content, salts and high chemical oxygen demand. The intensive release of dyes leads to diffuse contamination of non target environments. For instance contamination of ground water, nearby irrigation land and surface water bodies threaten human, animals and plants health. The primary objective of this study is to explore potential of rice husk as an agricultural waste and biochar of rice husk as natural adsorbent to sorb color from effluent and efficiency of constructed wetlands (CWs) in dye contaminated water treatment. The experiment was divided into four levels. Study 1 was a lab scale study in which we study the adsorption of reactive black dye on rice husk and biochar. Study 2 was done to determine comparative dye removal in constructed wetland system. Study 3 was done to evaluate the dye removal ability of Ks-23, Ks-26 and I-15 in laboratory. Study 4 was taken in constructed wetland for evaluating the microbial assisted dye removal efficiency of the system.the results of study reveal that there was significant reduction in COD of the systems leading up to 40% to 50% with maximum reduction in constructed wetland containing microbes and biochar as medium. Color is the major problem regarding wastewater textile effluents. A considerable color reduction was observed in CWs. The color removal increases with the passage of time. 70% to 90% color removal was observed in this study with an HRT of 30.24hours. A strong negative correlation was observed between COD and color removal. Maximum removal was observed at the end of the two months duration. Rice husk system has a COD of 95 mg/l with 78.74% color removal ( ) compared to 93mg/L COD and 75% color removal ( ) in Biochar system. Similar trend was observed in systems containing microbes along with rice husk and biochar. Ricehusk+ks-23 have a COD of 373mg/L with % color removal ( ) and biochar+ks-23 have 358mg/L COD and % color removal ( ). Keywords: rice husk, constructed wetlands, reactive black, dye removal 1. Introduction Industrial activities are releasing a number of hazardous chemical species polluting wastewater effluents. Dyes must be treated as they account for the most part of perilous chemical present in industrial effluents lessening light penetration, hence disturbing the photosynthetic activity and biological processes of aqueous flora [1]. The technologies available for dye removal such as physicochemical and biological are very expensive and resulted in sludge formation which cause secondary pollution. Adsorption is the most appropriate and proficient method for dye removal in effluents [2]-[4]. A significant role was played by phytoremediation in contaminant removal through filtration, adsorption, cation exchange, and throughout plant-induced chemical transformations in rhizosphere [5]. Plants generally have optimistic outcome on decontamination and play a promising role in CWs [6]. Rice husk is a key agricultural waste throughout the world; therefore its appropriate management is very indispensable to lessen its effect on environment. Rice Corresponding Author: Tel.: ; fax: address: beenishsaba@uaar.edu.pk 13

8 husk, rice bran and rice ash was used to destroy a number of dyes including methylene blue [3], crystal violet [7], Brilliant Vital Red [8], Direct Red-31 and Direct Orange-26 [9] and Congo red [10]. So a combination of microbes, plants and constructed wetland would establish itself as an efficient system. The objectives of the study will be as follows. 1. To compare rice husk media and biochar on treatment of wastewater. 2. To evaluate adsorption, removal and degradation of the dye related contaminants 3. To assess dye removal efficiency at different level of studies 4. To reuse the treated wastewater for irrigation purpose 2. Materials and Methods In this study two systems of wetland were constructed and assigned as system 1 and 2. System 1 consisted of mixture of soil and ricehusk in 1:1 and system 2 contained of soil and biochar of ricehusk in the same ratio. Wetland encompassed of plastic boxes with a height and volume of 28cm and 15.12L respectively. Prescaria barbata was planted in both systems and acclimatize with water before application of synthetic dye wastewater. Plant density in both systems was 12 plants/system. The graduated influent container was positioned at height of 1 foot above these systems. The treated wastewater was collected in another container with a capacity of 10 liters. Wetland (system 1) was constructed with horizontal layer of gravel at the bottom, mixture of soil and ricehusk (1:1) as the main substratum and then again covers with fine gravel at the top. System 2 was also constructed in the same way having soil and biochar of ricehusk. Soil and ricehusk was sieved with a mesh size of 0.5 mm. Effluent samples were taken on every tenth day; on twelfth hour when dye wastewater was fed to the systems. Water samples are collected in plastic bottles and transferred to laboratory and kept at 4 C. 3. Result and Discussion This study was conducted to determine the degradation of synthetic dye wastewater by using constructed wetland system comprising of ricehusk and its biochar as a substratum. The COD of the influent was 167mg/L which was greatly reduced to 95mg/L in case of rice husk and 93mg/L in case of biochar. A significant reduction of COD was observed in the constructed wetland systems with the passage of time till end of two months (P< ). Similarly the COD of the influent and yeast was 672mg/L which was decreased up to 373mg/L in case of inoculated ricehusk wetland and 358mg/L in inoculated biochar system. A negative gradient was observed with the passage of time (p<0.006). There was significant reduction in COD of the systems leading up to 40 percent to 50 percent with maximum reduction in constructed wetland containing microbes and biochar as medium (figure 1). 60 % COD Removal Time (Days) Ricehusk Biochar Ricehusk+KS-23 Biochar+KS-23 Fig. 1: Comparison of percent COD removal of the systems 14

9 Color is the major problem regarding wastewater textile effluents. A considerable colour reduction was also observed in CWs. The color removal increases with the passage of time. 70 percent to 90 percent color removal was observed in this study with an HRT of hours (figure 2) with maximum colour removal was observed in biochar system assisted with KS-23. [11] also noted that the decolorization of Reactive Black was maximum by bacterial strains at 250 mg/l dye liquid medium when yeast extract was applied at the rate of 0.4 percent as a cosubstrate. % Color Removal Ricehusk Biochar Ricehusk+KS Biochar+KS-23 Time (days) Fig. 2: Colour removal rate of the four CWs at different time interval A strong negative correlation was observed between COD and color removal in all the four systems (figure 3). Maximum removal was observed at the end of the two months duration. Rice husk system has a COD of 95mg/L with percent color removal ( ) compared to 93mg/L COD and 75percent color removal ( ) in Biochar system. Similar trend was observed in systems containing microbes along with ricehusk and biochar. Ricehusk+ks-23 have a COD of 373mg/L with percent color removal ( ) and biochar+ks-23 have 358mg/L COD and percent color removal ( ). In another study maximum decolorization of Reactive Black was 80 to 100 percent in 24 hours by using selected strains of bacteria at 250 mg/l dye concentration assisted with 4 g/l yeast [12]. Another study showed a great COD reduction up to 88 percent when a combination of bacterial communities was used as compared to 36 percent and 48 percent for individual strains. He also analyzed that the consortium could nearly completely mineralize the dye with nontoxic residual metabolites evaluated by phyto toxicity and microbial toxicity tests. The Consortium was tested to decolorize and mineralize mixture of reactive dyes and actual dye wastewater shows significant efficiency in the color removal as well as the reduction of TOC and COD. The ability of consortium to utilize cheap cosubstrate such as rice husk and rice straw for dye decolorization represents an advantage for treatment of textile industry wastewaters [13]. COD (mg/l) % Color Removal COD Color Time (Days) Fig. 3: Comparison of COD and colour removal in effluents obtained from CW system containing Biochar+KS-23 15

10 The following conclusion can be drawn from this study: The dye removal rate is directly affected by ph, adsorbent dose, contact time, agitation rate and initial dye concentration. Highest efficiency was observed in a system containing microbes and biochar with 90 percent colour removal. A strong negative correlation was observed between COD and colour removal for all the systems. 4. References [1] Y. S. Al-Degs, M. I. El-Barghouthi, A. H. El-Sheikh and G. M. Walker Effect of solution ph, ionic strength, and temperature on adsorption behavior of reactive dyes on activated carbon. Dyes Pigments, 77(1): [2] M. Anbia, and S. Salehi Removal of acid dyes from aqueous media by adsorption onto amino-functionalized nanoporous silica SBA-3. Dyes Pigments, 94(1): 1-9. [3] M. N. Ashiq, M. Najam-Ul-Haq, T. Amanat, A. Saba, A. M. Qureshi and M. Nadeem Removal of methylene blue from aqueous solution using acid/base treated rice husk as an adsorbent. Desalination Water Treatment, 49(1-3): [4] W. Zhang, H. Yang, L. Dong, H. Yan, H. Li, Z. Jiang, X. Kan, A. Li and R. Cheng Efficient removal of both cationic and anionic dyes from aqueous solutions using a novel amphoteric straw-based adsorbent. Carbohydrate Polymers. [5] J. Nouri, N. Khorasani, B. Lorestani, M. Karami, A. Hassani and N. Yousefi Accumulation of heavy metals in soil and uptake by plant species with phytoremediation potential. Environmental Earth Sciences, 59(2): [6] L. Kong, Y. B. Wang, L. N. Zhao and Z. H. Chen Enzyme and root activities in surface-flow constructed wetlands. Chemosphere, 76(5): [7] T. Depci, A. R. Kul, Y. Onal, E. Disli, S. Alkan, Z. F. Tukmenoglu, Adsorption of crystal violet from aqueous solution on activated carbon derived from gölbaşi lignite. Physicochemical Problems in Mineral Processing, 48(1): [8] R. Rehman, J. Anwar, T. Mahmud, M. Salman and U. Shafique. 2011a. Influence of Operating Conditions on the Removal of Brilliant Vital Red Dye from Aqueous Media by Biosorption using Rice Husk. J. Chem. Soc. Pak., 33(4): 515. [9] Y. Safa, and H. N. Bhatti Kinetic and thermodynamic modeling for the removal of Direct Red-31 and Direct Orange-26 dyes from aqueous solutions by rice husk. Desalination, 272(1): [10] X. S. Wang, and J. P. Chen Biosorption of Congo Red from Aqueous Solution using Wheat Bran and Rice Bran: Batch Studies. Separation Science and Technology, 44(6): [11] D. Mahne, Combination of constructed wetland and TiO2 photocatalysis for textile wastewater treatment. Unpublished Doctoral Thesis. Univerza v Novi Gorici, podiplomski študij. [12] M. Shah, K. Patel, S. Nair and A. Darji Optimization of Environmental Parameters on Microbial Degradation of Reactive Black Dye. J. Bioremed. Biodeg, 4(183): 2. [13] R. Saratale, G. Saratale, J. Chang, and S. Govindwar Decolorization and biodegradation of reactive dyes and dye wastewater by a developed bacterial consortium. Biodegradation, 21(6):

11 2014 5th International Conference on Food Engineering and Biotechnology IPCBEE vol.65 (2014) (2014) IACSIT Press, Singapore DOI: /IPCBEE V65. 4 Effects of Bio-Based Ingredients on the Development and Quality of Food Wrapper from Jackfruit (Artocarpus heterophyllus Lam.) Seed Flour Mylene A. Anwar 1+ and Roberta D. Lauzon 2 1 Department of Food Science, College of Home Economics, Central Mindanao University, University Town, Musuan, Maramag 8710, Bukidnon, Philippines 2 Departmet of Food Science and Technology, College of Agriculture and Food Science, Visayas State University, Visca 6521-A, Baybay City, Leyte, Philippines Abstract. The use of edible food wrappers with antimicrobial properties is becoming popular nowadays. However, due to some complexities in its production and components, making it expensive, its use is often limited only to consumers who have the capacity to purchase at a relatively higher value. This can be answered by developing food wrapper using locally available bio-based ingredients known to exhibit antimicrobial properties. This study was conducted to develop food wrapper utilizing jackfruit seed flour and to assess its quality as affected by levels of malunggay leaf extract, cassava starch and garlic slurry as biobased ingredients. Level combination of 120 % malunggay leaf extract, 80 % garlic slurry and 40 g cassava starch is the optimum level combination that satisfies the optimum formulation requirement based on product s general acceptability, production cost and nutritional value. Aroma and general acceptability of food wrapper was significantly affected by malunggay leaf extract levels and the texture acceptability of the product by cassava starch levels. The food wrapper showed no cracks when subjected to folding test and has a water and oil absorption capacity of % and %, respectively. It has a microbial load of 7 x 10 1 cfu/g after 35 days storage at chilling condition and bacterial pathogens (Salmonella and E. coli) were not detected in the product. Overall consumer preference of 81% indicates that it has a strong potential to compete as a low cost healthy alternative with the existing food wrappers in the market. Keywords: bio-based, jackfruit seed flour, food wrapper 1. Introduction Consumers attitude towards bio-based food products and ingredients are in demand at present. High demand for these products is driven by the never-ending food-safety issues associated with synthetic chemical components along with environmental concerns. Furthermore, with the increase in knowledge on the benefits of bio-based ingredients, consumers nowadays are becoming more conscious on their consumption choices. Among these ingredients includes malunggay and garlic, which are known to contribute significant health benefits aside from being a natural antimicrobial agent. Cassava starch also is known to contribute in improving product quality in many food formulations. Though varieties of product forms are already available in the market which contains bio-based ingredients, a lot of consumers, especially those seriously affected by poverty cannot access these products due to high cost. Thus, there is a need to develop food products utilizing these ingredients without the need of sophisticated equipment for processing and that production can be done at home level. This study was conducted to maximize the use of jackfruit seeds and to utilize locally available biobased ingredients in the development of food wrapper that can serve as a healthy alternative to existing food wrappers in the market. + Corresponding author: Tel: (088) local 122/ address: myleneayod@rocketmail.com 17

12 2. Methods 2.1. Procurement of raw materials Jackfruit seeds were collected from the Department of Food Science and Technology vacuum and dehydrated jackfruit processing area for the production of jackfruit seed flour. Cassava starch was purchased from the PhilRootcrops Research and Training Center in Visayas State University, Visca, Baybay City, Leyte. All other raw materials needed including eggs, garlic, malunggay leaves and salt were purchased at Baybay City Public Market Variable screening Screening of variables was conducted using Plackett-Burman Design with seven (7) input variables for eight (8) runs [2] (Gacula, 1993). Response variables include the descriptive and acceptability of the products color, aroma, texture, pliability, taste, and general acceptability Experimental design for formulation optimization A 3 3 fractional factorial design, Central Composite Design (CCD), was employed with 15 treatments for experimental combinations Production of bio-based food wrapper for product optimization Filtrate from the malunggay leaves and the homogenized garlic slurry was gradually added to the previously sifted jackfruit seed flour and cassava starch in appropriate volumes. The mixture was added with constant amount of whole egg and iodized salt and was mixed thoroughly until a smooth consistency is achieved. Appropriate amount of the mixture was poured into a non-stick pan over medium heat. Doneness was determined when edges loosens from the pan and when surface looks completely dry. The final product was removed from the pan using spatula Sensory evaluation for product optimization Presentation of the samples was carried out following the Incomplete Block Design (IBD) set plan 13.7 laid out by [1] Cochran and Cox (1957) Statistical analysis and modeling Data obtained from sensory evaluation for variable screening was analyzed using STATISTICA version 6. Data from the optimization process was subjected to Response Surface Regression (RSReg) analysis using SAS Statistical Software to determine the effects of independent variables on the sensory qualities of the product Verification and consumer preference test Verification test was conducted using the optimum formulation and a treatment having level combination that falls outside the optimum region. Consumer preference test was carried out by subjecting the optimized food wrapper and a commercial counterpart to 100 randomly selected consumer panelists Physico-chemical and nutritional value analysis Physico-chemical analysis includes folding test, water and oil absorption capacity and the determination of the food wrapper s nutritional value, including fat, protein, total dietary fiber and food energy value by the Regional Standards and Testing Center (RSTC), Department of Science and Technology (DOST), Lahug, Cebu City Microbiological analysis and shelf life determination The microbiological quality of the food wrapper stored at chilling condition was periodically (every after seven days) examined for 35 days. A prepared vegetable for lumpia filling was wrapped using the food wrapper to assess its application. The product was stored at chilling temperature and was subjected to microbial analysis to determine its shelf life. 3. Results and Discussion 18

13 3.1. Variable screening Based on the result of variable screening as shown in Table 1, levels of cassava starch, malunggay leaves extract and garlic slurry are the variables that significantly affect the product s quality, thus, appropriate to be used in the optimization process. These variables are believed to improve the physico-chemical, sensory, nutritional and antimicrobial property of the food wrapper. Table 1. Statistical analysis of Plackett-Burman design expressed as effect estimates Parameter Parameter Estimates Color Aroma Texture Pliability Taste Gen. Accept. Mean/Interc ** ** ** ** ** ** JSF * * ns * ns ** CS ns * ns ns ns ns MLE ** ** ns ns ns ns Garlic ** ** ns ns ns ns Whole Egg ** ns ns ns ns ns Salt ns ns * ns ns ns JSF (jackfruit seed flour) MLE (malunggay leaves extract) CS (cassava starch) GS (garlic slurry) *significant (p<0.05) ** significant (p<0.01) ns not significant 3.2. Sensory qualities of the food wrapper Statistical analysis (Table 2) revealed that malunggay leaves extract affects the product s aroma and general acceptability. Texture acceptability is significantly affected by cassava starch level. Table 2. Parameter estimates for the response of sensory acceptability of the food wrapper Parameter Parameter Estimates Color Aroma Texture Pliability Taste Gen. Accept. MLE ns ** ns ns ns * CS ns ns ns ns ns ns GS ns ns ns ns ns ns MLE*MLE ns ns ns ns ns ** CS*MLE ns ns ns ns ns ns CS*CS ns ns * ns ns ns GS*MLE ns ns ns ns ns ns GS*CS ns ns ns ns ns ns GS*GS ns ns ns ns ns ns MLE (malunggay leaves extract) CS (cassava starch) GS (garlic slurry) *significant (p<0.05) ** significant (p<0.01) ns not significant 3.3. Optimum formulation Superimposed contour plots (Figure 1) showed that higher product acceptability is obtained at low level of cassava starch. Thus, a 40 grams cassava starch is used for the optimum formulation. Since the optimum shaded region for garlic is located within the set levels of the variables except at the region about %, a percentage level of 80% (64ml) garlic is used in the optimum formulation. This value maximizes the use of garlic and its contribution in enhancing the nutritional value of the product in addition to being a natural antimicrobial agent. To maximize also the use of malunggay leaves extract, a level beyond the set maximum value (beyond 80%) is used in the optimum formulation since product acceptability increases at levels below and beyond the set minimum and maximum values respectively. It is wise to utilize the high level of malunggay leaves extract in order also to enhance the nutritional value of the product. Thus, a level of 120% malunggay leaves extract is used in the optimum formulation. 19

14 Cassava Starch (g)/ Garlic Slurry (ml) Malunggay Leaves Extract (%)/ Garlic Slurry (ml) Fig. 1: Superimposed contour plots showing the optimum region 3.4. Verification and consumer preference test T-test analysis comparing the predicted and observed values (Table 3) showed that the observed sensory acceptability rating of the optimum formulation is significantly different from the predicted values. This, however, does not imply that the predicted values are not dependable but only shows that the level combination of variables used in the optimum formulation is the most acceptable as indicated by the increase in the sensory acceptability rating range from ( like moderately to like very much ) based on the 9-point hedonic scale. Table 3. T-test results for comparing predicted and observed sensory acceptability of the optimum treatment treatment outside the optimum region (Treatment 7) and Optimum Formulation Treatment 7 Parameter Sensory Acceptability Sensory Acceptability Predicted Observed Prob > T Predicted Observed Prob > T Color ** ns Aroma ** ns Texture * ** Pliability ** ** Taste * ** Gen. Accept ** ** N=28 *significant (p< 0.05) **significant (p< 0.01) ns not significant Range of scores: 9-like extremely 6-like slightly 3-dislike moderately 8-like very much 5-neither like nor dislike 2-dislike very much 7-like moderately 4- dislike slightly 1-dislike extremely The higher overall preference of the bio-based food wrapper (81%) than the commercial counterpart (19%) implies that the biobased food wrapper form jackfruit seed flour has great market potential and a healthy alternative to existing food wrappers that are commonly used especially in home cooking. It is also an ideal channel to introduce vegetable (the malunggay leaf) and spice (garlic) to individuals who do not usually consume it Physico-chemical properties The negative result of the folding test for the occurrence of cracks or any mechanical damage implies that pliability of the food wrapper in its optimum formulation is efficient enough to serve its purpose and to function well in wrapping foods. Result in water and oil absorption evaluation revealed that the food wrapper had the capacity to absorb 18.36% and 10.76% water and oil respectively. With this amount of water absorbed, the 20

15 food wrapper remains intact but its efficiency in wrapping decreases because texture and pliability are negatively affected Nutritional value Result of nutritional quality analysis showed that the food wrapper contains an appreciable amount of fat (0.647%), protein (4. 36%), total dietary fiber (2.27%), total carbohydrates (34.1%) and food energy value of 160 kcal/100g. This indicates that the food wrapper can serve as a healthy alternative to existing food wrappers in the market Microbiological analysis and shelf-life study Result in the microbiological analysis revealed that the food wrapper had 7x10 1 microbial count expressed as colony-forming units per gram of sample (cfu/ g) after 35 days of storage at chilling condition. The result indicates that the microbial load of the product is low and is within the set guideline level of 10 4 and 10 6 for determining the microbiological quality of ready-to-eat food [3] (NSW Food Authority, 2009). The microbial quality status of the product can be attributed to several factors, including the composition of the food product having garlic and malunggay leaves extract, which are known to exhibit antimicrobial activity, its storage condition, and most importantly the good manufacturing practices applied during the process. Result of pathogen test revealed that the bio-based food wrapper was free from pathogens, specifically Salmonella and E. coli. This implies that the bio-based food wrapper is safe for consumption, and that it does not pose a risk for any possible food borne illness caused by these pathogens. 4. Acknowledgements This study was funded by the Department of Science and Technology Science Education Institute (DOST-SEI) under the Accelerated Science and Technology Human Resource Development Program (ASTHRDP) through the National Science Consortium (NSC). Likewise, jackfruit seeds provided by the Department of Food Science and Technology of the Visayas State University are greatly appreciated and acknowledged. 5. References [1] W. G. Cochran and G.M. Cox. Experimental Designs. Second Edition. New York. 1957, p [2] M.C. Gacula, Design and Analysis of Sensory Optimization. Food and Nutrition Press, Trumbull, Connecticut, USA. pp [3] NSW Food Authority. Microbiological quality guide for ready-to-eat foods: a guide to interpreting microbiological results Retrieved September 12, 2009 from, 21

16 2014 5th International Conference on Food Engineering and Biotechnology IPCBEE vol.65 (2014) (2014) IACSIT Press, Singapore DOI: /IPCBEE V65. 5 Anti-inflammatory activities of cellulose nanofibers made from adlay and seaweed in an inflammatory bowel-disease model Kazuo Azuma 1, Shinsuke Ifuku 2, Tomohiro Osaki 1, Ichiro Arifuku 3, Yoshiharu Okamoto 1 1 Faculty of Agriculture, Tottori University, Koyama-minami Tottori, , Japan 2 Graduated School of Engineering, Tottori University, Koyama-minami Tottori, , Japan 3 Department of Applied Biotechnology, Food Developing Laboratory, Tottori Institute of Industrial Technology, Sakaiminato,Tottori , Japan Abstract. Inflammatory bowel disease (IBD) is one of the common diseases all over the world. In this study, we investigated the anti-inflammatory effects of cellulose nanofibers made from adlay (A-CNF) and seaweed (S-CNF) on colon inflammation using the mouse model of IBD. A-CNF and S-CNF improved the histological tissue injury in mice. A-CNF and S-CNF also suppressed activation of nuclear factor-kappa B in the colon. Furthermore, A-CNF and S-CNF suppressed myeloperoxidase activities of inflammatory cells such as leukocytes. On the other hand, cellulose nanofibers made from wood did not improve the histological tissue injury and colon inflammation in mice. These results revealed that A-CNF and S-CNF have suppressive effects on colon inflammation in an experimental IBD mouse model. Furthermore, our results indicate that A-CNF and S-CNF may be a potential source of dietary fiber for patients with IBD. Keywords: dietary fiber, cellulose nanofiber, inflammatory bowel disease, adlay, seaweed 1. Introduction Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gut [1]. Incidences of IBD have increased and it may be because of changes in dietary habits in recent years, particularly diets with low fiber content [2]. Recently, Abe et al. (2007) described an efficient method for isolation of cellulose nanofibers with a uniform width of approximately 15 nm from wood [3]. This nanofiber preparation method can be used to isolate cellulose nanofibers from any natural plant such as flax, sugarcane bagasse, wheat straw, and pear [4]-[6] Recently, we reported that nanofibrillated chitin has anti-inflammatory effects on IBD mouse model [7], [8]. Furthermore, we also reported the anti-inflammatory effects of cellulose nanofiber made from pear in IBD mouse model [9]. This result indicate that nanofibrillation confers beneficial and new aspects to materials. In this study, we evaluated cellulose nanofibers made from adlay and seaweed as new types of dietary fibers [10]. 2. Materials and Methods 2.1. Preparation of cellulose nanofibers Cellulose nanofibers from adlay (Coix lacryma-jobi) chaff (A-CNF) and Hijiki seaweed (Saragassum fusiforme) (S-CNF) were prepared by methods described previously [6] with some modifications. Briefly, dried Hijiki and adlay chaff were soaked in water and roughly crushed in a domestic blender. The suspensions were passed through a grinder (MKCA6-3; Masuko Aangyo Co. Ltd., Saitama, Japan) set at 1500 rpm. Grinding was performed with a clearance gauge of 1.5 (corresponding to a 0.15 mm shift) from the zero position, which was determined by the point of slight contact between the grinding stones. The samples were placed in a pressure-tight glass vessel and hydrothermally treated at 150 C for 120 min in a Corresponding author. Tel.: ; fax: address: kazu-azumamuses.tottori-u.ac.jp. 22

17 high-pressure cooker (VS-2416; Koyo Engineering Corp., Saitama, Japan) to break down the matrix substances such as hemicellulose polysaccharides, the pectin matrix, and phenolic polymer lignin embedded in the cellulose nanofibers of the cell wall. The thermally treated wet samples were then passed through the Star Burst system (Star Burst Mini, HJP-25001S; Sugino Machine Co., Ltd.) equipped with a ball-collision chamber. The slurry was ejected from a small nozzle with a diameter of 100 μm under high pressure (245 MPa) and collided with a ceramic ball with a diameter of 12.7 mm. The suspensions were passed through 10 (adlay chaff) or 5 (Hijiki seawood) mechanical treatments. Concentrations of A-CNF and S-CNF homogeneous slurries were 3 and 1 wt%, respectively. Before the oral administration experiment, A-CNF and S-CNF were diluted to 0.1 w% of homogeneous slurries in water. Each 0.1 w% diluted A-CNF and S- CNF were used for the oral administration experiment. Isolation of cellulose nanofibers from wood (W-CNF) was performed according to a previous report (Abe et al., 2007). Wood powder from Radiata Pine (Pinus radiata D. Don) was used for this study. Before the oral administration experiment, A-CNF, S-CNF and E- CNF were diluted to 0.1 w% of homogeneous slurries in water Animals and reagents Twenty-five C57BL/6 mice (female, 5 weeks old) were purchased from CLEA Japan (Osaka, Japan). The animals were maintained under conventional conditions. Mice were used in experiments after 7 days of acclimation. Animal procedures were approved by the Animal Research Committee of Tottori University. DSS (molecular weight: kda; reagent grade) was purchased from MP Biomedicals LLC (Solon, OH, USA) Animals and reagents Mice (n = 25) were randomized into 5 groups: the control ( ) group was administered tap water (n = 5), the control (+) group was administered 3% DSS dissolved in tap water (n = 5), the A-CNF (+) group was administered A-CNF and 3% DSS dissolved in tap water (n = 5), the S-CNF (+) group was administered S- CNF and 3% DSS dissolved in tap water (n = 5), and the wood cellulose nanofiber (W-CNF) (+) group was administered W-CNF and 3% DSS dissolved in tap water (n = 5). To elicit colitis, mice were administered with 3% DSS ad libitum for 5 days. A-CNF, S-CNF, and W-CNF were diluted to 0.1 w% in water. The A- CNF (+), S-CNF (+), and W-CNF (+) groups, 0.1 w% diluted A-CNF, S-CNF, and W-CNF were also administered ad libitum for 5 days. Colon sampling was performed at day 5 in all groups Histological evaluation of colitis Colon tissues were fixed in 10% buffered formalin. Thin sections (3 μm) were prepared from each sample for histological observation after hematoxylin-eosin staining. Each section was examined microscopically, and histological scoring was performed as described by us [7]. Histological scoring was performed in 10 fields at 100 magnification using 3 mice in each group. The mean score for 30 fields was considered as the histological score for each group. Counting of MPO-positive cells in the submucosal layer was performed as described previously [7]. Immunohistochemical detection of NF-κB was performed by methods described previously [8]. Quantitative digital morphometric analyses of NF-κB-positive areas of colonic sections were performed according to methods described previously [8] Statistical analysis The data are expressed as the mean ± S.E. Statistical analyses were performed using Steel-Dwass test. A p-value of <0.05 was considered statistically significant. 3. Results 3.1. Effects of A-CNF and S-CNF on histological changes in IBD model mice The damage of the intestinal mucosa was microscopically evaluated by histological scoring. In control (+) and W-CNF (+) groups, we observed erosions, shorting or destruction of the crypts, and edema. In A-CNF (+) and S-CNF (+) groups, we observed some erosion and marked suppression of shorting or destruction of the crypts, and slight suppression of edema. The results of the histological scoring are shown in Figure 1. In the control (+) group, the histological scores were significantly higher than those in the non-treated control 23

18 (control ( )) group were (p < 0.01). In addition, histological scores of the A-CNF (+) and S-CNF (+) groups were significantly lower than those of the control (+) and W-CNF (+) groups were (p < 0.01). Fig. 1: Effects of A-CNF and S-CNF on histological changes in IBD model mice. Data represent the mean ± S.E. in each group. Statistical analysis was performed with the Steel-Dwass test. **p < Effects of A-CNF and S-CNF on colon MPO-positive cells in IBD mouse model The results of the numbers of MPO-positive cells are shown in Figure 2. In the control (+) group, the numbers of MPO-positive cells were significantly higher than those of the non-treated control (control ( )) group were (p < 0.01). In A-CNF (+) and S-CNF (+) groups, the numbers of MPO-positive cells were significantly lower than those of the control (+) and W-CNF (+) groups were (p < 0.01). Fig. 2: Effects of A-CNF and S-CNF on colon MPO-positive cells in IBD mouse model. Data represent the mean ± S.E. in each group. Statistical analysis was performed with the Steel-Dwass test. **p < Effects of A-CNF and S-CNF on NF-κB expression in the colon epithelium In the control (+) group, the positive areas of NF-κB were significantly increased compared with those of the non-treated control (control ( )) group (p < 0.01). In A-CNF (+) and S-CNF (+) groups, the positive areas of NF-κB were significantly decreased compared with those of the control (+) and W-CNF (+) groups (p < 0.01) (Figure 3). 4. Discussion Intake of dietary fiber reduces the risk of developing certain gastrointestinal disorders [11]. In this study, we evaluated the potential of A-CNF and S-CNF as new types of dietary fiber using the IBD mouse model. In A-CNF (+) and S-CNF (+) groups, histological scores were significantly lower than those of the control (+) and W-CNF (+) groups were. Being a marker of oxidative stress, high MPO activities were observed in a 24

19 IBD mouse model [12], [13]. In the A-CNF(+) and S-CNF (+) groups, MPO-positive cells were significantly fewer than in the control(+) and W-CNF(+) groups. Therefore, we can infer that P-CNF suppresses the inflammation caused by acute UC by decreasing the MPO activation of inflammatory cells such as leukocytes. On the other hand, W-CNF did not suppress the clinical symptoms and colon inflammation in the IBD mouse model. These data indicated that A-CNF and S-CNF suppressed colon damage in the experimental IBD mouse model. NF-κB is a critical transcription factor needed to express genes associated with proinflammatory responses [14]. It stimulates expression of cyclooxygenase-2, prostaglandin E 2, and pro-inflammatory cytokines (IL-6, TNF-α, and monocyte chemotactic protein-1) [15]. In A-CNF (+) and S- CNF (+) groups, positive areas of NF-κB in colon epithelia were significantly decreased compared with those of the control (+) and W-CNF (+) groups. Our results indicated that A-CNF and S-CNF had antiinflammatory effects via suppression of NF-κB activation in the IBD mouse model. Fig. 3: Effects of A-CNF and S-CNF on NF-κB expression in the colon epithelium. Data represent the mean ± S.E. in each group. Statistical analysis was performed with the Steel-Dwass test. **p < In conclusion, A-CNF and S-CNF suppress shortening of colons and increases the colon weight/length ratio. A-CNF and S-CNF also suppress colon inflammation. Our data indicate that the cellulose nanofibers made from adlay and seaweed have the potential to be considered new types of beneficial dietary fibers for IBD patients. 5. Acknowledgements This work was supported by a Tottori prefecture-financed aid project for beauty & health products ( ). 6. References [1] G. Morrison, B. Headon, P. Gibson. Update in inflammatory bowel disease. Aust. Fam. Physician. 2009, 38, [2] D.J. Rose, M.T. DeMeo, A. Keshavarzian, B.R. Hamaker. Influence of dietary fiber on inflammatory bowel disease and colon cancer: importance of fermentation pattern. Nutr. Rev. 2007, 65, [3] K. Abe, S. Iwamoto, H. Yano. Obtaining cellulose nanofibers with a uniform width of 15 nm from wood. Biomacromolecules. 2007, 8, [4] K. Abe, H. Yano. Comparison of the characteristics of cellulose microfibril aggregates of wood, rice straw and potato tuber. Cellulose. 2009, 16, [5] K. Abe, H. Yano. Comparison of the characteristics of cellulose microfibril aggregates isolated from fiber and parenchyma cells of Moso bamboo (Phyllostachys pubescens). Cellulose. 2010, 17, [6] S. Ifuku, M. Adachi, M. Morimoto, H. Saimoto. Fabrication of cellulose nanofiber from parenchyma cells of pear and apple. Seni Gakkaishi. 2011, 67,

20 [7] K. Azuma, T. Osaki, T. Wakuda, S. Ifuku, H. Saimoto, T. Tsuka, T. Imagawa, Y. Okamoto, S. Minami. Beneficial and preventive effect of chitin nanofibrils in a dextran sulfate sodium-induced acute ulcerative colitis model. Carbohydr. Polym. 2012, 87, [8] K. Azuma, T. Osaki, S. Ifuku, H. Saimoto, T. Tsuka, T. Imagawa, Y. Okamoto, S. Minami. α-chitin nanofibrils improve inflammatory and fibrosis responses in mice with inflammatory bowel disease. Carbohydr. Polym. 2012, 90, [9] K. Azuma, T. Osaki, S. Ifuku, M. Morimoto O. Takashima, T. Tsuka, T. Imagawa, Y. Okamoto, H. Saimoto, S. Minami. Anti-inflammatory effects of cellulose nanofiber made from pear in inflammatory bowel disease model. Bioactive Carbohydrate and Dietary Fibre, 2014, 3, [10] K. Azuma, T. Osaki, S. Ifuku, H. Maeda, M. Morimoto O. Takashima, T. Tsuka, T. Imagawa, Y. Okamoto, H. Saimoto, S. Minami. Suppressive effects of cellulose nanofibers made from adlay and seaweed on colon inflammation in an inflammatory bowel-disease model. Bioactive Carbohydrate and Dietary Fibre, 2013, 2, [11] L. Petruzziello, F. Iacopini, M. Bulajic, S. Shah, G. Costamagna. Review article: uncomplicated diverticular disease of the colon. Aliment. Pharmacol. Ther. 2006, 23, [12] Y. Naito, T. Takagi, T. Yoshikawa. Neutrophil-dependent oxidative stress in ulcerative colitis. J. Clin. Biochem. Nutr. 2007, 41, [13] R.K. Schindhelm, L.P. van der Zwan, T. Teerlink, P.G. Scheffer. Myeloperoxidase: a useful biomarker for cardiovascular disease risk stratification? Clin. Chem. 2009, 55, [14] C.O. Elson, Y. Cong, V.J. McCracken, R.A. Dimmitt, R.G. Lorenz, C.T. Weaver. Experimental models of inflammatory bowel disease reveal innate, adaptive, and regulatory mechanisms of host dialogue with the microbiota. Immunol. Rev. 2005, 206, [15] T. Karrasch, T. Jobin. NF-κB and the intestine: friend or foe? Inflamm. Bow. Dis.2008, 14,

21 2014 5th International Conference on Food Engineering and Biotechnology IPCBEE vol.65 (2014) (2014) IACSIT Press, Singapore DOI: /IPCBEE V65. 6 Suppressive Effects of Onion Peel Extract Tea in Experimental Obese Mice Yoshiharu Okamoto 1, Kazuo Azuma 1, Tomohiro Osaki 1,Norihiko Itoh 1 and Mayumi Watanabe 2 1 Faculty of Agriculture, Tottori University, Tottori , Japan 2 FINARL co. Inc., Tottori , Japan Abstract. In this study, it was examined the effects of onion peel tea (OPT) in a high-fat-diet-induced obese mouse model. Mice were fed a high-fat diet for 3 weeks, then a normal diet with or without OPT for 28 days. OPT suppressed the increase in body weight and level of epididymal fat tissue; it also significantly reduced the serum concentrations of total-cholesterol on day 14 and that of glucose and leptin on day 28. Our results indicate that OPT has anti-obesity effects in an experimental high-fat-diet-induced obese mouse model. Keywords: Onion peel, tea, anti-obesity, leptin, functional food 1. Introduction Obesity is a growing health problem worldwide, and it has been associated with metabolic syndrome (MetS), diabetes, cardiovascular disease, hypertension, and cancer [1]. The increasing incidence of obesity suggests that this epidemic will worsen in the future [2]. Animal models are useful tools to evaluate the efficacy of potential compounds for the prevention and treatment of obesity. It has been reported that rodents fed a high-fat diet are excellent models of obesity, in which the dietary environment is a major contributor [3]. It has been previously reported that some foods are beneficial for the suppression or prevention of MetS, including tea [4]. Green tea is already a popular beverage and can be easily incorporated as part of a diet designed to mitigate or prevent the symptoms of MetS [4]. Catechins, in particular, are one of the major polyphenolic compounds in tea and are beneficial for the treatment of the main MetS conditions, including obesity, type-2 diabetes, and cardiovascular risk factors [5]. Another potentially beneficial food is onion. Onion has the capacity to regulate lipid metabolism and suppress hyperglycemia and diabetes [6]. Many reports have attributed anti-obesity effects to quercetin, one of the flavonoids present in onion peel [7]-[9]. Tea extracted from onion peel (onion peel tea; OPT) could thus be expected to have beneficial effects for MetS. In this study, we evaluated the anti-obesity effects of OPT in mice that were fed a high-fat diet. We also examined the effects of OPT on blood parameters in these mice [10]. 2. Materials and Methods 2.1. Onion peel tea Freeze dried OPT containing 1.15 mg/g quercetin (Saratto Tamatya, Fainaru, Tottori, Japan) was used in this study Animals and diets Corresponding author. Tel: ; fax: address:yokamoto@muses.tottori-u.ac.jp 27

22 Twenty BALB/c mice (male, 4 weeks old) were purchased from CLEA Japan (Osaka, Japan). The animals were maintained under conventional conditions. The use of these animals and the procedures they were subjected to were approved by the Animal Research Committee of Tottori University. Throughout the experimental period, the mice had unrestricted access to food and water Study design Mice were randomized into 2 groups; a control group and an OPT group (n = 10 mice per group). After habituation, all mice were fed a high-fat diet (HFD: High Fat Diet-32, CLEA Japan, Osaka, Japan) from day -21 to day 0. The control group was then fed a normal powdered diet (CE-2, CLEA Japan, Osaka, Japan) from day 0 to day 28, while the OPT group was fed a normal powdered diet supplemented with 5% (w/w) OPT. The mice were weighed every 7 days from day -21 to day 28. Blood and epididymal fat tissue were harvested on days 14 and 28 (n = 5 at each time-point). Blood was collected via cardiac puncture under isoflurane inhalation anesthesia. After 1 h at room temperature, serum was recovered by centrifugation of the blood at 1,000 g for 10 min at 4 C. The serum samples were stored at -80 C prior to analysis. After blood collection, animals were immediately sacrificed by cervical dislocation, and their epididymal fat tissue was harvested and weighed Blood chemical analysis Blood chemicals were measured using a blood chemical auto analyzer (DRY-CHEM 7000, FUJIFILM Inc., Tokyo, Japan). Serum triglyceride (TG), total-cholesterol (T-cho), glucose (Glu), alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), geranylgeranyltransferase (GGT), and albumin (Alb) levels were measured Measurement of serum leptin concentration A sandwich enzyme-linked immunosorbent assay kit (MIoBS Inc., Yokohama, Japan) was used to measure leptin, in accordance with the manufacturer s instructions Statistical analysis Statistical analyses were performed on all results by using Student s t-test or one-way ANOVA and the Tukey Kramer test. All data are reported as mean ± S.D. A p value of <0.05 was considered statistically significant. 3. Results 3.1. Effects of dietary OPT on body weight and epididymal fat tissue In the OPT group, mice were fed 5 6 mg/kg/day quercetin during the experimental period. During the experimental periods, the body weights of mice increased in both the control and OPT groups (Figure 1). In the OPT group, however, the gain in body weight was mitigated compared to that of the control group. On days 14 and 21 particularly, body weights in the OPT group were significantly lower than those of the control group (p < 0.05). The epididymal fat tissue weights were evaluated. On day 14, there was no significant difference between the control group (0.2 ± 0.0 g) and the OPT group (0.2 ± 0.1 g). On day 28, however, the mean epididymal fat tissue weight of the OPT group (0.3 ± 0.1 g) was significantly lower than that of the control group (0.5 ± 0.0 g) (p < 0.01) Effects of OPT on blood chemical parameters On day 28, the mean serum Glu concentration in the OPT group was significantly lower than that of the control group (p < 0.01). On day 14, however, there was no significant difference in serum Glu concentration between the groups. On day 14, the mean serum concentration of T-cho in the OPT group was significantly lower than that of the control group (p < 0.05). On day 28, however, there was no significant difference in mean serum T-cho concentration between the groups. On day 28, the mean serum ALP concentration in the OPT group was significantly higher than that of the control group (p < 0.01), although there was no 28

23 difference on day 14. In the OPT group, mean serum TG concentrations were lower than those of the control group on days 14 and 28, although these differences were not statistically significant. There were no statistically significant differences between the groups with regard to serum concentrations of ALT, AST, GGT, or Alb, on days 14 or 28. Fig. 1: Effects of OPT on body weight changes in diet-induced obesity model. Data were shown by mean ± S.D. n=5, **:p<0.01 compared with the control group by student s t-test Effects of OPT on serum leptin levels The leptin results are shown in Figure 2. On day 14, there was no significant difference between the control group (1.9 ± 0.5 ng/ml) and the OPT group (1.6 ± 0.2 ng/ml). The mean serum leptin concentration in the control group was significantly higher on day 28 than that on day 14 (p < 0.01). On day 28, the mean serum leptin concentration in the OPT group (2.7 ± 0.4 ng/ml) was significantly lower than that of the control group (4.9 ± 1.0 ng/ml) (p < 0.05). Fig. 2: Effect of OPT on serum leptin concentration. Data were shown by mean ± S.D. n=5, **:p<0.01 by Turkey-Kramer s test. 4. Discussion In this study, dietary OPT evidently suppressed the increase in body weight and level of epididymal fat tissue in an experimental mouse model. It has been reported that in rodents, a high-fat diet is a major contributor to obesity [3]. Our data indicated that dietary OPT can reduce body weight in an experimental high-fat-diet-induced obesity model. Previous reports indicate that adipocytes in adipose tissue secrete a variety of proteins known as adipocytokines, including tumor necrosis factor-α, interleukin-6, resistin, leptin, and adiponectin [11]. Plasma leptin concentrations are positively correlated with adiposity (excessive body fat) and body weight changes in humans and rodents [12]. Adiponectin contributes to insulin sensitivity and fatty acid oxidation, 29

24 and circulating concentrations of adiponectin are inversely correlated with body mass [13]. Our results indicate that OPT suppresses the secretion of leptin from adipocytes. Suppression of the secretion of leptin may have contributed to the reduction in body weight and the weight of epididymal fat tissue observed in the OPT group. Quercetin is a major flavonol that is abundant in plant products, particularly onions, and it has been reported to possess antioxidative, anti-inflammatory, and lipid-regulating properties [14]. Numerous studies involving human clinical investigation, animal trials, and in vitro experiments have demonstrated that phenolic substances, including quercetin have important anti-inflammatory and anti-obesity properties [14]. OPT is rich in quercetin (1.15 mg/g). One possible mechanism by which OPT exerts anti-obesity effects may be the action of quercetin. In the previous reports, experimental animals were fed more amounts of quercetin than our study [9]. Our data suggest that another mechanism of action of OPT may exist. To understand additional mechanisms of action of OPT, analysis of all of the components of it may be required. In conclusion, OPT suppressed the increase in body weight and epididymal fat tissue weight normally associated with a high-fat-diet-induced obesity model. It also significantly reduced serum levels of totalcholesterol and glucose. Furthermore, in the OPT group, the level of serum leptin on day 28 was significantly reduced. Collectively, our results indicate that OPT may be a potent functional food for the treatment, management, or prevention of obesity. 5. References [1] I.Vucenik, J.P. Stains JP. Obesity and cancer risk: evidence, mechanisms, and recommendations. Ann. N Y Acad. Sci. 2012, 1271: [2] D.W. Haslam, W.P. James. Obesity. Lancet. 2005, 366 (9492): [3] M. Bullo, P. Casas-Agustench, P. Amigo-Correig, J. Jranceta, Salas-Salvado. Inflammation, obesity and comorbidities: the role of diet. Public. Health. Nutr. 2007, 10 (10A): [4] S. Sae-tan, K.A. Grove, J.D. Lambert. Weight control and prevention of metabolic syndrome by green tea. Pharmacol. Res. 2011, 64 (2): [5] F. Thielecke, M. Boschmann. The potential role of green tea catechins in the prevention of the metabolic syndrome - a review. Phytochemistry (1): [6] M. Corzo, N. Corzo, M. Villamiel. Biological properties of onions and garlic. Trends. Food. Sci. Technol. 2007, 18 (12): [7] J. Ahn, H. Lee, S. Kim, J. Park, T. Ha. The anti-obesity effect of quercetin is mediated by the AMPK and MAPK signaling pathways. Biochem. Biophys. Res. Commun. 2008, 373 (4): [8] F.R. Seiva, L.G. Chuffa, C.A. Braga, J.P. Amorim, A.A. Fernande. Quercetin ameliorates glucose and lipid metabolism and improves antioxidant status in postnatally monosodium glutamate-induced metabolic alterations. Food. Chem. Toxicol. 2012, 50 (10): [9] O.Y. Kim, S.M. Lee, H. Do, J. Moon, K.H. Lee, Y.J. Cha, M.J. Shin. Influence of quercetin-rich onion peel extracts on adipokine expression in the visceral adipose tissue of rats. Phytother. Res. 2012, 26 (3): [10] S. Matsunaga, K. Azuma, M. Watanabe, T. Tsuka, T. Imagawa, T. Osaki, Y. Okamoto. Onion peel extract tea ameliorates obesity and affects blood parameters in high-fat-diet-induced obese mice. Exp. Ther. Med. 2013, in press [11] M. Fasshauer, R. Paschke. Regulation of adipocytokines and insulin resistance. Diabetologia. 2003, 46: [12] M. Gnacińska, S. Małgorzewicz, M. Stojek, W. Łysiak-Szydłowska, K. Sworczak. Role of adipokines in complications related to obesity: a review. Adv. Med. Sci. 2009, 54: [13] E.D. Rosen, B.M. Spiegelman. Adipocytes as regulators of energy balance and glucose homeostasis. Nature. 2006, 444: [14] L. Rivera, R. Morón, M. Sánchez, A. Zarzuelo, M. Galisteo. Quercetin ameliorates metabolic syndrome and improves the inflammatory status in obese Zucker rats. Obesity (Silver Spring) 2008, 16:

25 2014 5th International Conference on Food Engineering and Biotechnology IPCBEE vol.65 (2014) (2014) IACSIT Press, Singapore DOI: /IPCBEE V65. 7 Strength Parameters of Packaged Roma Tomatoes at Peak Point under Compressive Loading F.A. Babarinsa 1 and M. T. Ige 2 1 Nigerian Stored Products Research Institute, P.M B. 1489, Ilorin, Nigeria 2 Obafemi Awolowo University, Ile-Ife, Nigeria Abstract. Compression test was conducted to investigate the peak stress and deformation induced in packaged Roma tomatoes under compressive loading and the effects of ripeness stage, vibration level and type of container on the two strength parameters. Tomatoes of three ripeness stages: unripe (5.6 Brix%), halfripe (3.9 Brix%) and full-ripe (3.2 Brix%), were packed in plastic crate and raffia basket. Using a laboratory vibrator, the fruit bulks were subjected to three levels of vibration: non-vibrated, low vibration (frequency 3.7 Hz) and high vibration (frequency 6.7 Hz). These were then compressed in a Universal Testing Machine at a loading rate of 2.50mm/min and deformation and stress at peak point in the fruit bulk were measured. Level of vibration significantly (P=0.001) reduced maximum deformation and the corresponding stress at peak point. Stage of ripeness, however, showed no significant effects on both deformation and stress at peak. Rather, it induced minimal overall differences in stress, ranging from E-03 N/mm 2 to 9.956E-03 N/mm 2. Effects of container types on stress were significant (P=0.001) but were not significant on deformation. Average peak deformation of the fruit ranged from N to 50183N while peak stress ranged from 5.917E-03 N/mm 2 to 6.936E-02 N/mm 2. The three levels of vibration exhibited stress values ranging from 1.274E-02 N/mm 2 to 8.988E-03 N/mm 2. Keywords: strength parameter, packaged Roma tomatoes, peak point, compressive loading, 1. Introduction The tomato (Lycopersicon esculentum Mill.) is a tender and compression-sensitive fruit. The fruit contains a considerable amount of water and other liquid-soluble materials surrounded by semi-solid cell wall and pectic middle lamella materials. It is thus susceptible to mechanical damage, especially compression injury, during handling and road transportation, in Nigeria, as the handlers sometimes subject the packaged produce to various forms of compressive loading in lorry truck. In Nigeria raffia baskets are the most used packaging containers in commercial transportation of fresh tomato fruit but other packaging materials such as fiberboard cartons are used. The handlers usually stack these containers one over the other whereby greater part of the compression load is transmitted directly into the fruit via packaging. Much compression damage is therein encountered due to cracking and squeezing of the fruit in multi-layers. This contributes much to the mechanical damage inflicted on the fresh fruit in transit. Mechanical properties such as compressive strength are important engineering data needed to study fruit resistance to cracking and breaking. The key measurements of mechanical behavior under compression force can be made in terms of three strength parameters: maximum load, deformation and stress. These are measured at three points of deformation - bioyield, break and peak points. These basic strength parameters have been measured and studied in Roma tomatoes at the bioyield point and break point [1], [2]. Other important strength parameters studied include energy absorption capacity and Young s modulus [3], [4] of the fruit. The strength parameters at the peak point denote the properties leading to the point of maximum load sustained by the vegetative tissues. The evaluation of strength parameters of tomatoes at this point is Corresponding author: F.A. Babarinsa. Tel.: address: fababarinsa@yahoo.com 31

26 essential for a clear understanding of the maximum stress which the tissues can withstand. It is useful therefore to study these properties from compression testing. This work investigated the peak (maximum) stress and corresponding deformation induced in packaged Roma tomatoes under compressive loading. Compression test was conducted to study the effects of ripeness stage, vibration level and type of container on the two strength parameters. 2. Materials and Method 2.1. Experimental material Fresh tomatoes of the Roma variety used were hand-harvested at three stages of maturity/ripeness from a local market farm in the suburb of Ilorin, Nigeria. The three stages The unripe stage is the mature green/breaker (or green pink) stage, consisting of the first point of skin colour change from complete green to about 30% pink. The half-ripe stage will consist of 30-70% pink to red skin while the ripe (or table ripe) stage with of % red skin but still firm. Stages of tomato ripeness were determined subjectively by skin colour rating [5]-[7] and objectively (using digital hand-held refractometer), as described in our previous work [2], Table.1: Statistical Analysis of Variance (ANOVA) of data on deformation at peak of Roma tomato fruit under compression Source Type III Sum Df Mean Square F Sig. of Squares Corrected Model 884,971 a 11 80, Intercept , , , Vibration 446, ,404 2, Container 35, , Ripeness 24, , Vibration*Container 172, ,457 1, Vibration*Ripeness 155, , Container*Ripeness.000 0,,, Vibration*Container*Ripeness.000 0,,, Error 3534, ,155 Total , Corrected Total Compression testing Compression tests were conducted, in a 2 x 3 2 factorial experiment, with a Testometric Universal Testing Machine installed in the Engineering Material Testing Laboratory at the National Center for Agricultural Mechanization (NCAM), Ilorin, Nigeria. This was applied in studying the effects of three ripening stages, three vibration levels and two containers on load, deformation and stress at peak point of Roma tomatoes under compressive loading. Roma tomatoes of three ripeness stages of 1) unripe (5.6 Brix%), 2) half-ripe (3.9 Brix%) and 3) full-ripe (3.2 Brix%), were packed in plastic crate [6]-[8], and raffia basket. A laboratory vibrator was used to apply vibration onto the fruits. The three levels of applied vibration are 1) non-vibrated 2) low vibration (frequency 3.7 Hz) and 3) high vibration (frequency 6.7 Hz). The basic methodology for compression testing of the packaged tomatoes (at a speed of 2.50mm/min) to obtain measurements of deformation (mm) and stress was described by Babarinsa and Ige [1], The measured values, at the peak point, were recorded on a computerized PC data acquisition system in a personal computer. Load-deformation plots were obtained directly as produced with the aid of the PC during compression Statistical Analysis 32

27 Data collected from compression test runs were subjected to statistical analysis using SPSS 110 software package in a randomized complete block design based on a 3 2 x 2 factorial experiment. Treatment means were compared using Duncan s Multiple Range Test (P< 0.05). Table 2: Statistical Analysis of Variance (ANOVA) of data on Stress at peak of Roma tomato fruit under compression. Source Type III Sum Df Mean Square F Sig. of Squares Corrected Model 9,562E-04 a 11 8,693E-05 8, Intercept 4,351E ,351E , Vibration 3,619E ,809E-04 17, Container 4,322E ,322E-04 41, Ripeness 3,565E ,782E-04 17, Vibration*Container 1,174E ,870E-05 5, Vibration*Ripeness 1,142E ,856E-05 2, Container*Ripeness.000 0,,, Vibration*Container*Ripeness.000 0,,, Error 4,322E ,029E-05 Total 5,730E Corrected Total 1,388E Results and Discussion 3.1. Load-deformation curve Force-deformation curves yielded by the compression test for all stages of ripeness, levels of vibration and containers indicated that deformation for the bulk is non-linear viscoelastic. The generated curves show sharp peaks at the end of each compression, rather than rounded peaks. This observed behavior in compression has been attributed to soft, weak brittle materials [9]. It is particularly noted that the point of maximum force or rupture could also occur at bioyield point Effects of stage of ripeness Fruit ripeness is an important factor that affects tomato compression tolerance. Stress was highest in the unripe stage regardless of containers and levels of vibration. Pereira & Calbo [10] reported that the riper the fruit the bigger is the effects of fruit compression, because ripe fruits have lager plasticity and elasticity. However the compressive measurements in this study demonstrated that ripeness stage did not have significant effect on deformation at peak (Table 1) of fruit at the three ripeness stages tested. Average deformation of the fruit at peak point showed minimal or no overall differences within comparative treatments. This was quite at variance with reported response of deformation in tomato when measured at both bioyield point [1] and break point [2], whereby deformation (at both bioyield and break points) reduced significantly with stage of ripeness. An apparent little or no variation in measured deformation with stage of ripeness may be explained by the fact that application of force (hence stress) beyond the peak point leads to rupture rather than further deformation. A study of ripening-related changes of the mechanical properties of the tomatoes by Andrews et al. [11]) shows that the epidermal cell walls contribute to a large extent to the mechanical properties of the tomato fruit exocarp. The epidermis also presumably plays a major role in resistance to turgor-driven tomato fruit growth ([6]-[11). The observed ripening-related changes of the mechanical properties of the skin seem to be determined mainly by modifications of the chemical composition of the cuticular membrane. Several studies have been carried out ([6]-[11]) noting that the mechanical properties of tomato fruit exocarp strips resulted from increasing fruit age, especially during ripening. A macromolecular explanation for the biomechanical behavior of fruit CM describes (i) greater stiffness associated with a glass state below the transition ripeness and (ii) plastic characteristics, being associated with a more viscous state, above the transition ripeness [12]. The strength properties at peak point of whole tomato fruit can be recognized as a limiting point to effect of imparted force in increasing deformation in the fruit. This point marks the beginning of energy dissipation 33

28 towards structural failure in the form of fracture leading to bruising, cracking or cutting ([6]-[11]). The amount of deformation of to mm, stress at peak point ranged from E-03 N/mm 2 to 9.956E-03 N/mm 2 at the three levels of ripeness. In most cases, the packaged tomatoes became cosmetically unacceptable, at the point of peak and this determined failure. The lowering of mechanical strength is an important part of the ripening process in tomatoes, as changes in cell walls accompany fruit softening [13]. For example, tomato fruit ripening is accompanied by significant degradation of cell wall pectin [14]. At both bioyield and break points ([1], [2]), deformation and stress reduced significantly with stage of ripeness, but the strength parameters at the peak point were unaltered by stage of ripening Effects of Vibration The increasing level of vibration caused an apparently little variation in measured deformation ranging from mm to mm with minimal differences among treatments. Increase in deformation recorded following the application of low-vibration to fruits was higher than increase in deformation caused by subsequent application of high-vibration. This is probably because the initially measured deformation seemed to be driven, in part, by the existence of interspaces (void) volumes within the bulk, the larger amount of which has been removed during the initial application of low-vibration. The analysis of results presented in Table 2 indicates that the level of vibration had highly significant (p = 0.001) effects on peak strength (or stress at peak). Stress at peak point decreased with increasing level of vibration level, with values ranging from 5.917E-03 to 1.274E-02 N/mm 2. Level of vibration, thus, displayed a tendency to decrease for both peak stress and deformation in unripe to fully ripe fruits. The decrease in stress at peak and increase in deformation at peak at high level vibration might be influenced by the breaking (or cracking) of fruit skin resulting from weakening of the cuticle at high level of vibration. The tissues of vibrated tomatoes had earlier undergone yielding, in which its ability to resist applied load was drastically reduced [1]. In particular, the observed relationship here between vibration and strength would indicate the effect of vibration on the structural relation of skin components. It has been reported that strength in a composite biomaterial, like tomato fruit cuticular membrane (CM), depends on the biomaterial components [12], [13], Bargel and Neinhuis [15] and Mattas et al. [14] particularly reported that CM contributes to the resistance of Cherry tomato fruit to tension mechanically. Application of vibration to bulk tomatoes, caused settlement to set in as the immediate effect of the rotational and relocational movement of the respective fruit in multilayer in vibration. Vibration force then relocates the individual fruits relative to other fruits in the bulk, leading to an initial compaction of the fruit bulk. Thus, application of external load (or force) first and foremost causes contact compression. The resulting self compaction increases the number of contact points, and eventually changes the distribution of energy dissipated into the fruit layers during subsequent inter-layer compression. 4. Conclusion Compression testing was used to characterize peak strength of the packaged tomatoes. This work has demonstrated that strength parameters at peak of the Roma tomato fruit depend more on level of imparted vibration and less on stage of ripeness, which both affected the structural component. Maximum deformation at peak was found to be constant at the range of ripeness and vibration tested. The peak strength parameters lead to a point where further compressive loading does not increase deformation. Our result demonstrates that the combined effect of ripeness and vibration seems critical in explaining the biomechanics of the fruit, and provides the basis for explaining mechanical failure such as fruit bruising and skin cracking. These findings can provide useful information about the influence of fruit properties (for example ripeness) on mechanical damage susceptibility, whereby bruise prediction, for example, can be made for cultivar Roma. Recorded test results could provide expected control limits for tomato fruit strength and provide a good baseline for material or design modifications. They would provide an accurate way of assessing the overall strength of a fruit bulk in filled container. 5. References [1] F. A. Babarinsa and M. T. Ige, Strength parameters of packaged Roma tomatoes at bioyield under compressive 34

29 loading, Food Science and Quality Management, vol. 4, pp , 2012a. [2] F. A. Babarinsa and M. T. Ige. Strength parameters of packaged Roma tomatoes at break point under compressive loading, International Journal of Scientific and Engineering Research, 3 (10): b. [3] F. A. Babarinsa and M. T. Ige. Energy absorption capacity of packaged Roma tomatoes under compressive loading, International Journal of Scientific and Engineering Research, 3 (10) 17-23, 2012c. [4] F. A. Babarinsa and M.T. Ige, Young's Modulus for Packaged Roma Tomatoes under Compressive Loading International Journal Of Scientific & Engineering Research, 3 (10): Research Volume 3, Issue 10, October -2012d. [5] N. N. Mohsenin, Physical Properties of Plant and Animal Materials. Gordion and Breach Science Publ., N. Y., [6] A. K. Thompson, Fruit and Vegetables. Harvesting, Handling and Storage. (2 nd Edn). Blackwell Publishing Ltd. Oxford, UK, pp 33-34, [7] W. B. McGlasson, B.B. Beattie, and E.E. Kavanagh, Tomato ripening guide, Agfact, H8.45, NSW Department of Agriculture, W. B. McGlasson, B.B. Beattie, and E.E. Kavanagh, Tomato ripening guide, Agfact, H8.45, NSW Department of Agriculture, [8] NSPRI, Storing Your Produce, Advisory Booklet No.4: Fruits and Vegetables. Nigerian Stored Products Research Institute, Ilorin, Nigeria, pp , [9] P. J. Fellows, Food Processing Technology, Principles and Practice. Woodhead Publishing Company Limited, Great Abington Press, San Diego, CA, pp , [10] A. V. Pereira, and A.G. Calbo, Elastic stresses and plastic deformations in Santa Clara tomato fruits caused by box dependent compression. Pesquisa Agropecuária Brasileira, Brasília, 35(12): , [11] J. Andrews, S. R. Adams, K. S. Burton, and R. N. Edmondson (2002). Partial purification of tomato fruit peroxidase and its effect on the mechanical properties of tomato fruit skin. Journal of Experimental Botany 53, [12] J. Matas, E. D., Cobb, J. A Bartsch,. D. J. Paolillo, and K. J. Niklas, Biomechanics and anatomy of Lycopersicon Esculentum fruit peels and enzyme treated samples. Am. J. Bot., 91(3): , [13] K.J., Nunan, I.M. Sims., A. Bacic, S.P. Robinson, and G.B. Fincher, Changes in cell wall composition during ripening of grape berries. Plant Physiol. 118, , [14] Errington, J. N. R. Mitchell, and G.A. Tucker, Changes in the force relaxation and compression responses of tomatoes during ripening: The effect of continual testing and polygalacturonase activity. Postharvest Biol. Technol. 11, , [15] H. Bargel, and C. Neinhuis Altered tomato (Lycopersicon esculentum Mill.) fruit cuticle biomechanics of a pleiotropic non ripening mutant. J Plant Growth Regul. 2004, 23:

30 2014 5th International Conference on Food Engineering and Biotechnology IPCBEE vol.65 (2014) (2014) IACSIT Press, Singapore DOI: /IPCBEE V65. 8 Monitoring Mango Fruit Ripening after Harvest using Electronic Nose (znose TM ) Technique Farhad Gholizadeh Nouri 1, Zhiyuan Chen 1 and Mehdi Maqbool 2 1 School of Computer Science, Faculty of Science, The University of Nottingham Malaysia Campus, Jalan Broga, Semenyih, Selangor Darul Ehsan, Malaysia 2 Crops For the Future Research Centre, Level 2 Block B, The University of Nottingham Malaysia Campus, Jalan Broga, Semenyih, Selangor Darul Ehsan, Malaysia Abstract. Over the past few years, electronic nose technology is offering a non-destructive method to sense aroma, which can be used to determine fruit ripening stages after harvesting. The objective of this study was to monitor mango fruit ripening after harvest using electronic nose (znose TM ). Data acquisition was started using an electronic nose for a total of one hundred locally grown mangoes cv. Chokanan. The fruits were divided into two different groups, unripe and ripe mangoes. Concentration of volatiles was measured using electronic nose for each of the two groups during storage. However, to be able to classify the mangoes into three classes, namely unripe, ripening and ripe, the experiment was carried out with the unripe samples for three more days, every 48 hrs from the starting day. Besides, observation of new volatiles from the unripe mangoes as they were ripening indicated trend of volatiles during mango ripening, proving the efficacy of electronic nose for the formation of climacteric crops profiles in terms of volatiles liberated during ripening. Keywords: Maturity analysis, climacteric fruits, Non-destructive method, znosetm 1. Introduction To monitor the ripening process in fruits during storage has become a very important issue to manage fruits because the quality of fruits and other properties are dependent on the ripening stages. There have been many methods or techniques to monitor the ripening of fruits during storage and most of them are basically dependent on firmness and texture [1]. Some of the other techniques require destructive analysis of the samples which is one the major limitation or disadvantage and therefore, they are not practically feasible to use. Thus, the estimation of the right ripening stage is completely dependent on hands-on experience or visual observations such as colour change, which is sometimes less correlated with the actual ripening. Although testing firmness or measuring starch, sugar and acid content of fruits are among available methods of maturity determination, they are all considered as destructive methods [2]. However, an excessive research has been carried out to develop non-destructive methods for measuring fruit maturity. In fact, it has been reported that an alternative method to determine maturity level of fruits during their ripening stages is the utilization of electronic olfactory systems to sense the volatiles liberated [3]. Being classified as climacteric fruit, mango undergoes some chemical reactions during its ripening stages and therefore, liberate certain volatile organic compounds (VOCs) that can be measured non-destructively using electronic nose technology [4]. Based on this idea, a relatively different electronic nose (znose ) has been used in this study to measure the concentration of volatile compounds liberated by mango during ripening stages. The main objective of this study was to gather volatile compounds data in mangoes using electronic nose and then decide the right time of harvest so that the fruits can stay longer in the market without losing the important nutrients. Corresponding author. Tel.: + 6 (03) ; fax: +6 (03) address: mehdi.maqbool@cffresearch.org. 36

31 2. Materials and Methods 2.1. Fruit material A total of one hundred local mango fruits (cv. Chokanan) were divided into two groups: 50 unripe mangoes with an average weight of g, and 50 ripe mangoes with an average weight of g. Using an electronic nose (znose ), concentration of volatiles was measured for each of the two groups during two separate days. However, to be able to classify the mangoes into three classes, namely unripe, ripening, and ripe, the experiment was carried out with the unripe samples for three more days, every 48 hours from the beginning day, so as to measure the concentration of volatiles liberating during mango ripening stages Electronic nose Electronic nose systems are sensor arrays which mimic the operation of a human nose. When an atmosphere loaded with volatile components flows over it, each sensor generates a signal. The combined signal of all sensors is then statistically related to, e.g. the response of a human taste panel. Sensors that rely on chemical properties of the target molecule, whether it can adsorb at a particular surface or be oxidized or reduced, have been developed for a variety of analytes. Popular at present are sensors based on the conduction of semiconductors, such as in tin oxide, or polymers such as polypyrrole [5] How znose TM works Unlike other electronic noses whose detector unit contain an array of metal oxide semi-conductor sensors, each being responsible for detecting one or more volatiles [3], znose Model 4200 used in this project uses an electronic detector. The system consists of a sensor head, a support chassis, and a system controller and its basis is (gas) chromatography Kovats Retention Indices Kovats index of a sample component is defined as a number, obtained by interpolation (usually logarithmic), relating the adjusted retention volume (time) or the retention factor of the sample component to the adjusted retention volumes (times) of two standards eluted before and after the peak of the sample component [6] (Table 1). Table 1: List of volatile compounds verified during different ripening stages along with their corresponding Kovats Retention Index for DB-5 column [7]. Volatile Organic Compounds (VOCs) Kovats Retention Index Volatile Organic Compounds (VOCs) Kovats Retention Index Ethanol 668 α-pinene 934 Toluene 773 β-pinene 990 Hexanal Carene 1011 Myrcene 994 α-terpinene 1018 Limonene 1036 γ-terpinene 1074 cis-3-hexenal 795 o-cymene 1020 p-cymene 1033 α-terpinolene 1096 Octanal 1006 Heptenal 957 cis-3-hexenol 858 Decanal 1209 Methyle Decanoate 1328 α-copaene 1391 β-caryophyllene 1467 α-humulene 1452 Cedrol Results and Discussion 3.1. Trend of VOCs liberated Results obtained for unripe, ripe, and ripening mangoes indicate that a total of 17 volatiles out of the 23 expected volatiles were liberated during five days of measurements (Fig. 1). The most probable reason for observing less VOCs than expected is that VOCs listed in table one are collectively liberated from different types of mangoes, whereas the mangoes tested in this experiment were all of one local type, i.e. Chokanan. 37

32 Although some of the volatiles were uniquely liberated from the ripe and unripe mangoes (on the first day measurements), results obtained on the following days indicated that as unripe mangoes were ripening, some of the VOCs liberated from the ripe mangoes were also liberated from the ripening mangoes (Table 2). Fig. 1: Number of VOCs liberated based on sample maturity. Table 2: List of VOCs liberated based on samples ripeness. VOC name Samples Ripe Unripe Ripening γ-terpinene α-terpinolene Decanal α-humulene Octanal 3-Carene Cedrol Methyle Decanoate α-copaene β Caryophyllene β-pinene Ethanol α-pinene Myrcene Toluene p-cymene As one would expect, the range of maximum peaks observed for each of the VOCs was quite wide (Fig. 2). In fact, the least maximum concentration measured with a value of 170 was the maximum amount of Ethanol liberated from a ripe mango. Whereas the most maximum concentration measured was the amount of Decanal liberated from an unripe mango. It is not quite obvious from the graph, γ-terpinene is the prominent VOC among the ripe mangoes, whereas Decanal and α-terpinolene are the most prominent VOCs among the unripe mangoes. While α-terpinolene was never observed in the ripe mangoes, Decanal was observed in all ripe samples; but with a drastic decrease in value. Values of γ-terpinene and Decanal in the data acquired indicated that the value of Decanal among the unripe samples is always (much) more than the value of its corresponding γ-terpinene value for each of the samples. However, this situation reverses in all but three of the ripe samples. Although the number of ripe mangoes volatiles is roughly twice as much as that of the unripe mangoes, of the eight volatiles observed 38

33 specifically in the results obtained for the ripe samples, only Methyle Decanoate was present in all the 50 mangoes (Fig. 3). Fig. 2: Maximum amount of each of the VOCs liberated from ripe and unripe mangoes. Fig. 3: Ripe-Only Volatiles Count. Finally, comparison of the first and third columns of table 2 reveals that during the ripening stages of the mangoes, six more volatiles (that were not present on first day measurements) were liberated. However, the bar charts (Fig. 4), indicates that the peaks of some of the volatiles liberated from the ripe mangoes, such as methyle decanoate or beta-pinene, are by no means comparable to those of the ripening mangoes. 4. Conclusion Fig. 4: Comparison of the peaks of common volatiles in ripe and ripening samples. It can be concluded from the results that the trend of liberating VOCs during ripening using znose TM could potentially be used to predict mango fruit maturity which can help to harvest the fruits on a right maturity stage. However, using reverse engineering method, znose can be used to form an initial list of volatiles for any unripe, ripening, and ripe crop. Once the list is ready, it can be verified using chemical methods and if correctly distinguished, the volatiles can be used in an experiment similar to the one used in this study for the predication of any crop s maturity and help farmers plan for optimal harvest time. 39

34 5. Acknowledgements The authors would like to thank the Crops For the Future Research Centre for providing partial financial support for the studies and Mr Tibby Lim from Ameritech Sdn Bhd for his guidance to understand the working mechanism of electronic nose (znose TM ). 6. References [1] J. Wang, B. Teng, Y. Yu. Pear dynamic characteristics and firmness detection, Eur. Food Res. Technol. 2004, 218 (3): [2] A. Kader. Postharvest Technology of Horticultural Crops. 3 rd ed., University of California Publication, [3] A.H. Gómez, G. Hu, J. Wang, A.G. Pereira. Evaluation of tomato maturity by electronic nose. Comput. Electron. Agric. 2006, 54(1): [4] M. Lebrun, A. Plotto, K. Goodner, M. Ducamp, E. Baldwin. Discrimination of mango fruit maturity by volatiles using the electronic nose and gas chromatography. Postharvest Biol. Technol. 48(1): [5] W.J. Florkowski, S.E. Prussia, R.L. Shewfelt, B. Brueckner. Postharvest Handling. 2nd ed. A Systems Approach (Food Science and Technology) Academic Press, [6] A.D. McNaught, A. Wilkinson. IUPAC Compendium of Chemical Terminology. 2nd ed. International Union of Pure and Applied Chemistry, [7] NIST Chemistry WebBook

35 2014 5th International Conference on Food Engineering and Biotechnology IPCBEE vol.65 (2014) (2014) IACSIT Press, Singapore DOI: /IPCBEE V s rrna Gene Sequencing and Analysis of Marine Bacterium for Biomedical Applications C.Chellaram 1, R.Sivakumar 2, G.Murugaboopathi 3, A. Alex John 1 and M.M.Praveen 1 1 Dept. of Biomedical Engineering, 2 Dept. of Electrical and Electronic Engineering, 3 Dept. of Information Technology, Vel Tech Multitech Dr.Rangarajan Dr.Sakunthala Engineering College, Chennai, Tamilnadu. India Abstract. A marine epibiotic bacterial strain A4 was isolated from the coral Subergorgia suberosa from Tuticorin coast, Gulf of Mannar region, south east coast of India. Phylogenetic identification based on comparative sequence analysis of 16S rrna gene indicated that the stain A4 fell under the genera Marinobacterium. The initial screening using agar overlay method the Marinobacterium strain A4 was found to exhibit broad spectral activity against Escherichia coli and Candida albicans.the highest zones of 8mm and 9mm were noted against the strain Escherichia coli and Candida albicans respectively. The culture broth was ethanol precipitated, and the activity was noted in the precipitate (crude extract). This present study highlights the importance of epibiotic bacteria associated with corals as a potential source for the discovery of novel antimicrobial and other natural products. Keywords: bacillus, Epibiotic, Subergorgia suberosa, Candida albicans, Antimicrobial activity 1. Introduction Surface-attached bacteria grow on submerged biotic and abiotic surfaces in the marine environment [1], [2]. Marine invertebrates in particular have diverse communities of attached bacteria on their surfaces [3], [4]. The microbial compounds are most prominent source for discover and production for new drugs [5], [6]. Antibiotics from marine microorganisms have been reported, including loatins from Bacillus produce both antibiotics and several bioactive substances [7]. The ocean remains as an unexploited source for many drugs and for the pharmacologically active substances [8]. The Marine organisms that are present in the environments are extremely rich in source of bioactive compounds [9]-[11].Marine bacteria are important for maintenance of carbon dynamics in marine ecosystem. The presence of variety of heterotrophic bacteria and their importance is very well recognized for sustained ecological and biogeochemical cycle in marine environment [12]-[14]. In this study, we have isolated and identified a marine bacterium from sea coral Subergorgia suberosa, in Gulf of mannar, Southeast coastal region waters of India. The strain was found to possess antimicrobial compounds against E.coli and Candida albicans. Initial screening was done against five human pathogens by Agar overlay method. Ehanol extract of the strain was obtained and tested against the test organisms by well diffusion method. 2. Materials and Methods 2.1. Collection of coral samples The bacterial strains were collected from surface of gorgonian coral Subergorgia suberosa by SCUBA Isolation of bacteria Corresponding author. Tel.: ; fax: address:chellarampublications@gmail.com 41

36 The cotton swab was then directly swabbed on to Zobell marine agar plates. Plates were incubated at room temperature for 7 days and from the 5th day on colonies of different morphotypes were isolated and repeatedly streaked on to Zobell marine agar plates to obtain pure cultures. The pure cultures were then stored at 4 C in marine agar slants until further studies Screening for antibiotic production Antibiotic production by marine bacteria was carried out by following the standard agar-overlay method. Initially the marine strain were spotted on Zobell marine agar plates and allowed to grow for 5 days. Test strains E. coli, K.pnemoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans were gently overlaid using soft agar over the marine strain. The soft agar was prepared by inoculating 1ml of test strain in 100 ml of soft agar (0.75% agar) and mixing thoroughly. For marine strains 1.5% NaCl was added to the soft agar. The overlaid plates were then incubated at 37 C for 24h and the zones of inhibition (measured from the edge of the colony to the edge of the clear zone) were recorded Cold-ethanol precipitation The cold ethanol precipitation of the culture broth was carried out following the slightly modified method of Schubert and Finn (1981). The Bacillus strainsg3culture was prepared as mentioned above. To the supernatant two volumes of ice-cold ethanol was added gradually simultaneously agitating with a magnetic stirrer. When the solvent addition was complete, the culture was agitated at 4 C for at least 60min. The culture was then placed in an ice bucket and left overnight inside a cold room (4 C). The precipitate was separated from the supernatant by centrifugation at 7000rpm for 30min in 4 C. The precipitate was dried in room temperature to remove the ethanol and then dissolved in 5ml of MilliQ water. The antimicrobial activity of the ethanol precipitate was carried out using agar-well diffusion method Agar well diffusion assay The agar well diffusion assay was carried out using the modified Stein et al., (2002) method. Tryptic Soy Agar (TSA) was used as the assay medium. TSA was prepared by adding 3g Tryptic Soy broth powder (Himedia, Mumbai, India) and1g of low electro end osmosis (EEO) Agarose in 100ml of double distilled water. Hundred micro liters of the extracts (ethanol precipitate/ crude biofilm) were poured into wells (6-mm diameter) of TSA plates previously seeded with the test strains. Plates were placed at 4 C for 4 to 6h to allow diffusion of the substance into the agar, and their contents were subsequently incubated for 12 to 18 h at 37 C. The presence or absence of inhibition zones around the wells was recorded Molecular identification and phylogenetic analysis Single isolated colony of the strain was taken from the agar plate and suspended in 50μl of lysis buffer (10mM Tris-HCl, ph 7.5; 10mM EDTA and 50μl/ml of proteinasek). The reaction mixture was then incubated at 55 C for 15min followed by proteinasek inactivation at 80 C for 10 min. The reaction mixture was then centrifuged at 15,000 rpm at 4 C for 15 min. The supernatant that contains genomic DNA was directly used as template in PCR reaction. PCR amplification of almost full-length16ss rrna gene was carried out with eubacteria specific primer set 16F27N (5 -CCAGAGTTTGATCMTGGCTCAG-3 ) and 16R1525XP (5 -TTCTGCAGTCTAGAAGGAGGTGWTCCAGGC-3 ) (Pidiyar et al., 2002). A 25 l reaction volume PCR was performed using about 10ng of the genomic DNA, 1X reaction buffer (10mM Tris-HCL, ph 8.8 at 25 C, 1.5mM MgCl2, 50mM KCl and 0.1% Triton X-100), 0.4mM deoxynucleoside triphosphates (Invitrogen), 0.5U DNA Polymerase (New England Labs, UK). The PCR was performed in an automated Gene Amp PCR system 9700 thermal cycler (Applied Biosystems, Foster City, USA) under the following conditions. The amplification conditions were as follows: 94 C for 1 min (denaturation), 55 C for 1 min (annealing), 72 C for 1.30 min (elongation) at and 72 C for 10 min final elongation. Expected PCR product of around 1.5 Kb was checked by electrophoresis with 5μl of the PCR product on 1% agarose gel in 1X TBE buffer and stained with ethidium bromide 0.5 μl/ml. The PCR product was precipitated by PEG- NaCl (20% PEG in 2.5 M NaCl) precipitation at 37 C for 30 min. The reaction mixture was centrifuged at 12,000 rpm for 30 min at room temperature. The supernatant was discarded and the pellet was washed twice with 70% ethanol. After drying the pellet was resuspended in 5μl of sterile nuclease-free water. One microliter (~ 50ng) of purified PCR product was sequenced as described earlier (Pidiyaret al., 2002). The analysis of the sequence was done at NCBI server ( whereas the 42

37 alignment of the sequence was done using CLUSTALW programmed at European Bioinfomatics site ( Trees were constructed using the MEGA Software version Results 3.1. Bioactivity The Bacillus strain A4 was found to exhibit broad-spectral activity (agar-overlay method), inhibiting the growth of Escherichia coli and Candida albicansstrain. The bacteria secreted metabolites that were both antibacterial (Figure.1) as well as antifungal in activity. Maximum antibacterial activity was found against Candida albicans (Figure 2) Fig. 1: Strain Serinicoccus marinus Fig. 2: Activity of A4 strain against E.coli 3.2. Ethanol extract The crude extract (ethanol) was found to be active against E. coli and Candida albicans Molecular identification and phylogeny The strain initially designated as A4 when isolated, was identified in the genus Marinobacterium, employing 16Ss rrna gene sequence method. Phylogenetic analysis based on comparative analysis of the sequenced 16Ss rrna indicated that the strain was closely related to Serinicoccus marinus strain (Figure 3). 4. Discussion The discovery of new antibiotics is so important due to the increasing incidence of multiple resistant pathogenic microorganisms to drugs that are currently using in clinical treatment. In the marine environ-ment, 90% of bacteria are Gram-negative with different characteristics and the Gram-negative cell wall is better adapted for survival in the marine environment [15-16]. We have successfully culture a strain designated A4, isolated from the surface of a coral. Using molecular phylogeny tools such as 16s rrna sequencing, which confirmed that the strain A4 was closely related to Serinicoccusmarinusstrain. We have shown that the recently discovered marine natural product A4 possesses potent in vitro antimicrobial activity against strain of E.coli and Candida albicans.future studies will further re-find the activity of A4 analogs from marinederived actinomycetes for characterization and optimization of activity toward further preclinical development.further study is necessary for purification of compound using High PerformanceLiquid Chromatography (HPLC) and for structure and functional group elucidation of thecompound by using Nuclear Magnetic Resonance (NMR) and Infra-red (IR) spectroscopy, inorder to practice it for pharmacological advantage. 43

38 5. Acknowledgements Fig. 3: Phylogenetic tree of the strain A4 Authors deliver their sincere gratitude to SERB-DST (Young Scientist Award, No.SR/FT/LS-23/2010) Govt. of India for financial support. 6. References [1] C. Chellaram, T. Prem Anand, G. Kuberan, A. Alex John, G. Priya, B. Arvind Kumar. Anti-inflammatory and analgesic effects of coral reef associated gastropod, Trochus tentorium from Tuticorin coastal waters, Southeastern India. African Journal of Biotechnology, 2012, 11(80): [2] WM. Dunne. Bacterial adhesion: seen any good biofilms lately. Clinical Microbiology Reviews. 2002, 15(2): [3] F. Rohwer, V. Seguritan, F. Azam, N. Knowlton. Diversity and distribution of coral-associated bacteria. Marine Ecology Progress Series. 2002, 243: [4] C. Chellaram, R. S. Sreenivasan, T. P. Anand, S. Kumaran, D. Kesavan, G. Priya. Antagonistic Bacteria From Live Corals, Tuticorin Coastal Waters, Southeastern India. Pakistan Journal of Pharmaceutical Science. 2011, 24: [5] C. Chellaram, R. S. Sreenivasa, S. Jonesh, T. Prem Anand, JKP. Edward. In vitro Antibiotic bustle of coral reef associated gastropod, Drupa margariticola (Broderip 1832) of Tuticorin coastal waters, Southeastern India, Biotechnology. 2009, 8 (4), [6] W. Zhang, ZY. Li, XL. Miao, FL. Zhang. The screening of antimicrobial bacteria with diverse novel nonribosomal peptide synthetase (NRPS) genes from South China sea sponges. Mar Biotechnol. 2009, 11: [7] JM.Gerard, P.Haden, MT.Kelly, RJ. Andersen. Loloatins A-D, cyclic decapeptide antibiotics produced in culture by a tropical marine bacterium. J. Nat. Prod. 1999, 62: [8] K. Sivasubramanian, S.Ravichandran, M. Vijayapriya. Antagonistic activity of marine bacteria Pseudo alteromonas tunicate against microbial pathogens. Afr J Microbiol Res. 2011, 5: [9] C. Chelllaram, T. Prem Anand, C. Felicia Shanthini, B. Arvind Kumar. Sidharth P Sarma. Bioactive peptides from epibiotic Pseudoalteromonas strain P1, APCBEE Procedia, 2012, 2: [10] R. Solanki, M. Khanna, R. Lal. Bioactive compounds from marine actinomycetes. Indian J Microbiol : [11] K. Hong, AH. Gao, QY. Xie, H. Gao, L. Zhuang, HP. Lin. Actinomycetes for marine drug discovery isolated from mangrove soils and plants in China. Mar Drugs. 2009, 7: [12] WB. Whitman, DC. Coleman, WJ. Wiebe, Prokaryotes: the unseen majority. Proc Natl Acad Sci. 1999, 95:

39 6583. [13] MC. Austen, PJD. Lambshead, PA. Hutchings, G. Boucher, PVR. Snelgrove. Biodiversity links above and below the marine sediment water interfacethat may influence community stability. Bio divers Conserv. 2002, 11: [14] L. Zinger, LA. Amaral-Zettler, JA. Fuhrman, MC. Horner-Devine, SM. Huse. Global patterns of bacterial betadiversity in seafloor and seawater ecosystems. Plos one. 2011, 6: e [15] AR. White, Effective antibacterials: at what cost? The economics of antibacterial resistance and its control. J Antimicrob Chemother. 2011, 66: [16] C. Chellaram, TP. Anand, MM. Praveen, G. Murugaboopathi, R. Sivakumar, B. Arvind Kumar, S. Krithika. Selflife Studies on an Underutilized Sea Food from Southeast Coast of India, International Conference on Agriculture and Animal Sciences, (CAAS 2013), APCBEE Procedia, 2013, 8: In Press. 45

40 2014 5th International Conference on Food Engineering and Biotechnology IPCBEE vol.65 (2014) (2014) IACSIT Press, Singapore DOI: /IPCBEE V Neuroprotective Effects of Soybean Oligopeptides (SOPs) Against H 2 O 2 -induced Oxidative Stress in PC12 Cells Jingbo Liu 1, Wenchao Liu 1, Dan Liu 1, Menglei Xu 1 and Yan Zhang 1 Laboratory of Nutrition and Functional Food, Jilin University, Changchun, , P.R. China Abstract. In the study, soybean protein isolate was hydrolyzed with Alcalase, hydrolysates were separated by membrane ultrafiltration to obtain SOPs. We examined the antioxidant properties of SOPs, including Fe 2+ chelating capacity, free radical scavenging activities against 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), oxygen radical absorbance capacity (ORAC), and inhibiting the autoxidation of linoleic acid capacity. Then the neuroprotective effect of SOPs against H 2 O 2 -induced lipid peroxidation and cell death in PC12 cells was evaluated. The PC12 cell line pretreated with different concentrations (0.1, 0.5, and 1 mg/ml) of the SOPs and then treated with H 2 O 2 to induce lipid peroxidation and neurotoxicity. The neurotoxicity was assessed by cell viability, lactate dehydrogenase (LDH) release and morphological characteristics. The results indicated that SOPs exhibited potent antioxidant activities, and pretreatment of PC12 cells with SOPs, prior to H 2 O 2 exposure, significantly increased the survival of cells, and reduced the levels of LDH. These findings suggest that SOPs can protect PC12 cells against H 2 O 2 -induced lipid peroxidation and cell death as a neuroprotective agent. Keywords: neuroprotective, PC12 cells, Soybean oligopeptides, Oxidative stress 1. Introduction The number of people suffering from neurological disorders has been increased worldwide, especially in the developed countries [1]. Oxidative stress as a result of aberrant production of reactive oxygen species (ROS) is the major culprit in neuronal cell degeneration observed in neurodegenerative diseases, such as Alzheimer s disease, Parkinson s disease [2]. The imbalance between oxidants and antioxidants leads to disruption of redox signaling leading to accumulation of ROS. Excessive generation of free radicals causes damage to all kinds of biomolecules, such as lipids, proteins and DNA, in addition, lipid peroxidation is the main nerve toxic [3]. Therefore, current attention has been dedicated to find natural neuroprotective agents that can reduce oxidative stress in neurons, might be an appropriate choice for the treatment of neurodegenerative diseases [4]. Soybean based food has a long lasting tradition, especially in Asia. Moreover, soybean protein is a potential dietary source of bioactive peptides, enzymatic hydrolysis of proteins is one effective approach that can be used to release various bioactive peptides [5]. The soybean protein-derived peptides have been reported to possess a wide variety of biological activities such as antihypertensive, antithrombotic, opioid, antiamyloid and antioxidant activities. Previous studies have also shown that SOPs through Alcalase have relatively higher antioxidant activities and antiamyloid effects [6], and indicate that SOPs may reduce oxidative stress in neurons as neuroprotective agents. However, the direct effect of SOPs on the neuroprotective efficacy has not been investigated. Accordingly, the present study aimed to evaluate the potential neuroprotective effect of SOPs on H 2 O 2 -induced oxidative stress and neurotoxicity and in PC12 cells. 2. Materials and methods Corresponding author. Tel.: ; fax: address: @163.com. 46

41 2.1. Materials The soybean protein isolate was obtained from Harbin Hi-Tech Soybean Food co.,ltd (Harbin China). Alcalase 2.4 L (2.4 AU/g) was purchased from Novo Nordisk (Bagsvaerd, Denmark). The lactate dehydrogenase (LDH) assay kits was purchased from Beyotime Institute of Biotechnology (Haimen, China). Dulbecco's Modified Eagle's Medium (DMEM) and Fetal bovine serum (FBS) was purchased from Gibco BRL, Life Technologies (USA). EDTA, DPPH, Fluorescein, AAPH, Trolox were purchased from Sigma- Aldrich (USA). MTS from Promega (Msdiso, USA) and other chemicals were purchased from Beijing Chemical Plant (Beijing, China) Preparation of SOPs Soybean protein isolate was dispersed in distilled water to obtain 10% protein slurry (w/v) was hydrolyzed in a 1000 ml reactor under the controlled temperature and ph. The mixture was heated to 90, for 10 min, in order to denature the protein. The ph value was adjusted to 8.0 using 1 mol/l NaOH and incubated in 55 water bath. The protein hydrolysis was initiated by the addition of a AU/g dosage of Alcalase based on protein content and stirred for 3 h. After the hydrolysis reaction was terminated by boiling for 10min and the solutions were centrifuged at rpm for 15min at 4 to obtain soybean protein isolate hydrolysates. The hydrolysates were ultra-filtered through a membrane with 1 kda nominal molecular weight limit to obtain the soybean oligopeptides. The SOPs were freeze-dried and stored at -20 before further analysis DPPH radical-scavenging activity The DPPH radical-scavenging activity of SOPs was measured according to the previously reported method with some modifications [7]. An aliquot of 100 μl 0.1 mm DPPH (dissolved in 95% ethanol) was mixed with the same volume of sample solution. The mixture was shaken and left for 30 min at room temperature. The absorbance of the resulting solution was measured at 517 nm. The blank substituted 100 μl of buffer solution instead of the sample, and Trolox was used as a standard Metal ion-chelating activity The ability of SOPs to chelate Fe 2+ ions was evaluated according to the previously reported method with some modifications [8]. Briefly, 50 μl sample solution was mixed with 50 μl of 0.2 mm ferrous chloride solution. After 3 min the reaction was initiated by the addition of 200μL of 0.5 mm ferrozine. The mixture was shaken vigorously and left at room temperature for 10 min. Absorbance was measured at 522 nm to determine chelating activity using EDTA as a standard Measurement of oxygen radical absorbance capacity (ORAC) The ORAC assay was conducted to kinetically measure the peroxyl radical scavenging activity of SOPs with Trolox as the antioxidant standard [9]. Fluorescein was used as the fluorescent probe and the peroxyl radicals were generated from AAPH in 75 mmol/l phosphate buffer (ph 7.4). Excitation and emission wavelengths were 485 and 530 nm, respectively. Trolox equivalents (TE) were calculated using the relative area under the curve for samples compared to a Trolox standard curve prepared under the same experimental conditions Inhibition of linoleic acid autoxidation The lipid peroxidation inhibition activity of SOPs was measured in a linoleic acid model system according to the method of Osawa and Namiki [10] with some modifications. The sample dissolved in 5 ml of 50 mm phosphate buffer (ph 7.0) was added to a solution of 65 μl of linoleic acid and 5 ml of 99.5% ethanol. The total volume was then adjusted to 12.5 ml with distilled water. The mixture was incubated in at 60 ± 1 for 7 days in a dark room. The degree of oxidation of linoleic acid was measured using the ferric thiocyanate method. Briefly, 0.1mL of the reaction mixture was mixed with 4.7 ml of 75% ethanol, 0.1 ml of 300 g/l ammonium thiocyanate and 0.1 ml of 20 mm ferrous chloride solution in 35% HCl. After 3 min of incubation the colour development, which represents linoleic acid oxidation, was measured at 500 nm Cell culture 47

42 The PC12 cell line was obtained from the Institute of Biochemistry and Cell Biology, SIBS, CAS (Shanghai, China). Cells cultured in DMEM supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin in an incubator aerated with 5% CO 2 at 37 C Analysis of cell survival The metabolic status of the mitochondria of PC12 cells was analyzed by the MTS assay. The cells were seeded ( cells/800 μl/well) and cultured in 24-well plates. The cells were then pre-incubated with SOPs for 24 h followed by incubation with or without 200 μm H 2 O 2 for 12 h. After that, 100 μl of MTS (0.5 mg/ml) was added to each well and then incubated for 1h at 37, the absorbance was determined at 490 nm using a microplate reader. The value for cell viability was converted to the percentage of control culture value. The morphology of the cells was also observed and photographed under a microscope LDH leakage The LDH leakage was detected with an assay kit (Beyotime, Haimen, China) according to the manufacturer s protocol. The culture medium was collected and retained for LDH determination. The adherent cells were washed twice with PBS and then lysed by the cell lysis buffer to release the intracellular LDH of the living cells into the new supernatant. After the reaction, each sample was measured at wavelength of 450 nm. LDH leakage was calculated as the ratio of released LDH to total LDH Statistical analysis The results were expressed as means ± SD. Differences among different experimental groups were tested for significance using one-way analysis of variance, taking **P < 0.01 as significant. 3. Results and Discussion 3.1. Antioxidant capacity of SOPs The antioxidant activities of SOPs were estimated by various antioxidant methods, including DPPH scavenging, Fe 2+ chelating, ORAC and inhibition of linoleic acid autoxidation assays. As shown in Fig. 1A Fig. 1: Antioxidant activities of SOPs, DPPH scavenging activity (A), Fe 2+ chelating capacity (B), ORAC (C), inhibiting the autoxidation of linoleic acid capacity (D). The results are expressed as mean ± SD of three experiments. and B, SOPs exhibited DPPH radical-scavenging activity and Fe 2+ -chelating activity, in addition, the activity increased 48

43 with increasing peptide concentration. The ORAC value of SOPs was μmol Trolox equivalents per gram dried weight (Fig. 1C). As shown in Fig. 1D, SOPs showed lipid peroxidation-inhibitory activity in a linoleic acid model system. Lower absorbance at 500 nm indicated higher lipid peroxidation inhibition. Overall, the results suggest SOPs exhibited potent antioxidant activity Protective effect of SOPs against H 2 O 2 induced cytotoxicity As the major ROS, H 2 O 2 has been extensively used to induce oxidative stress, resulting in apoptosis or necrosis of PC12 cells. In our preliminary experiments, different concentrations of H 2 O 2 ( μm) induced injury on PC12 cells was assessed by MTS assay. As shown in Fig. 2A, the H 2 O 2 treatment decreased the cell viability in a dose-dependent manner with 31.5 % viability at 200 μm H 2 O 2 challenge which was used for further experiments. Then the neuroprotective effects of SOPs were investigated. The cells pretreated with different concentrations of SOPs (0.1, 0.5, 1 mg/ml) for 24 h before 200 μm H 2 O 2 treatment (12 h) showed significant improvement in cell survival up to 49.2 ± 3.1% with 1 mg/ml of SOPs, as shown in Fig. 2B. These data shows that SOPs could increase cell viability and offer the protection against H 2 O 2 -induced cell death. Fig. 2: A Cytotoxic effects of H 2 O 2 on PC12 neuronal cell. B Dose dependent protective effect of pretreatment of SOPs for 24 h on 12 h treatment of 200 μm H 2 O 2 -induced cytotoxicity in PC12 cells, the cell viability was determined by MTS assay. C The plasma membrane damage was analysed by LDH leakage and D H 2 O 2 -induced morphological alterations in PC12 neurons by phase contrast microscopy. The data are represented as mean ± SD of three independent experiments, *P<0.05 versus 200 μm H 2 O 2 treated group, **P<0.01 versus 200 μm H 2 O 2 treated group Protective effect of SOPs against plasma membrane damage The cytotoxicity of H 2 O 2 and the protective activity of SOPs were further evaluated by LDH assay. PC12 cells were pretreated with 1 mg/ml of SOPs for 24 h, before treatment with 200 μm H 2 O 2 for 12 h (Fig. 2C). The results show that the release of LDH of 71.4 % of total enzyme with 200 μm H 2 O 2 challenge which 49