Natural products from tomato peels (Lycopersicon esculentum variety “Hybrid Rome”)

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Abstracts / Journal of Biotechnology 131S (2007) S23–S31

6. Production of xylooligosaccharides from cotton and tobacco stalk Ozlem Akpinar 1,∗ , Kader Bakir 2 , Levent Yilmaz 2

Erdogan 1 , Ozlem

Ak 2 , Ufuk

1 Gaziosmanpasa 2 Middle

University, Tokat, Turkey East Technical University, Ankara, Turkey

Xylooligosaccharides (XOs) are non digestible oligosaccharides which have many beneficial health effects. XOs are produced from xylan rich materials. For production of XOs, among the xylan rich materials, the residual origin such as forestal, agricultural or industrial wastes of lignocellulosic nature were special interest since utilization agricultural biomass as raw material provides economic and ecological benefits. The driving force of this study was to test the suitability of cotton and tobacco stalks for production of XO. These are the residue remaining after the harvest. After cotton and tobacco stalks were dried and grounded, their chemical structure were characterized. After xylan was extracted from cotton and tobacco stalk with alkali, they were converted into XOs with enzymatic process. The hydrolysis condition was optimized by using different temperatures, substrate and enzyme concentrations. The results showed that XOs concentration increased as the reaction times increased. XOs concentration was characterized by a rapid increase in the reducing sugar depending on the substrate concentration. The time XOs production was shortened depending on enzyme concentrations. The xylan was hydrolyzed to a variety of oligosaccharides, xylopentaose and xylohexose being the major products followed by xlobiose and xylotriose. This study demostrated that cotton and tobacco stalk, which has no economical value, could be easily converted to more valuable product, XOs, by enzyme and these oligosaccharides could be used for further application. doi:10.1016/j.jbiotec.2007.07.042 7. Natural products from tomato peels (Lycopersicon esculentum variety “Hybrid Rome”) New challenges and new opportunities of application: Chemical, biotechnological and pharmacological Tommonaro Giuseppina 1 , De Stefano Daniela 2 , Pulsinelli Marianna 1 , Carnuccio Rosa 2 , Nicolaus Barbara 1 , Poli 1,∗ Annarita 1 Istituto di Chimica Biomolecolare, CNR Pozzuoli, Naples, Italy

others are utilised as industrial food additivies taking advantage of their chemical-physical properties such as emulsifying, viscoelasticity, polyelectrolyte, adherence, bio-compatible, stabilizer, etc., (Nicolaus et al., 1999; Poli et al., 2006). One of the main problems of food industry is the management of waste and their conversion in products of higher value. Modern technologies eco-compatible, promote the use of food waste to obtain biopolymers that can be re-used in the same sector of the raw materials (Strazzullo et al., 2003). We have studied the purification procedures of polysaccharide fraction coming from products and by-products of tomato industry (Lycopersicon esculentum). Its chemical composition, rheological properties and partial primary structure were determined on the basis of spectroscopic analyses (UV, IR, 1 H NMR). Moreover, the anticytotoxic activity of the polysaccharide by brine shrimp assay was also reported. In addition, we have investigated the effect of the polysaccharide on nitrite and ROS production in LPS-stimulated J774 macrophages for 24 h. The tomato peel polysaccharide inhibited in a concentrationdependent manner nitrite and ROS production as well as iNOS protein expression induced by LPS. Incubation of cells with polysaccharide determined a significant decrease of NFkB/DNA binding activity which was correlated with a marked reduction of iNOS mRNA levels. These results show that the polysaccharide inhibits NF-kB activation and iNOS gene expression by preventing the reactive species production, and suggest for this compound a role for controlling oxidative stress and/or inflammation (De Stefano et al., in preparation). The achievement of biodegradable and thermoplastic materials by using chemical and enzymatic processes from polysaccharides are also reported. The realization of three and more manufactured articles, for example flowerpots or rigid packing vases, film for agriculture use and ensiling film will be in progress. Tomato and tomato products represent a major natural source of several nutrients with beneficial effect on human health and their ability in the prevention of some major chronic diseases such as some types of cancer and cardiovascular disorders. Tomatoes represent an effective way to supply nutrients such as folate, vitamin C, and potassium, but the peculiar compounds of these vegetables are carotenoids, particularly beta-carotene and lycopene (Balestrieri et al., 2004). In order to assess the nutritional value of the waste material, the concentration of major carotenoids, lycopene and beta-carotene, contained in lipophilic extract, was also determined. The lipophilic antioxidant activity was evaluated by ABTS (2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) method (Strazzullo et al., 2007).

2 Dipartimento di Farmacologia Sperimentale, Universit` a degli

Studi di Napoli “Federico II”, Naples, Italy

References

The polysaccharides from natural sources have remarkable interest among several biotechnological products and they show a large range of applications in industrial field due to their commercial purposes. Some of them, for example, showing strong anti-genic and pathogenic activities, are employed successfully by the pharmaceutical industry for the formulation of vaccines,

Balestrieri, M.L., De Prisco, R., Nicolaus, B., Pari, P., Schiano Moriello, V., Strazzullo, G., Iorio, E.L., Servillo, L., Balestrieri, C., 2004. Free Radic. Bio. Med. 36 (8), 1058–1067. De Stefano, D., Tommonaro, G., Simeon, V., Poli, A., Nicolaus, B., Carnuccio, R. FEBS Lett., in preparation. Nicolaus, B., Panico, A., Lama, L., Romano, I., Manca, M.C., De Giulio, A., Gambacorta, A., 1999. Phytochemistry 52, 639–647.

Abstracts / Journal of Biotechnology 131S (2007) S23–S31 Poli, A., Manca, M.C., De Giulio, A., Strazzullo, G., De Rosa, S., Nicolaus, B., 2006. J. Nat. Prod. 69 (4), 658–661. Strazzullo, G., Schiano Moriello, V., Poli, A., Immirzi, B., Amazio, P., Nicolaus, B., 2003. J. Food Technol. 1, 102–105. Strazzullo, G., De Giulio, A., Tommonaro, G., La Pastina, C., Saturnino, C., Poli, A., Nicolaus, B., De Prisco, R., 2007. Int. J. Food Properties 10, 1–9.

doi:10.1016/j.jbiotec.2007.07.043 8. Enzyme-coupled procedure for xylose conversion into biotechnologically important intermediates Georgiana Petrareanu, Mihaela Balasu, Stefan Szedlacsek ∗ Institute of Biochemistry, Bucharest, Romania The great majority of the actual research studies dedicated to finding new ways to valuate (ligno)cellulosic residues are directed to fermentative procedures. Less attention has been paid so far to the discovery of processes leading to high-value compounds. To overcome this shortcoming, we suggest a coupled enzymatic reaction procedure which can convert xylose – one of the most important pentose in lignocellulosic wastes – to high value biosynthetic intermediates. Specifically, xylose is initially isomerized to xylulose by xylose isomerase and then xylulose is phosphorylated to xylulose 5-phosphate by xylulose kinase. Finally, xylulose 5-phosphate is converted by dxylulose 5-phosphate phosphoketolase to acetyl phosphate and glyceraldehyde 5-phosphate, both important intermediates for biosynthetic technologies. While the first two enzymes have been cloned and are well documented, cloning of phosphoketolase has been relatively recently reported (Meile et al., 2001). Therefore, we cloned the hypothetical phosphoketolase genes from four different organisms: Lactococcus lactis, Leuconostoc mesenteroides, Synechocystis sp. and Pseudomonas aeruginosa. Each gene was subsequently inserted in a prokaryotic vector, then expression was optimized and expressed proteins were purified. Enzyme kinetic characterization of purified phosphoketolases necessitated setting up of a small scale procedure for preparation of xylulose 5-phosphate substrate (commercially not available). Thus, enzyme stability data, optimal reaction conditions and enzyme kinetic parameters were used in a comparative study aimed to find the optimal phosphoketolase for our purpose. Finally, all three enzymes of the coupled reaction were immobilized on inert supports in three individual columns, at laboratory scale. Preliminary experiments performed on the serially connected columns evidenced the successful conversion of xylose to acetyl phosphate and glyceraldehyde 5-phosphate. Reference Meile, L., Rohr, L.M., Geissmann, T.A., Herensperger, M., Teuber, M., 2001 May. Characterization of the d-xylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase gene (xfp) from Bifidobacterium lactis. J. Bacteriol. 183 (9), 2929–2936.

doi:10.1016/j.jbiotec.2007.07.044

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9. Construction of amylolytic nuclear petite Saccharomyces cerevisiae strains for bioethanol production by SSF 1 , Stephen G. ¨ Doga Arslan 1 , Ozlem Ates 2,∗ , Ebru Toksoy Oner 3 4 Oliver , Bet¨ul Kirdar 1 Department

of Chemical Engineering, Marmara University Goztepe 34722, Istanbul, Turkey 2 Department of Chemical Engineering, Yeditepe University Kayisdagi 34755, Istanbul, Turkey 3 School of Biological Sciences, University of Manchester, Manchester, United Kingdom 4 Department of Chemical Engineering, Bo˘ gazi¸ci University Bebek 34342, Istanbul, Turkey The energy intensive high temperature cooking process followed by enzymatic hydrolysis of starch to fermentable sugars in the current bioethanol production process warrants novel and innovative new technology for reducing the total manufacturing cost. The aim of the present study was to develop a novel noncooking fermentation system utilizing amylolytic microorganisms that are capable of hydrolyzing starch into fermentable sugars by enabling depolymerization of starch to glucose in a simultaneous saccharification and fermentation (SSF) process for bioethanol production. Respiratory-deficient nuclear petite Saccharomyces cerevisiae strains hold a great potential for the commercial production of bioethanol. Therefore, for the construction of the amylolytic yeast strains, the nuclear petite S. cerevisiae FY23
pet191 strain was used as host. This 100% respiratory-deficient strain was generated using polymerasechain-reaction (PCR)-mediated disruption of the PET191 gene which encodes a protein required for the assembly of the polypeptide subunits that constitute the active cytochrome c oxidase holoenzyme (Hutter and Oliver, 1998). The constructed yeast strains were found to excrete a bifunctional fusion protein that contains both the Bacillus subtilis alpha-amylase and ¨ the Aspergillus awamori glucoamylase activities (Toksoy Oner et al., 2005). The fermentation performances of these strains pointed out the fact that in order to use these strains on industrial scale, both the copy number and stability of the genes encod¨ ing the amylolytic enzymes had to be improved (Toksoy Oner, 2006). In the light of this, a cloning strategy was developed and used in order to integrate multiple copies of the fusion protein gene into the ribosomal DNA (rDNA) locus of Saccharomyces cerevisiae that consists of 100–200 tandem copies of a 9.1 kb repeat on the right arm of chromosome XII. 2.7 kb long fragment containing the gene for 25S rRNA was amplified by PCR and cloned into pJET1 cloning vector to construct pJRD. In order to construct the PJBG plasmid, the DNA fragment containing the genes encoding for the amylolytic enzymes together with the LEU2 selective marker region was amplified by PCR and cloned into the unique HpaI site within the rDNA region of the pJRD. In order to avoid integrating unnecessary antibiotic marker or vector sequences, the fusion gene and marker region flanked by the rDNA sequences was amplified by PCR from the pJBG plasmid and directly introduced into S. cerevisiae FY23
pet191 cells by electroporation. Among