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GMO Traceability Using Very Short, Locked Nucleic Acid TaqMan Probes
S336
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
structures. The results showed a relationship between extent of cross-linking, apparent viscosity and CWP ratio of the yoghurt gels. During storage for up to 4 weeks at 4 ◦ C, no changes in rheological properties for stirred yoghurt gels prepared from cross-linked milk proteins.
aspects concerning performance characteristics, and the estimation of detection and quantification limits of LNA probes will be presented.
doi:10.1016/j.jbiotec.2010.09.349
[P-F.108]
[P-F.106]
Antioxidant Constituents of Functional Animal Protein
Effect of phenolic compounds on the growth and L-malic acid metabolism of Oenococcus oeni M. Iorizzo ∗ , S.J. Lombardi, T. DI Renzo, B. Testa, R. Coppola, E. Sorrentino Distaam - Universita’ Del Molise, Italy Keywords: Wine; Oenococcus Oeni; Phenolic; L-Malic acid In this work the effect of the most common phenol carboxylic acids, (gallic, caffeic and ferulic acids) and flavonoids (catechin and quercetin) on the growth and L-malic acid metbolism of Oenococcus oeni was investigated. A total of 143 strains, isolated from different wines, were assayed in a synthetic medium added with ethanol (12% v/v) and malic acid (5 g/L) and different amounts of each compound under analysis. The three carboxyl acids showed an inhibitory effect on growth of Oenococcus oeni at high concentrations; 1000 mg/L inhibited the growth, and inhibition by ferulic acid was greater than that registered for caffeic acid. As for gallic acid its presence in the medium at 500 mg/L stimulated the growth of assayed strains while higher concentrations, until 1000 mg/L did not inhibit the growth. Catechin had a stimulating effect, which increased with concentration. While quercetin showed a slight stimulating effect only at low concentrations. Flavonoids also stimulated the Malolactic fermentation. doi:10.1016/j.jbiotec.2010.09.350 [P-F.107] GMO Traceability Using Very Short, Locked Nucleic Acid TaqMan Probes Sergio Salvi ∗ , Fabio D’orso, Giorgio Morelli National Research Institute on Food and Nutrition (Inran), Italy Keywords: GMO; Real-time PCR; Locked nucleic acid (LNA); Standard reference plasmids Many countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms (GMO). Real-time quantitative PCR based upon the TaqMan probe chemistry has become the mostly used technology to support these regulations, and event-specific PCR is the preferred method in GMO detection because of its high specificity based on the flanking sequence of the transgenic integrant. The aim of this study was to evaluate the use of 8-nucleotide long locked nucleic acid (LNA) probes in 5 -nuclease PCR assays for the detection and quantification of GMO. Conventional TaqMan and LNA probes were compared for the analysis of the maize MON810 transgene. The performance of the two types of probes was tested on the CaMV 35S promoter, the hsp70/cryIA(b) construct as well as for the eventspecific 5 integration junction of MON810, using the maize hmga as endogenous reference gene. Specific plasmids, developed in our laboratory, were used as standard reference molecules. Several
doi:10.1016/j.jbiotec.2010.09.351
I.V. Nikolaev 1,∗ , F. Lambertini 2 , V.P. Khotchenkov 1 , S. Sforza 2 , O.V. Koroleva 1 1 A.N. Bach Institute of Russian Academy of Sciences, Russian Federation 2 Department of organic and industrial chemistry of University of Parma, Italy Keywords: antioxidant capacity; TEAC assay; ORAC assay; functional animal protein
Introduction: Protein hydrolysates are considered to be a valuable source of antioxidant components involved into maintaining of human well-being and prevention of age-related diseases. Biocatalytic technology for poultry meat&bone residues conversion into functional animal protein (FAP) possessing tailored antioxidant properties had been recently developed (Nikolaev et al., 2008; Semenysheva et al., 2009). Therefore, the purpose of the study was to identify the most abundant antioxidant constituents of FAP. Methods: Two antioxidant capacity methods TEAC assay (Pellegrini et al., 2003) and ORAC assay (Ou et al., 2001) were validated for FAP analysis. FAPˇıs produced under different hydrolysis conditions were subsequently fractionated by SE-HPLC and IE-HPLC. Components of most active fractions were identified by MS/MS analysis on Varian 500-MS Ion Trap Mass Spectrometer. Antioxidant properties of selected amino acids and peptides were confirmed using the corresponding standards. Results: Antioxidant capacity (AOC) of FAP determined by ORAC and TEAC assays varied in the range of 407-529 and 428511 mol/g respectively. Also FAP AOC increased when higher enzyme dosage and water to raw material ratio were used for raw material conversion. Two most active fractions were obtained after SE-HPLC fractionation. The first was composed by amino acids (Y, R, M, K, H) and dipeptides (AF, GF, SF, anserine and carnosine), although the second contained only free tryptophan. Direct determination of AOC of these compounds revealed that tyrosine, tryptophan and carnosine exhibited antioxidant activity under both TEAC and ORAC assays. In the meantime anserine, histidine and methionine were active only against peroxyl radicals. Conclusions: FAPˇıs AOC values determined by both methods were quite comparable (r = 0.91). Low molecular weight (Mw < 3 kDa) components contributed more then 95% of FAPˇıs AOC. The results on AOC of individual amino acids and selected peptides clearly discriminate reactivity of ABTS radical cation and peroxyl radical. Acknowledgements: The research has received funding from European Commission 7-th Framework Program FP7/2007-2011 under grant agreement No 212696. References Nikolaev I. V., Stepanova E. V., Eremeev N. L., Ismailova D. Yu., Zaichik B. Ts., Ruzhitskii A.O., Khotchenkov V. P., Kostyleva E.V., Sinitsyn A. P., Volik V. G., Koroleva O. V. (2008) Optimization of enzymatic hydrolysis of animal raw material for obtaining functional meat protein preparation. Biotechnology (Moscow), 5, 5967. Semenysheva, A.I., Nikolaev, I.V., Mashentseva, N.G., Stepanova, E.V., Mitaseva, L.F., Koroleva, O.V., 2009. Approach for rational conversion of raw materials of poultry processing industry. Fleischwirtschaft International Russia 2, 50–53.
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
structures. The results showed a relationship between extent of cross-linking, apparent viscosity and CWP ratio of the yoghurt gels. During storage for up to 4 weeks at 4 ◦ C, no changes in rheological properties for stirred yoghurt gels prepared from cross-linked milk proteins.
aspects concerning performance characteristics, and the estimation of detection and quantification limits of LNA probes will be presented.
doi:10.1016/j.jbiotec.2010.09.349
[P-F.108]
[P-F.106]
Antioxidant Constituents of Functional Animal Protein
Effect of phenolic compounds on the growth and L-malic acid metabolism of Oenococcus oeni M. Iorizzo ∗ , S.J. Lombardi, T. DI Renzo, B. Testa, R. Coppola, E. Sorrentino Distaam - Universita’ Del Molise, Italy Keywords: Wine; Oenococcus Oeni; Phenolic; L-Malic acid In this work the effect of the most common phenol carboxylic acids, (gallic, caffeic and ferulic acids) and flavonoids (catechin and quercetin) on the growth and L-malic acid metbolism of Oenococcus oeni was investigated. A total of 143 strains, isolated from different wines, were assayed in a synthetic medium added with ethanol (12% v/v) and malic acid (5 g/L) and different amounts of each compound under analysis. The three carboxyl acids showed an inhibitory effect on growth of Oenococcus oeni at high concentrations; 1000 mg/L inhibited the growth, and inhibition by ferulic acid was greater than that registered for caffeic acid. As for gallic acid its presence in the medium at 500 mg/L stimulated the growth of assayed strains while higher concentrations, until 1000 mg/L did not inhibit the growth. Catechin had a stimulating effect, which increased with concentration. While quercetin showed a slight stimulating effect only at low concentrations. Flavonoids also stimulated the Malolactic fermentation. doi:10.1016/j.jbiotec.2010.09.350 [P-F.107] GMO Traceability Using Very Short, Locked Nucleic Acid TaqMan Probes Sergio Salvi ∗ , Fabio D’orso, Giorgio Morelli National Research Institute on Food and Nutrition (Inran), Italy Keywords: GMO; Real-time PCR; Locked nucleic acid (LNA); Standard reference plasmids Many countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms (GMO). Real-time quantitative PCR based upon the TaqMan probe chemistry has become the mostly used technology to support these regulations, and event-specific PCR is the preferred method in GMO detection because of its high specificity based on the flanking sequence of the transgenic integrant. The aim of this study was to evaluate the use of 8-nucleotide long locked nucleic acid (LNA) probes in 5 -nuclease PCR assays for the detection and quantification of GMO. Conventional TaqMan and LNA probes were compared for the analysis of the maize MON810 transgene. The performance of the two types of probes was tested on the CaMV 35S promoter, the hsp70/cryIA(b) construct as well as for the eventspecific 5 integration junction of MON810, using the maize hmga as endogenous reference gene. Specific plasmids, developed in our laboratory, were used as standard reference molecules. Several
doi:10.1016/j.jbiotec.2010.09.351
I.V. Nikolaev 1,∗ , F. Lambertini 2 , V.P. Khotchenkov 1 , S. Sforza 2 , O.V. Koroleva 1 1 A.N. Bach Institute of Russian Academy of Sciences, Russian Federation 2 Department of organic and industrial chemistry of University of Parma, Italy Keywords: antioxidant capacity; TEAC assay; ORAC assay; functional animal protein
Introduction: Protein hydrolysates are considered to be a valuable source of antioxidant components involved into maintaining of human well-being and prevention of age-related diseases. Biocatalytic technology for poultry meat&bone residues conversion into functional animal protein (FAP) possessing tailored antioxidant properties had been recently developed (Nikolaev et al., 2008; Semenysheva et al., 2009). Therefore, the purpose of the study was to identify the most abundant antioxidant constituents of FAP. Methods: Two antioxidant capacity methods TEAC assay (Pellegrini et al., 2003) and ORAC assay (Ou et al., 2001) were validated for FAP analysis. FAPˇıs produced under different hydrolysis conditions were subsequently fractionated by SE-HPLC and IE-HPLC. Components of most active fractions were identified by MS/MS analysis on Varian 500-MS Ion Trap Mass Spectrometer. Antioxidant properties of selected amino acids and peptides were confirmed using the corresponding standards. Results: Antioxidant capacity (AOC) of FAP determined by ORAC and TEAC assays varied in the range of 407-529 and 428511 mol/g respectively. Also FAP AOC increased when higher enzyme dosage and water to raw material ratio were used for raw material conversion. Two most active fractions were obtained after SE-HPLC fractionation. The first was composed by amino acids (Y, R, M, K, H) and dipeptides (AF, GF, SF, anserine and carnosine), although the second contained only free tryptophan. Direct determination of AOC of these compounds revealed that tyrosine, tryptophan and carnosine exhibited antioxidant activity under both TEAC and ORAC assays. In the meantime anserine, histidine and methionine were active only against peroxyl radicals. Conclusions: FAPˇıs AOC values determined by both methods were quite comparable (r = 0.91). Low molecular weight (Mw < 3 kDa) components contributed more then 95% of FAPˇıs AOC. The results on AOC of individual amino acids and selected peptides clearly discriminate reactivity of ABTS radical cation and peroxyl radical. Acknowledgements: The research has received funding from European Commission 7-th Framework Program FP7/2007-2011 under grant agreement No 212696. References Nikolaev I. V., Stepanova E. V., Eremeev N. L., Ismailova D. Yu., Zaichik B. Ts., Ruzhitskii A.O., Khotchenkov V. P., Kostyleva E.V., Sinitsyn A. P., Volik V. G., Koroleva O. V. (2008) Optimization of enzymatic hydrolysis of animal raw material for obtaining functional meat protein preparation. Biotechnology (Moscow), 5, 5967. Semenysheva, A.I., Nikolaev, I.V., Mashentseva, N.G., Stepanova, E.V., Mitaseva, L.F., Koroleva, O.V., 2009. Approach for rational conversion of raw materials of poultry processing industry. Fleischwirtschaft International Russia 2, 50–53.