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Extraction strategies for selenium in selenium-enriched plants and determination by GC–MS–MS
S724
Abstracts / Journal of Biotechnology 136S (2008) S717–S742
VIII1-P-012 Extraction strategies for selenium in selenium-enriched plants and determination by GC–MS–MS Shengfang Wu 1 , Junliang Sun 2 , Hao Zhang 2,∗ , Haizhen Mo 2 1
Centre for Analysis, Southern Yangtze University, Wuxi 214122, Jiangsu Province, China 2 School of Food Science, Henan Institute of Science and Technology, Xinxiang, 453003, China Selenium component extraction method and speciation in selenium-enriched edamame and cabbage were studied. Through HCl buffer and ultrasound-assisted extraction method, selenium component in cabbage and edamame can be extracted. Results show that the main selenium form in cabbage is in free state, and main selenium form in edamame is protein binding. Using 0.01 HCl buffer, assisted with 120 s ultrasonic probe, most selenium in cabbage can be extracted. For edamame selenium extraction, it is necessary to adopt proteolytic processing. Ultrasound can be used to deal with selenium edamame protein to shorten the enzyme process to 180 s. Derivative of selenium amino acid in cabbage and edamame was done with ethyl chloride formate. Then, they were detected with gas chromatography mass spectrometry. Chemical analysis of selenium showed that main selenium species in cabbage was Se-methyl-selenocysteine although selenomethionine and selenocystein were also detected. For edamame, the main selenium species was selenomethionine. Keywords: Selenium speciation; Extraction; GC–MS–MS References Infante, H.G., O’Connor, G., et al., 2004. Selenium speciation analysis of seleniumenriched supplements by HPLC with ultrasonic nebulisation ICP-MS and electrospray MS/MS detection. J. Anal. At. Spectrom. 19 (12), 1529–1538. Mo, H.Z., Zhang, M., Zhu, Y., 2006. Selenium enrichment pattern in flowering Chinese cabbage, cabbage and asparagus. Agro Food Ind. Hi-tech. 17 (2), 39–42. Yang, L., et al., 2004. Comparison of extraction methods for quantitation of methionine and selenomethionine in yeast by species specific isotope dilution gas chromatography-mass spectrometry. J. Chromatogr. A 1055 (1–2), 177–184.
doi:10.1016/j.jbiotec.2008.07.1722 VIII1-P-013 Isolation and antigenicity evaluation of -lactoglobulin form buffalo milk Xin Li 1,2,∗ , Zengling Luo 1,2 , Hongbing Chen 1,2 , Yusheng Cao 1,2 1
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, China 2 Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang, China E-mail address: [email protected] (X. Li). A simple and reproducible method was developed for isolating -lactoglobulin from buffalo milk while preserving its antigenicity. Buffalo -lactoglobulin in phosphate buffer (0.02 M, pH 6.8) was adsorbed on DEAE-Sepharose Fast Flow gel, and eluted with a linear gradient of NaCl (0–0.5 M) in 0.02 M phosphate buffer, pH 6.8 (Hahn et al., 1998). A further purification was performed on Sephadex G-75 gel by loading a concentrated and dialyzed fraction of samples containing buffalo -lactoglobulin from ion-exchange chromatography, and separating at a flow rate of 0.15 ml/min in 0.02 M phosphate buffer, pH 6.8 (Nevestani et al., 2003). Milligrams level of purified buffalo -lactoglobulin was obtained from each run of certain volume of buffalo milk. The purity of the
isolated buffalo -lactoglobulin was above 90% in comparison to the standard bovine -lactoglobulin by SDS-PAGE, and shown only one band by loading 50 g concentrated protein sample, and IEF-PAGE. The antigenicity of the buffalo -lactoglobulin was evaluated by indirect ELISA, Western-blotting and inhibition ELISA with anti-buffalo and anti-bovine -lactoglobulin rabbit serum. The results showed that buffalo -lactoglobulin could be separated and purified by anion-exchange chromatography combined with gel filtration chromatography, and with a well-preserved antigenicity. Acknowledgement The work was supported by “National Natural Science Foundation of China”, project no. 30560096, and “Program for Changjiang Scholars and Innovative Research Team in University”, project no. IRT0540. References Hahn, R., Schulz, P.M., Schaupp, C., Jungbauer, A., 1998. Bovine whey fractionation based on cation-exchange chromatography. J. Chromatogr. A 795, 277–287. Nevestani, T.R., Djalali, M., Pezeshki, M., 2003. Isolation of alpha-lactalbumin, betalactoglobulin, and bovine serum albumin from cow’s milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity. Protein Exp. Purif. 29, 202–208.
doi:10.1016/j.jbiotec.2008.07.1723 VIII1-P-014 Production of carnation pigments in submerged culture of Monascus spp. Mian-hua Chen ∗ , Lu Hao, Zhi-liang Fu, Chang-lu Wang Tianjin Key Laboratory of Food Nutrition and Safety, College of Food Science & Bioengineering, Tianjin University of Science & Technology, Tianjin 300457, PR China E-mail address: chen [email protected] (M.-h. Chen). Monascus pigments are heat-stable colorant commonly applied in meat, fish and drinking sectors (Laurent et al., 2005). There are a number of reports on red and yellow pigments produced by monascus molds (Hassan et al., 2000; Suchada et al., 2004). In this paper we report a monascus mold which is capable of producing water soluble carnation pigments (max = 505 nm) instead of red pigments (max = 488 nm). Various factors affecting carnation pigments production have been examined. It was found that the Monascus spp. only produces pigments at low temperature; the carnation pigments would transfer to black color if the fermentation was not terminated in time. The maximal amount of pigment under the optimal conditions is 60 U/ml, and the content of citrinin in the pigments is 0.99 mg/ml. References Hassan, H., Philippe, B., Evelyne, G., Jean, L.U., Gerard, G., Pascal, L., 2000. Kinetic analysis of red pigment and citrinin production by Monascus ruber as a function of organic acid accumulation. Enzyme Microbial. Technol. 27, 619–625. Laurent, D., Patrick, G., Anina, Y., Shoshana, M.A., Philippe, B., Kotamballi, N., Chidambara, M., Gokare, A.R., 2005. Microorganisms and microalgae as sources of pigments for food use: a scientific oddity or an industrial reality? Trends Food Sci. Technol. 16, 389–406. Suchada, J., Prasat, K., Busaba, Y., Rapepol, B., Somboon, T., Nattapat, L., Yodhathai, T., 2004. Azaphilone pigments from a yellow mutant of the fungus Monascus kaoliang. Phytochemistry 65, 2569–2575.
doi:10.1016/j.jbiotec.2008.07.1724
Abstracts / Journal of Biotechnology 136S (2008) S717–S742
VIII1-P-012 Extraction strategies for selenium in selenium-enriched plants and determination by GC–MS–MS Shengfang Wu 1 , Junliang Sun 2 , Hao Zhang 2,∗ , Haizhen Mo 2 1
Centre for Analysis, Southern Yangtze University, Wuxi 214122, Jiangsu Province, China 2 School of Food Science, Henan Institute of Science and Technology, Xinxiang, 453003, China Selenium component extraction method and speciation in selenium-enriched edamame and cabbage were studied. Through HCl buffer and ultrasound-assisted extraction method, selenium component in cabbage and edamame can be extracted. Results show that the main selenium form in cabbage is in free state, and main selenium form in edamame is protein binding. Using 0.01 HCl buffer, assisted with 120 s ultrasonic probe, most selenium in cabbage can be extracted. For edamame selenium extraction, it is necessary to adopt proteolytic processing. Ultrasound can be used to deal with selenium edamame protein to shorten the enzyme process to 180 s. Derivative of selenium amino acid in cabbage and edamame was done with ethyl chloride formate. Then, they were detected with gas chromatography mass spectrometry. Chemical analysis of selenium showed that main selenium species in cabbage was Se-methyl-selenocysteine although selenomethionine and selenocystein were also detected. For edamame, the main selenium species was selenomethionine. Keywords: Selenium speciation; Extraction; GC–MS–MS References Infante, H.G., O’Connor, G., et al., 2004. Selenium speciation analysis of seleniumenriched supplements by HPLC with ultrasonic nebulisation ICP-MS and electrospray MS/MS detection. J. Anal. At. Spectrom. 19 (12), 1529–1538. Mo, H.Z., Zhang, M., Zhu, Y., 2006. Selenium enrichment pattern in flowering Chinese cabbage, cabbage and asparagus. Agro Food Ind. Hi-tech. 17 (2), 39–42. Yang, L., et al., 2004. Comparison of extraction methods for quantitation of methionine and selenomethionine in yeast by species specific isotope dilution gas chromatography-mass spectrometry. J. Chromatogr. A 1055 (1–2), 177–184.
doi:10.1016/j.jbiotec.2008.07.1722 VIII1-P-013 Isolation and antigenicity evaluation of -lactoglobulin form buffalo milk Xin Li 1,2,∗ , Zengling Luo 1,2 , Hongbing Chen 1,2 , Yusheng Cao 1,2 1
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, China 2 Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang, China E-mail address: [email protected] (X. Li). A simple and reproducible method was developed for isolating -lactoglobulin from buffalo milk while preserving its antigenicity. Buffalo -lactoglobulin in phosphate buffer (0.02 M, pH 6.8) was adsorbed on DEAE-Sepharose Fast Flow gel, and eluted with a linear gradient of NaCl (0–0.5 M) in 0.02 M phosphate buffer, pH 6.8 (Hahn et al., 1998). A further purification was performed on Sephadex G-75 gel by loading a concentrated and dialyzed fraction of samples containing buffalo -lactoglobulin from ion-exchange chromatography, and separating at a flow rate of 0.15 ml/min in 0.02 M phosphate buffer, pH 6.8 (Nevestani et al., 2003). Milligrams level of purified buffalo -lactoglobulin was obtained from each run of certain volume of buffalo milk. The purity of the
isolated buffalo -lactoglobulin was above 90% in comparison to the standard bovine -lactoglobulin by SDS-PAGE, and shown only one band by loading 50 g concentrated protein sample, and IEF-PAGE. The antigenicity of the buffalo -lactoglobulin was evaluated by indirect ELISA, Western-blotting and inhibition ELISA with anti-buffalo and anti-bovine -lactoglobulin rabbit serum. The results showed that buffalo -lactoglobulin could be separated and purified by anion-exchange chromatography combined with gel filtration chromatography, and with a well-preserved antigenicity. Acknowledgement The work was supported by “National Natural Science Foundation of China”, project no. 30560096, and “Program for Changjiang Scholars and Innovative Research Team in University”, project no. IRT0540. References Hahn, R., Schulz, P.M., Schaupp, C., Jungbauer, A., 1998. Bovine whey fractionation based on cation-exchange chromatography. J. Chromatogr. A 795, 277–287. Nevestani, T.R., Djalali, M., Pezeshki, M., 2003. Isolation of alpha-lactalbumin, betalactoglobulin, and bovine serum albumin from cow’s milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity. Protein Exp. Purif. 29, 202–208.
doi:10.1016/j.jbiotec.2008.07.1723 VIII1-P-014 Production of carnation pigments in submerged culture of Monascus spp. Mian-hua Chen ∗ , Lu Hao, Zhi-liang Fu, Chang-lu Wang Tianjin Key Laboratory of Food Nutrition and Safety, College of Food Science & Bioengineering, Tianjin University of Science & Technology, Tianjin 300457, PR China E-mail address: chen [email protected] (M.-h. Chen). Monascus pigments are heat-stable colorant commonly applied in meat, fish and drinking sectors (Laurent et al., 2005). There are a number of reports on red and yellow pigments produced by monascus molds (Hassan et al., 2000; Suchada et al., 2004). In this paper we report a monascus mold which is capable of producing water soluble carnation pigments (max = 505 nm) instead of red pigments (max = 488 nm). Various factors affecting carnation pigments production have been examined. It was found that the Monascus spp. only produces pigments at low temperature; the carnation pigments would transfer to black color if the fermentation was not terminated in time. The maximal amount of pigment under the optimal conditions is 60 U/ml, and the content of citrinin in the pigments is 0.99 mg/ml. References Hassan, H., Philippe, B., Evelyne, G., Jean, L.U., Gerard, G., Pascal, L., 2000. Kinetic analysis of red pigment and citrinin production by Monascus ruber as a function of organic acid accumulation. Enzyme Microbial. Technol. 27, 619–625. Laurent, D., Patrick, G., Anina, Y., Shoshana, M.A., Philippe, B., Kotamballi, N., Chidambara, M., Gokare, A.R., 2005. Microorganisms and microalgae as sources of pigments for food use: a scientific oddity or an industrial reality? Trends Food Sci. Technol. 16, 389–406. Suchada, J., Prasat, K., Busaba, Y., Rapepol, B., Somboon, T., Nattapat, L., Yodhathai, T., 2004. Azaphilone pigments from a yellow mutant of the fungus Monascus kaoliang. Phytochemistry 65, 2569–2575.
doi:10.1016/j.jbiotec.2008.07.1724