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Bitterness reduction of limonin by selective adsorption onto new polymeric resins
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
on this loss, no studies assessing quantitatively and systematically the effect of these tags on the recovery, purification and stability of these fusion proteins have been reported. In this work, we studied the effect of hydrophobic polypeptide tag fusion upon production and purification process of several model proteins (cellulase, cutinase and xylanase). We defined preliminary criteria allowing the identification of which tag type should be fused to purify a target protein. Such criteria are based on quantitative parameters: hydrophobicity of polypeptide tags, hydrophobicity increase in tagged proteins and hydrophobicity of the target protein. In conclusion, using these quantitative criteria it will be possible to choose the best hydrophobic tags in a systematic way to allow an optimum production and purification process for the target protein. doi:10.1016/j.nbt.2009.06.736
2.5.35 Encapsulation of probiotic for food application — a review F. Lisboa ∗ , J.C. Andrade, M.B. Oliveira Pharmacy Faculty of University of Porto, Porto, Portugal
Foods are no longer considered by consumers only in terms of taste and immediate nutritional needs but also in terms of their ability to provide specific benefits above and beyond their basic nutritional value. Probiotic bacteria are ‘defined, live microorganisms which, administered in adequate amounts, confer a beneficial physiological effect on the host’. In the probiotic approach, the ingested bacteria are selected to survive gastrointestinal transit and arrive viable and able to contribute positively to the activity of the intestinal microbiota, and thus, the health of the host. As the market for functional foods continues to expand, research in the development of food products containing bifidobacteria and other probiotic bacteria will also continue to grow. Future technological prospects exist in innovations finding solutions for the stability and viability of probiotics in new food environments. Current research on novel probiotic formulations and microencapsulation technologies exploiting biological carrier and barrier materials and systems for enteric release provides promising results. Encapsulation of probiotics in biodegradable polymer matrix has a number of advantages. Probiotics present two sets of problems when considering microencapsulation: their size (typically between 1 and 5 m diameter), and the fact that they must be kept alive. This latter aspect has been crucial in selecting the appropriate microencapsulation technology. To date, the research on the encapsulation of probiotics has focused mainly on maintaining the viability of the probiotic bacterial cells at low pH and high bile concentrations. Textural and sensorial properties of food products in which encapsulated probiotics are added are also matter of study. In this review, different perspectives of probiotic encapsulation are explored and the most recent advances of encapsulation technologies applied to food market are highlighted. doi:10.1016/j.nbt.2009.06.737
S186
www.elsevier.com/locate/nbt
2.5.36 Hydrophilic magnetic nonporous microspheres for bacterial DNA isolation 1,2 ˇ ˇ Trachtová 2 , D. Horák 3 , I. Syslová 2 , S. B. Rittich 1,2,∗ , A. Spanová 1
Masaryk University, Faculty of Science, Institute of Experimental Biology, Tvrdého 14, CZ-602 00 Brno, Czech Republic 2 Brno University of Technology, Faculty of Chemistry, Institute of Food Science ˇ and Biotechnology, Purkynova 464/118, CZ-612 00 Brno, Czech Republic 3 Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsk´y Sq. 2, CZ-162 06 Prague, Czech Republic
Polymerase chain reaction (PCR)-based methods are specific and sensitive tools for identification of microorganisms. The occurrence of false negative results is a great problem in the analysis of real samples. A rapid, small-scale DNA isolation method is needed to take full advantage of the speed potential of the PCR technology. For this reason DNA isolation is an essential step in sample preparations. Carboxyl group-containing magnetic nonporous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) microspheres were used for the isolation of bacterial DNA. P(HEMA-co-GMA) microspheres were prepared by the dispersion polymerization in toluene/2-methylpropan-1-ol mixture in the presence of magnetite nanoparticles obtained by coprecipitation of Fe(II) and Fe(III) salts with ammonium hydroxide. The method proposed was used for bacterial DNA isolation from different dairy products (cheese, fermented dairy products) containing Lactobacillus cells. DNA was adsorbed on particles in the presence of 16 wt.% of poly(ethylene glycol) (PEG 6000) and 2 M NaCl concentrations. A special attention was focused on the optimization of the conditions of DNA elution. The presence of target DNA and the quality of isolated DNA were checked by conventional and real-time polymerase chain reaction (PCR, RT-PCR) with specific primers. doi:10.1016/j.nbt.2009.06.738
2.5.37 Bitterness reduction of limonin by selective adsorption onto new polymeric resins S.I. Vieira 1,∗ , S. Ferreira-Dias 2 , M.H. Ribeiro 1 1
2
University of Lisbon, Lisbon, Portugal Technical University of Lisbon Tapada da Ajuda, Lisbon, Portugal
Citrus fruits are accepted for their nutritive and medicinal value as well as for providing distinctive flavor to a wide variety of food products, making it very popular among food product designers. However, a worldwide major problem in the citrus industry is the formation of bitterness in citrus juice and products after juice extraction. This bitterness occurs in certain varieties of oranges, grapefruits and lemons showing a significant negative commercial impact. The conversion of limonate-A-ring lactone into limonin has been reported as the reason for delayed bitterness of citrus juice. This reaction proceeds under acidic conditions below pH 6.5 and is accelerated by the enzyme limonin-D-ring lactone hydrolase. The presence of limonin and
New Biotechnology · Volume 25S · September 2009
nomilin has been the main cause of delayed bitterness of citrus fruits. In this study, the selective removal of limonin from orange juice by batch adsorption to different microstructured resins was investigated. Since the removal of reducing sugars, pigments and vitamin C may also occur, the eventual adsorption of these compounds was also investigated. The following synthetic neutral resins, from Rhom and Haas, were tested: Amberlite XAD-8, XAD-16N and XAD-1600. The experiments were performed in a microscale, using 24 wells microplates. Different amounts of these sorbents, 0.5—25 g L—1 were added to 2 mL Navel orange juice (pH 3—3.5) previously centrifuged at 4000 rpm. The highest adsorption efficiency for the bitter compound was observed when the synthetic neutral resin, Amberlite XAD-16N, was used, followed by XAD-18. The adsorption of sugars, pigments and vitamin C to the resins were also evaluated. Both Freundlich and Langmuir isotherm models were fitted to the experimental data of adsorption of limonin to the resins used. The uptake of limonin, throughout the time, to the sorbents XAD-16N, XAD-18 and XAD-1600 was tested. doi:10.1016/j.nbt.2009.06.739
2.5.38 Improvement of specific monoclonal antibody (mAb) activity by reduction of the mAb heterogeneity using continuous chromatography (MCSGP) T. Müller-Späth 1,∗ , L. Aumann 1 , G. Ströhlein 1 , M. Morbidelli 2 1
Institute for Chemical and Bioengineering - ChromaCon AG, Zürich, Switzerland 2 Institute for Chemical and Bioengineering
Monoclonal antibodies (mAbs) are among the most important therapeutic proteins in development and production by the biopharmaceutical industry. However, mAbs actually represent mixtures of different molecules themselves: due to post-translational modifications and degradative processes, the mAbs display a large heterogeneity. Different glycoforms, deamidated variants, lysine variants, etc. have been reported. Although it is known that the variants may have different biological activities, a separation of the variants has not been performed on a preparative scale yet due to a lack of suitable technologies. It has been shown that with multicolumn countercurrent solvent gradient purification it is possible to separate monoclonal antibody charge variants with high yield and purity using a cost-effective cation exchange stationary phase. Thus, by means of MCSGP the specific activity of the drug may be increased by removal of less active charge variants. This aspect may be of particular interest with respect to life cycle management of mAbbased drugs. In the work presented, the variant separation using MCSGP is shown for a commercially available mAb including process development, modeling and experimental data.
ABSTRACTS
2.5.39 Sulphate-reducing bacteria from mining environments for metals bioremediation S.M. Paixão ∗ , M.C. Sàágua, L. Baeta-Hall, M.A. Barreiros, A.M. Anselmo, J.C. Duarte INETI, Lisboa, Portugal
Toxic heavy metals and metalloids constitute an international pollution problem that not only impacts public health but also is of environmental and economic importance. Several conventional treatment technologies for removing metals are available. These techniques, based on chemical methods of neutralisation and precipitation, even though quick and effective, present several disadvantages, such as the need for building additional treatment plants, the high cost of the chemical reagents used and the generation of an important volume of sludge which need further treatment. Prokaryotes with physiological activity of sulphate reduction are found in several environmental sites containing metals and these microorganisms have developed several different strategies for detoxification and resistance to toxic elements that are potentially useful for bioremediation. Since sulphate-reducing bacteria (SRB) are found in a large number of contaminated sites containing toxic metals, it is apparent that these organisms have a functional defence system that enables them to persist and even grow under metal stress. The enzymatic metal reduction by SRB offers an alternative to chemical processes to remediate environments containing metals. Acid mine drainage (AMD) is one of the most important source of heavy metal environmental pollution. AMD is characterised by its high acidity (≤3), high concentration of metals (e.g. Cu, Fe, Zn, A1, Pb, As and Cd) and high concentration of dissolved sulphates (≥3000 ppm). Taking advantage of the fact that SRB are present in these mining environments, several samples were collected from S. Domingos abandoned mine (Portugal) and screened for their ability to sulphate reduction, metal resistance and bioremediation. In this context, the most promising consortium of SRB (SRB no. 6) was grown in matrices column reactors, using lactate and ethanol as an electron donor source for the production of H2 S from sulphates. Metal resistance was tested in batch using different metal concentration for Cu, Zn and Fe. The results obtained for this SRB inoculum, showed 97% of sulphate reduction (3750—350 mg/l) in the reactors, and a metal tolerance to Fe, Cu and Zn, at concentrations until 260 mg/l, 40 mg/l and 80 mg/l, respectively, in the batch assays, being observed a sulphate reduction (2000—0 mg/l) and a significant metals concentration decrease in the growth medium. Further studies are being carried out to apply this mining inoculum for the design of reactors for AMD bioremediation. doi:10.1016/j.nbt.2009.06.741
doi:10.1016/j.nbt.2009.06.740
www.elsevier.com/locate/nbt S187
ABSTRACTS
on this loss, no studies assessing quantitatively and systematically the effect of these tags on the recovery, purification and stability of these fusion proteins have been reported. In this work, we studied the effect of hydrophobic polypeptide tag fusion upon production and purification process of several model proteins (cellulase, cutinase and xylanase). We defined preliminary criteria allowing the identification of which tag type should be fused to purify a target protein. Such criteria are based on quantitative parameters: hydrophobicity of polypeptide tags, hydrophobicity increase in tagged proteins and hydrophobicity of the target protein. In conclusion, using these quantitative criteria it will be possible to choose the best hydrophobic tags in a systematic way to allow an optimum production and purification process for the target protein. doi:10.1016/j.nbt.2009.06.736
2.5.35 Encapsulation of probiotic for food application — a review F. Lisboa ∗ , J.C. Andrade, M.B. Oliveira Pharmacy Faculty of University of Porto, Porto, Portugal
Foods are no longer considered by consumers only in terms of taste and immediate nutritional needs but also in terms of their ability to provide specific benefits above and beyond their basic nutritional value. Probiotic bacteria are ‘defined, live microorganisms which, administered in adequate amounts, confer a beneficial physiological effect on the host’. In the probiotic approach, the ingested bacteria are selected to survive gastrointestinal transit and arrive viable and able to contribute positively to the activity of the intestinal microbiota, and thus, the health of the host. As the market for functional foods continues to expand, research in the development of food products containing bifidobacteria and other probiotic bacteria will also continue to grow. Future technological prospects exist in innovations finding solutions for the stability and viability of probiotics in new food environments. Current research on novel probiotic formulations and microencapsulation technologies exploiting biological carrier and barrier materials and systems for enteric release provides promising results. Encapsulation of probiotics in biodegradable polymer matrix has a number of advantages. Probiotics present two sets of problems when considering microencapsulation: their size (typically between 1 and 5 m diameter), and the fact that they must be kept alive. This latter aspect has been crucial in selecting the appropriate microencapsulation technology. To date, the research on the encapsulation of probiotics has focused mainly on maintaining the viability of the probiotic bacterial cells at low pH and high bile concentrations. Textural and sensorial properties of food products in which encapsulated probiotics are added are also matter of study. In this review, different perspectives of probiotic encapsulation are explored and the most recent advances of encapsulation technologies applied to food market are highlighted. doi:10.1016/j.nbt.2009.06.737
S186
www.elsevier.com/locate/nbt
2.5.36 Hydrophilic magnetic nonporous microspheres for bacterial DNA isolation 1,2 ˇ ˇ Trachtová 2 , D. Horák 3 , I. Syslová 2 , S. B. Rittich 1,2,∗ , A. Spanová 1
Masaryk University, Faculty of Science, Institute of Experimental Biology, Tvrdého 14, CZ-602 00 Brno, Czech Republic 2 Brno University of Technology, Faculty of Chemistry, Institute of Food Science ˇ and Biotechnology, Purkynova 464/118, CZ-612 00 Brno, Czech Republic 3 Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsk´y Sq. 2, CZ-162 06 Prague, Czech Republic
Polymerase chain reaction (PCR)-based methods are specific and sensitive tools for identification of microorganisms. The occurrence of false negative results is a great problem in the analysis of real samples. A rapid, small-scale DNA isolation method is needed to take full advantage of the speed potential of the PCR technology. For this reason DNA isolation is an essential step in sample preparations. Carboxyl group-containing magnetic nonporous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) microspheres were used for the isolation of bacterial DNA. P(HEMA-co-GMA) microspheres were prepared by the dispersion polymerization in toluene/2-methylpropan-1-ol mixture in the presence of magnetite nanoparticles obtained by coprecipitation of Fe(II) and Fe(III) salts with ammonium hydroxide. The method proposed was used for bacterial DNA isolation from different dairy products (cheese, fermented dairy products) containing Lactobacillus cells. DNA was adsorbed on particles in the presence of 16 wt.% of poly(ethylene glycol) (PEG 6000) and 2 M NaCl concentrations. A special attention was focused on the optimization of the conditions of DNA elution. The presence of target DNA and the quality of isolated DNA were checked by conventional and real-time polymerase chain reaction (PCR, RT-PCR) with specific primers. doi:10.1016/j.nbt.2009.06.738
2.5.37 Bitterness reduction of limonin by selective adsorption onto new polymeric resins S.I. Vieira 1,∗ , S. Ferreira-Dias 2 , M.H. Ribeiro 1 1
2
University of Lisbon, Lisbon, Portugal Technical University of Lisbon Tapada da Ajuda, Lisbon, Portugal
Citrus fruits are accepted for their nutritive and medicinal value as well as for providing distinctive flavor to a wide variety of food products, making it very popular among food product designers. However, a worldwide major problem in the citrus industry is the formation of bitterness in citrus juice and products after juice extraction. This bitterness occurs in certain varieties of oranges, grapefruits and lemons showing a significant negative commercial impact. The conversion of limonate-A-ring lactone into limonin has been reported as the reason for delayed bitterness of citrus juice. This reaction proceeds under acidic conditions below pH 6.5 and is accelerated by the enzyme limonin-D-ring lactone hydrolase. The presence of limonin and
New Biotechnology · Volume 25S · September 2009
nomilin has been the main cause of delayed bitterness of citrus fruits. In this study, the selective removal of limonin from orange juice by batch adsorption to different microstructured resins was investigated. Since the removal of reducing sugars, pigments and vitamin C may also occur, the eventual adsorption of these compounds was also investigated. The following synthetic neutral resins, from Rhom and Haas, were tested: Amberlite XAD-8, XAD-16N and XAD-1600. The experiments were performed in a microscale, using 24 wells microplates. Different amounts of these sorbents, 0.5—25 g L—1 were added to 2 mL Navel orange juice (pH 3—3.5) previously centrifuged at 4000 rpm. The highest adsorption efficiency for the bitter compound was observed when the synthetic neutral resin, Amberlite XAD-16N, was used, followed by XAD-18. The adsorption of sugars, pigments and vitamin C to the resins were also evaluated. Both Freundlich and Langmuir isotherm models were fitted to the experimental data of adsorption of limonin to the resins used. The uptake of limonin, throughout the time, to the sorbents XAD-16N, XAD-18 and XAD-1600 was tested. doi:10.1016/j.nbt.2009.06.739
2.5.38 Improvement of specific monoclonal antibody (mAb) activity by reduction of the mAb heterogeneity using continuous chromatography (MCSGP) T. Müller-Späth 1,∗ , L. Aumann 1 , G. Ströhlein 1 , M. Morbidelli 2 1
Institute for Chemical and Bioengineering - ChromaCon AG, Zürich, Switzerland 2 Institute for Chemical and Bioengineering
Monoclonal antibodies (mAbs) are among the most important therapeutic proteins in development and production by the biopharmaceutical industry. However, mAbs actually represent mixtures of different molecules themselves: due to post-translational modifications and degradative processes, the mAbs display a large heterogeneity. Different glycoforms, deamidated variants, lysine variants, etc. have been reported. Although it is known that the variants may have different biological activities, a separation of the variants has not been performed on a preparative scale yet due to a lack of suitable technologies. It has been shown that with multicolumn countercurrent solvent gradient purification it is possible to separate monoclonal antibody charge variants with high yield and purity using a cost-effective cation exchange stationary phase. Thus, by means of MCSGP the specific activity of the drug may be increased by removal of less active charge variants. This aspect may be of particular interest with respect to life cycle management of mAbbased drugs. In the work presented, the variant separation using MCSGP is shown for a commercially available mAb including process development, modeling and experimental data.
ABSTRACTS
2.5.39 Sulphate-reducing bacteria from mining environments for metals bioremediation S.M. Paixão ∗ , M.C. Sàágua, L. Baeta-Hall, M.A. Barreiros, A.M. Anselmo, J.C. Duarte INETI, Lisboa, Portugal
Toxic heavy metals and metalloids constitute an international pollution problem that not only impacts public health but also is of environmental and economic importance. Several conventional treatment technologies for removing metals are available. These techniques, based on chemical methods of neutralisation and precipitation, even though quick and effective, present several disadvantages, such as the need for building additional treatment plants, the high cost of the chemical reagents used and the generation of an important volume of sludge which need further treatment. Prokaryotes with physiological activity of sulphate reduction are found in several environmental sites containing metals and these microorganisms have developed several different strategies for detoxification and resistance to toxic elements that are potentially useful for bioremediation. Since sulphate-reducing bacteria (SRB) are found in a large number of contaminated sites containing toxic metals, it is apparent that these organisms have a functional defence system that enables them to persist and even grow under metal stress. The enzymatic metal reduction by SRB offers an alternative to chemical processes to remediate environments containing metals. Acid mine drainage (AMD) is one of the most important source of heavy metal environmental pollution. AMD is characterised by its high acidity (≤3), high concentration of metals (e.g. Cu, Fe, Zn, A1, Pb, As and Cd) and high concentration of dissolved sulphates (≥3000 ppm). Taking advantage of the fact that SRB are present in these mining environments, several samples were collected from S. Domingos abandoned mine (Portugal) and screened for their ability to sulphate reduction, metal resistance and bioremediation. In this context, the most promising consortium of SRB (SRB no. 6) was grown in matrices column reactors, using lactate and ethanol as an electron donor source for the production of H2 S from sulphates. Metal resistance was tested in batch using different metal concentration for Cu, Zn and Fe. The results obtained for this SRB inoculum, showed 97% of sulphate reduction (3750—350 mg/l) in the reactors, and a metal tolerance to Fe, Cu and Zn, at concentrations until 260 mg/l, 40 mg/l and 80 mg/l, respectively, in the batch assays, being observed a sulphate reduction (2000—0 mg/l) and a significant metals concentration decrease in the growth medium. Further studies are being carried out to apply this mining inoculum for the design of reactors for AMD bioremediation. doi:10.1016/j.nbt.2009.06.741
doi:10.1016/j.nbt.2009.06.740
www.elsevier.com/locate/nbt S187