Publicação
Development of a bioprocess for the production of xylooligosaccharides
| Resumo: | The demand of prebiotic ingredients, such as xylooligosaccharides (XOS), has been growing over the years as consumers pay more attention to their health and well-being. XOS are considered emergent and competitively priced prebiotics, presenting high potential as food ingredients. As a result, the industry is focused on developing new approaches to improve production efficiency that can meet the increasing demand while reducing costs. Motivated by the industry needs, the main purpose of this thesis was to develop an integrated bioprocess, based on one-step fermentation, for the production of prebiotic XOS, towards the simplification and cost reduction of the process. The one-step fermentation of 13 agro-residues was performed using two Trichoderma species. The most promising results were found for T. reesei using brewers’ spent grain (BSG) as substrate. BSG is an inexpensive and abundant brewery agro-industrial residue that was herein proven interesting for the production of prebiotic arabino-xylooligosaccharides (AXOS). Under optimal conditions (3 days, pH 7.0, 30°C and 20 g/L of BSG), a production yield of 38.3 ± 1.8 mg/g (xylose equivalents/g of BSG) was achieved. AXOS with degree of polymerization (DP) from 2 to 5 were obtained. The onestep fermentation using T. reesei was compared with the application of a commercial xylanase from T. longibrachiatum. The first revealed to be a more attractive and low-cost strategy to hydrolyze sterilized raw BSG (without conventional pretreatment) and produce AXOS. In order to reduce the production time obtained with T. reesei (3 days), the Bacillus subtilis 3610 wild type (wt) was successfully used, for the first time, to produce AXOS through direct fermentation of BSG, reducing the production time to 12 h. Genetic engineering was used to further optimize the microorganism performance, by cloning the T. reesei xylanase gene coupled with a secretion tag into the B. subtilis chromosome (B. subtilis 3610 clone 2). After optimization by Box-Behnken experimental design, AXOS with a DP ranging from 2 to 6 were obtained at 12 h of fermentation with B. subtilis 3610 clone 2, using 20 g/L of BSG, at pH 7.0 and 45°C. The maximum production yield was 54.2 ± 1.1 mg/g (xylose equivalents/g of BSG), representing an increase of 33% comparing to the wt, and 29% comparing to the T. reesei. B. subtilis 3610 clone 2 was also selected for downscale production of XOS by direct fermentation of commercial beechwood xylan. The use of a commercial substrate allowed to better evaluate strategies to overcome common process limitations associated to the use of agro-residues as substrates, namely biomass measurement, mass transfer issues and substrate inhibition. The maximum production yield, 306 ± 4 mg/g (XOS/xylan), was achieved after 8 h of fermentation operating under one-time impulse fed-batch regimen. The optimal conditions found were pH 6.0 and 42.5°C, using 2.5 g/L of initial concentration of xylan increased up to 5.0 g/L at 3 h. A mixture of XOS with a DP ranging from 4 to 6 was obtained, presenting high stability after a static in vitro digestion (98.5 ± 0.2)%. Operating under fed-batch mode allowed to minimize the effect of substrate inhibition, which led to a great increase of the production yield. In vitro studies using human fecal inocula of two healthy donors were performed to evaluate and compare the potential prebiotic effect of commercial lactulose and the XOS herein produced through direct fermentation of beechwood xylan by B. subtilis clone 2. For both substrates, acetate was the main short-chain fatty acid (SCFA) produced during fermentation at 48 h. Lactulose led to the highest production of lactate, while XOS led to the maximum production of butyrate (9.0 ± 0.6 mM for donor 1 samples, and 10.5 ± 0.8 mM for donor 2 samples). The addition of XOS also resulted in the largest production of CO2. The significant increase in the production of SCFAs and CO2, added to the reduction of pH and ammonia concentration suggest that the XOS herein obtained hold potential functional properties for human health. The results gathered in this thesis are very promising, specially from the industrial perspective, providing important insights for the development of new integrated strategies for XOS production from agro-residues. |
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| Autores principais: | Amorim, Cláudia Catarina Oliveira |
| Assunto: | Prebiotic xylooligosaccharides brewers’ spent grain Bacillus subtilis Trichoderma reesei xylanase Prebiótico xiloligossacarídeos dreche Bacillus subtilis Trichoderma reesei xilanase |
| Ano: | 2019 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | The demand of prebiotic ingredients, such as xylooligosaccharides (XOS), has been growing over the years as consumers pay more attention to their health and well-being. XOS are considered emergent and competitively priced prebiotics, presenting high potential as food ingredients. As a result, the industry is focused on developing new approaches to improve production efficiency that can meet the increasing demand while reducing costs. Motivated by the industry needs, the main purpose of this thesis was to develop an integrated bioprocess, based on one-step fermentation, for the production of prebiotic XOS, towards the simplification and cost reduction of the process. The one-step fermentation of 13 agro-residues was performed using two Trichoderma species. The most promising results were found for T. reesei using brewers’ spent grain (BSG) as substrate. BSG is an inexpensive and abundant brewery agro-industrial residue that was herein proven interesting for the production of prebiotic arabino-xylooligosaccharides (AXOS). Under optimal conditions (3 days, pH 7.0, 30°C and 20 g/L of BSG), a production yield of 38.3 ± 1.8 mg/g (xylose equivalents/g of BSG) was achieved. AXOS with degree of polymerization (DP) from 2 to 5 were obtained. The onestep fermentation using T. reesei was compared with the application of a commercial xylanase from T. longibrachiatum. The first revealed to be a more attractive and low-cost strategy to hydrolyze sterilized raw BSG (without conventional pretreatment) and produce AXOS. In order to reduce the production time obtained with T. reesei (3 days), the Bacillus subtilis 3610 wild type (wt) was successfully used, for the first time, to produce AXOS through direct fermentation of BSG, reducing the production time to 12 h. Genetic engineering was used to further optimize the microorganism performance, by cloning the T. reesei xylanase gene coupled with a secretion tag into the B. subtilis chromosome (B. subtilis 3610 clone 2). After optimization by Box-Behnken experimental design, AXOS with a DP ranging from 2 to 6 were obtained at 12 h of fermentation with B. subtilis 3610 clone 2, using 20 g/L of BSG, at pH 7.0 and 45°C. The maximum production yield was 54.2 ± 1.1 mg/g (xylose equivalents/g of BSG), representing an increase of 33% comparing to the wt, and 29% comparing to the T. reesei. B. subtilis 3610 clone 2 was also selected for downscale production of XOS by direct fermentation of commercial beechwood xylan. The use of a commercial substrate allowed to better evaluate strategies to overcome common process limitations associated to the use of agro-residues as substrates, namely biomass measurement, mass transfer issues and substrate inhibition. The maximum production yield, 306 ± 4 mg/g (XOS/xylan), was achieved after 8 h of fermentation operating under one-time impulse fed-batch regimen. The optimal conditions found were pH 6.0 and 42.5°C, using 2.5 g/L of initial concentration of xylan increased up to 5.0 g/L at 3 h. A mixture of XOS with a DP ranging from 4 to 6 was obtained, presenting high stability after a static in vitro digestion (98.5 ± 0.2)%. Operating under fed-batch mode allowed to minimize the effect of substrate inhibition, which led to a great increase of the production yield. In vitro studies using human fecal inocula of two healthy donors were performed to evaluate and compare the potential prebiotic effect of commercial lactulose and the XOS herein produced through direct fermentation of beechwood xylan by B. subtilis clone 2. For both substrates, acetate was the main short-chain fatty acid (SCFA) produced during fermentation at 48 h. Lactulose led to the highest production of lactate, while XOS led to the maximum production of butyrate (9.0 ± 0.6 mM for donor 1 samples, and 10.5 ± 0.8 mM for donor 2 samples). The addition of XOS also resulted in the largest production of CO2. The significant increase in the production of SCFAs and CO2, added to the reduction of pH and ammonia concentration suggest that the XOS herein obtained hold potential functional properties for human health. The results gathered in this thesis are very promising, specially from the industrial perspective, providing important insights for the development of new integrated strategies for XOS production from agro-residues. |
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