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Abstract
Pectin is present in the cell walls of all dicotyledonous plants, but commercial extraction is performed mainly from citrus peel by subjecting it to low pH and high temperature. This process leaves behind large amounts of insoluble residues currently used in low value applications such as the production of biogas.
The work presented in this thesis aimed at providing an alternative process in order to improve the current high methoxyl pectin manufacturing processes, and utilize side streams, based on a specific process design from the company CP Kelco ApS. The extraction residue should be upgraded into high value functional products by enzymatic means while at the same time ensuring high pectin quality. This entailed a comprehensive mapping of the chemical structure of depectinized citrus residues and the development of novel enzymatic approaches to extract health promoting or otherwise useful carbohydrate structures from citrus biomass.
A thorough review of current literature on enzymatic degradation of plant cell walls was carried out with particular focus on targeted release of components beneficial to human health. This literature review highlighted the difficulties of targeted enzymatic degradation of the recalcitrant and complex citrus cell wall. In addition, it was also revealed that rhamnogalacturonan-I-like structures and xyloglucans may possess several health promoting effects, including prebiotic properties, effects promoting anti-adhesion of pathogens, and anti-cancer effects.
It was experimentally proven that pectin quality decreases significantly in subsequent extraction steps when using multiple consecutive extractions, which is often the case in current conventional pectin extraction facilities. For the specific high methoxyl pectin process studied, the decrease in quality was reflected in lower values of intrinsic viscosity, degree of esterification, and galacturonic acid content. Instead of compromising the pectin quality, the industrial depectinized citrus residue can be upgraded into high value functional products, thereby forming the basis of a new improved biorefinery process. Extensive monosaccharide analysis showed that many potential health promoting carbohydrate structures, and in particular fucosylated xyloglucans and pectic polysaccharides, were contained in the depectinized citrus residue. The structural elements were troublesome to release using targeted mono-active enzymes. Instead, use of preparations exhibiting a broad spectrum of enzyme activities (e.g., Cellic CTec3) was necessary.
Xyloglucan structures could successfully be extracted by both enzymatic as well as alkaline methods and converted into high value products, i.e., the fucosylated human milk oligosaccharide 2’- fucosyllactose. The conventional alkaline method resulted in a more specific release of hydrolysable fucose structures, whereas commercial cellulases provided a much less targeted release, essentially solubilizing the majority of the carbohydrate structures. However, the enzymatically extracted xyloglucans resulted in higher 2’-fucosyllactose yields due to their superiority as fucosyl donors compared to xyloglucans obtained through conventional alkaline extraction. Most likely, this can be attributed to structural differences between the xyloglucan structures – the enzymatically extracted xyloglucan structures were generally smaller in size (approx. 1 kDa), which stands in contrast to the more polydisperse distribution of the alkaline extracted xyloglucans (1-500 kDa). Another potential advantage of the cellulase treatment is that pectic polysaccharides are also solubilized and can be recovered later using appropriate separation techniques like membrane filtration, thus providing an additional potential value-added product.
It was also discovered that physicochemical pretreatment methods, including extrusion, steam explosion, and autoclaving, did not facilitate improved enzymatic reactions. On the contrary, these methods resulted in lower yields of hydrolysable fucose structures.
Four different cellulase preparations (Cellic CTec3, Cellic CTec2, ENZECO Glucanase PF, and ENZECO CE-3) were tested by use of multi-variate optimization in order to determine which enzyme produced the highest xyloglucan extraction yield and at what extraction conditions. Cellic CTec2 was preferred as it produced the highest yield of the tested cellulase preparations at a local maximum at concentration of 100 µL/g dry matter at 40 °C and pH 7. The main linear factor affecting the extraction was the enzyme concentration. Surprisingly, large amounts of enzyme resulted in lower yield, however, this was found to be due to the cellulase preparations also containing a variety of hemicellulolytic activities.
The results presented in this thesis describes the utilization and upgrading of an industrial side stream into structures relevant for further health-promoting investigation, by optimization of extraction and separation procedures. This may lead to a rethinking of pectin extraction as part of a larger biorefinery, rather than a separate chemical process.
The work presented in this thesis aimed at providing an alternative process in order to improve the current high methoxyl pectin manufacturing processes, and utilize side streams, based on a specific process design from the company CP Kelco ApS. The extraction residue should be upgraded into high value functional products by enzymatic means while at the same time ensuring high pectin quality. This entailed a comprehensive mapping of the chemical structure of depectinized citrus residues and the development of novel enzymatic approaches to extract health promoting or otherwise useful carbohydrate structures from citrus biomass.
A thorough review of current literature on enzymatic degradation of plant cell walls was carried out with particular focus on targeted release of components beneficial to human health. This literature review highlighted the difficulties of targeted enzymatic degradation of the recalcitrant and complex citrus cell wall. In addition, it was also revealed that rhamnogalacturonan-I-like structures and xyloglucans may possess several health promoting effects, including prebiotic properties, effects promoting anti-adhesion of pathogens, and anti-cancer effects.
It was experimentally proven that pectin quality decreases significantly in subsequent extraction steps when using multiple consecutive extractions, which is often the case in current conventional pectin extraction facilities. For the specific high methoxyl pectin process studied, the decrease in quality was reflected in lower values of intrinsic viscosity, degree of esterification, and galacturonic acid content. Instead of compromising the pectin quality, the industrial depectinized citrus residue can be upgraded into high value functional products, thereby forming the basis of a new improved biorefinery process. Extensive monosaccharide analysis showed that many potential health promoting carbohydrate structures, and in particular fucosylated xyloglucans and pectic polysaccharides, were contained in the depectinized citrus residue. The structural elements were troublesome to release using targeted mono-active enzymes. Instead, use of preparations exhibiting a broad spectrum of enzyme activities (e.g., Cellic CTec3) was necessary.
Xyloglucan structures could successfully be extracted by both enzymatic as well as alkaline methods and converted into high value products, i.e., the fucosylated human milk oligosaccharide 2’- fucosyllactose. The conventional alkaline method resulted in a more specific release of hydrolysable fucose structures, whereas commercial cellulases provided a much less targeted release, essentially solubilizing the majority of the carbohydrate structures. However, the enzymatically extracted xyloglucans resulted in higher 2’-fucosyllactose yields due to their superiority as fucosyl donors compared to xyloglucans obtained through conventional alkaline extraction. Most likely, this can be attributed to structural differences between the xyloglucan structures – the enzymatically extracted xyloglucan structures were generally smaller in size (approx. 1 kDa), which stands in contrast to the more polydisperse distribution of the alkaline extracted xyloglucans (1-500 kDa). Another potential advantage of the cellulase treatment is that pectic polysaccharides are also solubilized and can be recovered later using appropriate separation techniques like membrane filtration, thus providing an additional potential value-added product.
It was also discovered that physicochemical pretreatment methods, including extrusion, steam explosion, and autoclaving, did not facilitate improved enzymatic reactions. On the contrary, these methods resulted in lower yields of hydrolysable fucose structures.
Four different cellulase preparations (Cellic CTec3, Cellic CTec2, ENZECO Glucanase PF, and ENZECO CE-3) were tested by use of multi-variate optimization in order to determine which enzyme produced the highest xyloglucan extraction yield and at what extraction conditions. Cellic CTec2 was preferred as it produced the highest yield of the tested cellulase preparations at a local maximum at concentration of 100 µL/g dry matter at 40 °C and pH 7. The main linear factor affecting the extraction was the enzyme concentration. Surprisingly, large amounts of enzyme resulted in lower yield, however, this was found to be due to the cellulase preparations also containing a variety of hemicellulolytic activities.
The results presented in this thesis describes the utilization and upgrading of an industrial side stream into structures relevant for further health-promoting investigation, by optimization of extraction and separation procedures. This may lead to a rethinking of pectin extraction as part of a larger biorefinery, rather than a separate chemical process.
Original language | English |
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Place of Publication | Kgs. Lyngby, Denmark |
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Publisher | DTU Bioengineering |
Number of pages | 97 |
Publication status | Published - 2021 |
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New Process for production of high-value cell wall derived products form citrus peel
Biel-Nielsen, T. L. (PhD Student), Renard, C. M. G. C. (Examiner), Ulvskov, P. (Examiner), Holck, J. (Main Supervisor), Meyer, A. S. (Supervisor) & Sejberg, J. J. P. (Supervisor)
01/10/2018 → 09/05/2022
Project: PhD