Periodic Reporting for period 1 - RHAPPLE (Novel biotechnological route for the isolation and investigation of rhamnogalacturonan-I from apple side-streams as model systems)
Reporting period: 2024-09-01 to 2026-08-31
Globally, up to 40% of food is wasted, resulting in the loss of valuable nutrients and resources. Fruit and vegetable processing alone generates around 50% by-product waste, largely composed of plant cell wall polysaccharides such as pectin. Among pectic components, rhamnogalacturonan-I (RG-I) is particularly underexplored due to the difficulty of isolating it in its native, branched form. Yet, RG-I’s complex structure and its potential functional properties—such as gelling, emulsifying, and prebiotic activity—suggest a high value for food applications. Current extraction methods rely on harsh chemical or physical treatments that degrade RG-I, preventing both structural characterization and sustainable large-scale production.
RHAPPLE addresses this challenge by developing a biotechnological route for the selective isolation of intact RG-I from fruit and vegetable side-streams using a fermentative approach. The project uses the yeast Kluyveromyces (K.) lactis, which can be genetically modified to secrete specific enzymes that degrade other plant cell wall polysaccharides while leaving RG-I intact. This strategy replaces chemical extraction with a mild, low-resource, and scalable process.
The project’s objectives are to (1) establish K. lactis as a biotechnological platform for RG-I isolation, (2) engineer strains producing targeted enzyme sets, and (3) elucidate structure–function relationships of the isolated RG-I. By creating a sustainable process for producing high-quality RG-I and mapping its functional properties, RHAPPLE will unlock the potential of this polysaccharide as a multifunctional ingredient. The expected impact includes reducing food waste, promoting circular bioeconomy approaches, and fostering innovation in the European food and biotechnology sectors.
RHAPPLE addresses this challenge by developing a biotechnological route for the selective isolation of intact RG-I from fruit and vegetable side-streams using a fermentative approach. The project uses the yeast Kluyveromyces (K.) lactis, which can be genetically modified to secrete specific enzymes that degrade other plant cell wall polysaccharides while leaving RG-I intact. This strategy replaces chemical extraction with a mild, low-resource, and scalable process.
The project’s objectives are to (1) establish K. lactis as a biotechnological platform for RG-I isolation, (2) engineer strains producing targeted enzyme sets, and (3) elucidate structure–function relationships of the isolated RG-I. By creating a sustainable process for producing high-quality RG-I and mapping its functional properties, RHAPPLE will unlock the potential of this polysaccharide as a multifunctional ingredient. The expected impact includes reducing food waste, promoting circular bioeconomy approaches, and fostering innovation in the European food and biotechnology sectors.
During the first year, RHAPPLE focused on evaluating the suitability of Kluyveromyces lactis as a biotechnological platform for the selective isolation of the complex plant polysaccharide RG-I from apple pomace—a by-product of juice and cider production—serving as a model system (Work Package 1). The primary aims were to confirm that K. lactis can grow efficiently in rehydrated apple pomace and that it does not degrade RG-I or produce ethanol in concentrations that could interfere with its solubility and functionality. All generated data have been made publicly available through the ERDA repository (https://www.erda.au.dk/archives/6a4fd535633fa3c22de65bbe10a68281/published-archive.html(opens in new window)).
Growth performance and ethanol formation: K. lactis exhibited strong growth in rehydrated apple pomace medium after 24 h of incubation, reaching nearly 4 × 10⁷ cfu/mL, while effectively suppressing the growth of the native microbiota. Ethanol formation remained minimal (≈1.3% v/v after 48 h), indicating that fermentation did not compromise RG-I integrity.
RG-I stability and potential degradation: When incubated in a defined RG-I medium, the yeast caused only minor overall RG-I losses (≈8% after 48 h). Detailed carbohydrate profiling using high-performance anion exchange chromatography revealed that most monosaccharides remained stable. However, a slight decrease in galactose content suggested limited degradation of galactan side chains, potentially linked to endogenous β-galactosidase activity. Based on these findings, K. lactis was deemed suitable as a production platform, though further optimization—such as β-galactosidase knockout and shortened fermentation times—is recommended to preserve RG-I side-chain integrity.
Preparation for Work Package 2: Extensive literature research identified four well-characterized enzymes targeting non-RG-I plant cell wall polysaccharides for heterologous expression in K. lactis: endo-glucanase (Clostridium cellulovorans), β-glucosidase (Trichoderma reesei), xyloglucanase (Paenibacillus sp.), and polygalacturonase (Aspergillus aculeatus). Gene integration into the pKLAC2 vector for chromosomal insertion was designed using CLC Main Workbench, and plasmid maps were uploaded to ERDA. Necessary microbial DNA and synthetic genes were ordered, and the regulatory framework for performing genetic modifications was established. Therefore, a new biosafety level 1 laboratory for GMO work was successfully set up at Aarhus University’s Department of Food Science, providing the infrastructure to advance to the next project phase—engineering K. lactis for the enzymatic release of native RG-I from agro-industrial side-streams.
Growth performance and ethanol formation: K. lactis exhibited strong growth in rehydrated apple pomace medium after 24 h of incubation, reaching nearly 4 × 10⁷ cfu/mL, while effectively suppressing the growth of the native microbiota. Ethanol formation remained minimal (≈1.3% v/v after 48 h), indicating that fermentation did not compromise RG-I integrity.
RG-I stability and potential degradation: When incubated in a defined RG-I medium, the yeast caused only minor overall RG-I losses (≈8% after 48 h). Detailed carbohydrate profiling using high-performance anion exchange chromatography revealed that most monosaccharides remained stable. However, a slight decrease in galactose content suggested limited degradation of galactan side chains, potentially linked to endogenous β-galactosidase activity. Based on these findings, K. lactis was deemed suitable as a production platform, though further optimization—such as β-galactosidase knockout and shortened fermentation times—is recommended to preserve RG-I side-chain integrity.
Preparation for Work Package 2: Extensive literature research identified four well-characterized enzymes targeting non-RG-I plant cell wall polysaccharides for heterologous expression in K. lactis: endo-glucanase (Clostridium cellulovorans), β-glucosidase (Trichoderma reesei), xyloglucanase (Paenibacillus sp.), and polygalacturonase (Aspergillus aculeatus). Gene integration into the pKLAC2 vector for chromosomal insertion was designed using CLC Main Workbench, and plasmid maps were uploaded to ERDA. Necessary microbial DNA and synthetic genes were ordered, and the regulatory framework for performing genetic modifications was established. Therefore, a new biosafety level 1 laboratory for GMO work was successfully set up at Aarhus University’s Department of Food Science, providing the infrastructure to advance to the next project phase—engineering K. lactis for the enzymatic release of native RG-I from agro-industrial side-streams.
RHAPPLE demonstrated, for the first time, the feasibility of using the yeast Kluyveromyces lactis as a biotechnological platform for the selective isolation of the complex pectic polysaccharide RG-I from fruit-processing by-products. Using apple pomace as a model substrate, K. lactis showed robust growth under near-neutral conditions with negligible ethanol production, confirming its suitability for polysaccharide-based fermentations. Analytical profiling revealed that RG-I remained largely intact after fermentation, with only minor modification of galactan side chains, indicating that K. lactis mostly preserves the native structure of RG-I components.
Another major achievement was the establishment of a GMO level 1 laboratory at Aarhus University, enabling safe and efficient genetic modification of microorganisms within RHAPPLE and beyond. Preparatory work identified and designed the integration of four well-characterized genes encoding enzymes capable of degrading cellulose, hemicellulose, and homogalacturonan, setting the stage for the next phase—enzymatic release of intact RG-I directly from plant cell wall material.
To ensure further uptake and success, key needs include strain optimization to further reduce galactan degradation, integration of the four planned genes into K. lactis, demonstration of process scalability, detailed structure–function analysis of isolated RG-I, and later-on engagement with industrial stakeholders. The project has thus established a strong foundation for developing a sustainable, low-resource bioprocess for RG-I recovery, advancing circular bioeconomy and waste-valorisation goals.
Another major achievement was the establishment of a GMO level 1 laboratory at Aarhus University, enabling safe and efficient genetic modification of microorganisms within RHAPPLE and beyond. Preparatory work identified and designed the integration of four well-characterized genes encoding enzymes capable of degrading cellulose, hemicellulose, and homogalacturonan, setting the stage for the next phase—enzymatic release of intact RG-I directly from plant cell wall material.
To ensure further uptake and success, key needs include strain optimization to further reduce galactan degradation, integration of the four planned genes into K. lactis, demonstration of process scalability, detailed structure–function analysis of isolated RG-I, and later-on engagement with industrial stakeholders. The project has thus established a strong foundation for developing a sustainable, low-resource bioprocess for RG-I recovery, advancing circular bioeconomy and waste-valorisation goals.