New data about the valorized matrices characterization and high value products AND New characterized ingredients/materials for food, nutraceuticals, pharma and cosmetic applications [GRIP - D2.3.1 + D2.3.2]

NODES – Nord Ovest Digitale e Sostenibile FLAGSHIP PROJECT GRIP SPOKE 2 –GREEN TECHNOLOGIES AND SUSTAINABLE INDUSTRIES DELIVERABLE D2.3.1: New data about the valorized matrices characterization and high value products DELIVERABLE D2.3.2: New characterized ingredients/materials for food, nutraceuticals, pharma and cosmetic applications NB: D2.3.1 and D2.3.2 were merged REPORTING PERIOD Period covered: From M 8 to M 30 Periodic report date and version: Ver_1 Deliverable # Responsible UPO RM2 Responsible Arlorio This report is part of the project NODES which has received funding from the MUR – M4C2 1.5 of PNRR funded by the European Union - NextGenerationEU (Grant agreement no. ECS00000036) 1 Glossary Definition Hub Coordinator (HC) The Hub Coordinator represents the single point of contact for the implementation of the innovation ecosystem towards the MUR. It carries out the management and coordination activities of the innovation ecosystem, receives the fundings, verifies, and transmits to the MUR the reporting of the activities carried out by the Spoke and their affiliates. National Recovery and Resilience Plan (NRRP) This document uses the Italian acronym for the NRRP, which is PNRR (Piano Nazionale della Ripresa e Resilienza) Research Program Manager The person who will be the responsible for the overall scientific contents of the NODES project. The NODES will appoint the Research Program Manager. It refers to “Responsabile del Programma di Ricerca” in the MUR’s Call of proposal for “Ecosistemi di Innovazione” NODES’ Research and innovation program NODES’ Research and Innovation program is articulated in specific programs for each Spoke, with the aim to promote and support applied research on topics consistent with the Intelligent Specialization Strategy, with the guidelines of the 2021-2027 partnership agreement scheme, with regional operational plans and regional and national research and innovation priorities. Although NODES’ Spokes are concentrated on different themes, they will organize their activities and actions within a common framework – NODES’ Booster Methodoloy Spoke Coordinator The University in charge of coordinating the Spoke’s ecosystem. It refers to “Spoke” in the MUR’s Call of proposal for “Ecosistemi di Innovazione” Spoke Data Manager The person who will be the responsible for the monitoring and management of data generated at the Spoke level. The Spoke Coordinator will appoint the Spoke Data Manager. Spoke Partner The entity associated to the Spoke Coordinator. It can be an Innovation Cluster, Competence Center, Research Center related to the Spoke’s ecosystem and contributes to achieve objectives and impact under the Spoke’ leadership and management. It refers to “soggetti affiliati” in the MUR’s Call of proposal for “Ecosistemi di Innovazione”. Spoke Project manager The person who will be the responsible for the management, coordination and progress of the project at the Spoke level. The Spoke Coordinator will appoint the Spoke Project Manager. Spoke research and innovation program NODES’ Research and Innovation program is articulated in specific programs for each Spokes. The spoke will leverage a consolidated collaboration with leading private and public companies and will focus the applied research activity on technological domains and applications that can favour the integration of SMEs into new value chains. Spoke Scientific and Technical Manager The person who will be the responsible for the overall scientific contents of the project at the Spoke level. The Spoke Coordinator will appoint the Spoke Scientific and Technical Manager. 2 Spoke Stakeholders Committee (SC) Consultation structure formed by relevant stakeholders (Government, universities, companies, civil society, third sector, etc.) Spoke Thematic General target focus and domain of the Spoke research. Spoke Topics Specific areas/lines of development within the Spoke. Spoke Work Package Leader At the Spoke level, Work Packages (WPs) will be organized by WP leaders, who will be responsible for performance evaluation and reporting. Flagship Project Main research project at the Spoke level with the goal of prototyping, testing, demonstrating the research activities towards higher TRLs. 3 List of Deliverables D. No Name Type Disseminati on Level Due Date Delivery Date (actual) [number] [name] [R — Document, report] [DEM — Demonstrator, pilot, prototype] [DEC — Websites, patent filings, videos, etc] [DATA — data sets, microdata, etc] [DMP — Data Management Plan] [ETHICS] [SECURITY] [OTHER] [PU — Public] [SEN — Sensitive] [R] [C] [S] [month number] [dd/mm/yyyy] D2.1 Data about chemical and nutritional profiling of raw wastes and by products R PU M14 18/04/2024 D2.2.1 On line lab-scale technical multi-device for waste processing* Other (material) PU M18 25/10/2024 D2.2.2 Delivery of protocols (lab-scale) ready to scale-up the production of new high-value material (in collaboration with Companies) R PU M22 16/02/2025 D2.3.1 New data about valorized matrices characterization and high value products R PU M28 30/09/2025 D2.3.2 New characterized ingredients/materials for food, nutraceuticals, pharma and cosmetic applications Other (material) PU M30 30/09/2025 D2.4.1 New data about sustainable production of new materials from biomasses R PU M24 D2.4.2 New characterized materials from biomasses Other (material) PU M30 30/09/2025 D2.5.2 Porous sorbents from biomasses valorization Other (material) PU M30 29/09/2025 D2.6.1 New material produced at pilot scale ready to the formulation or co-formulation (in collaboration with Companies) Other (material) PU M32 D2.6.2 New well characterized process and formulated pilot-products (in collaboration with Companies) R/Other (Material) PU M32 4 D2.7 Report on the adsorption and/or catalytic performances of porous solids derived from biomasses valorization and non-recyclable plastic wastes R PU M32 29/09/2025 5 A) INTRODUCTION The partners involved in the activities related to the D2.3.1 and D2.3.2 (essentially related to the activities listed in Sub-Task 2.2.2) worked with the aim to characterize the materials/ingredients prepared at laboratory scale, as reported in M2.3.1. The principal aim of the material here described is related to the development of ingredients ready to formulate food supplements and cosmetic products, as well as to isolate bioactive compounds for pharmaceutical applications. ROLE OF PARTNERS The role of partners was distributed depending on i) the matrix and ii) the facilities availability, but particularly following the model process selected as best performing combined solutions useful to obtain sustainable new ingredients ready to be scaled-up and used in nutraceutical/cosmeceutical/food or pharmaceutical products. The Deliverable (here considered as previously planned as unique Deliverable merging D2.3.1 and D2.3.2) reports the characterization of the products as outcomes of the processes. As previously reported in D2.2.2, the four matrices selected were 1. Nervine foods 2. Rice by products 3. Fruit pomace and 4. Cow whey. EXPLANATION OF THE WORK CARRIED OUT AND OVERVIEW OF THE PROGRESS The overall work of described here was related to the chemical/functional characterization of the materials/ingredients produced. Following, the main materials valorized and characterized (and ready to be used to scale up and formulate new products, as expected) are reported and described. 6 DSF-UPO ▪ Apple pomace extract Fig. 1. Apple pomace after pre-processing (drying, milling) Apple pomace extract was obtained through heat-assisted extraction (parameters: 80 °C, 40 min, 1:10 matrix : solvent ratio) and subsequent freeze-drying. The optimized parameters for the pilot production were optimized thanks to the use of advanced mathematical modelling (Random Forest algorithm, data not shown). The extract showed a significant content of phloridzin (a target of the work depending on the bioactive properties of this compounds) when compared to the raw material, as well as a significantly high antioxidant activity. This product can be used both in food/nutraceutical area and cosmetic area. More precisely, the apple pomace was pre-processed drying the material at 40 °C overnight and then grounding it using a bead mill, in order to obtain an homogeneous material (depending on the variability of the raw material, containing also grape seeds). Apple pomace powder was then stored at -20°C until the analysis. The moisture content of AP, before the drying process was 82.8 ± 0.4 % (fresh weight). The result obtained is in agreement with what has been reported in literature. Moreover, on the fresh raw material, the lipid quantification was obtained through a Soxhlet apparatus, leading to obtain 5.20 ± 1.16 % (w/w) of lipid. Has expected, this relative high value was strictly related to the presence of seeds in this by-product. The proximate composition is reported in Table 1. Proximate composition Amount Moisture % (d.w.) 12.3 ± 0.3 Ashes % (d.w.) 3.28 ± 0.03 Protein % (d.w.) 4.32 ± 0.30 TDF % (d.w.) 33.5 ± 3.3 7 IDF SDF 23.9 ± 1.6 9.53 ± 0.21 Table 1. Proximate composition of apple pomace (AP): moisture content, ashes, protein and total dietary fiber (TDF) sum of insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) content. Results are expressed as average ± standard deviation of three replicates. Regarding the phenolic compound and the antioxidant activity, the analysis obtained from the material extracted using ethanol/water (70:30, v/v; solid/liquid ratio of 1:10) showed result in agreement with literature. Total phenolic content (TPC) was 2.48 ± 0.11 mg/g (d.w.) expressed as catechin equivalent, while antioxidant capacity (AC) measured with DPPH assay was 2.61 ± 0.01 mg/g (d.w.), expressed as Trolox equivalent. A preliminary extraction using only water as solvent showed result of 1.03 ± 0.07 mg/g for TPC and 2.05±0.02 mg/g Trolox equivalent for AC (measured with DPPH test). This result can be influenced by the present of sugars which are co-extracted with water. After the optimization of the phenolic’s fraction extraction (experimental design not reported here) the extract richer in phlorizin presented an amount of 50.3 µg/g, beside a significant content of hyperoside and rutin (evaluated as a sum of quercetin glycosides) with an amount of 95.2 µg/g. It was also possible to identify and quantified cyanidin-3-o-galactoside (4.93 µg/g), apparently the only anthocyanin presents in the sample. All these characteristics allow to conclude that the produced extract is useful both for nutraceutical and cosmetic applications, as well as ready – after powderization – to be included in food supplements formulae. ▪ Cocoa bean shells Cocoa bean shells, as reported in previous Deliverable, were processed using different methods from different Units of RM2. The following description is related to the processing selected by DSF-UPO. More precisely, cocoa bean shells were i) directlyenzymatically treat ed with Vyscozyme (1 % w/w), ii) pretreated with ultrasounds (15 minutes, 50 % amplitude, 50% sonication cycle) and iii) with a combination of both treatments. Fig. 2 shows the aspect of the different fiberand polyphenols-rich materials obtained with different single or combined approaches. 8 Fig. 2. Cocoa bean shells fibers (before and after treatments) Table 2 shows the different parameters measured in the same processed samples, particularly focusing on fiber fraction, polyphenolic fraction, antioxidant properties and sugar content. CBS CBS+US CBS+ENZ CBS+US+ENZ Total dietary fiber (%) ~66,5% (insolubile ~57,5%; solubile ~9,0%) ~48,4% (insolubile ~40,3%; solubile ~8,2%) ~50,5% (insolubile ~43,1%; solubile ~7,4%) ~49,4% (insolubile ~42,0%; solubile ~7,4%) Total phenolic content (mg GAE/g) 12,98 ± 0,17 5,43 ± 0,25 5,72 ± 0,13 5,85 ± 0,03 DPPH (mg TE/g, frazione solubile) 13,91 ± 0,25 9,28 ± 0,27 9,56 ± 0,17 9,59 ± 0,17 FRAP (mg TE/g, frazione solubile) 17,02 ± 0,30 11,01 ± 0,01 11,12 ± 0,07 11,52 ± 0,66 DPPH QUENCHER (mg TE/g) 2,59 ± 0,06 5,70 ± 0,31 10,57 ± 0,09 10,95 ± 1,31 Total monomeric sugars (mg/g) 1,68 ± 0,03 1,29 ± 0,04 8,53 ± 0,85 9,19 ± 0,09 Table 2. Composition of the cocoa bean samples processed and ready to be used in model nutraceuticals/cosmetics The data reported in the table 2 highlight significant differences between untreated CBS and those subjected to enzymatic and/or ultrasound treatment, as expected. First, the value of the total dietary fiber is significantly higher in the untreated sample (~66% w/w), consisting mainly of insoluble fiber. The treatments cause a marked reduction in fiber content: both the enzyme and 9 Fig. 6. Comparison among the VOC’s profiles of AKO and PKO using HS-GC-IMS. POLITO The activities described in this report were carried out in the framework of Project RM2, Sub-Task 2.2.2, which aims to isolate high-value ingredients and compounds from waste matrices, with the objective of obtaining up-cycled ingredients for applications in the food, nutraceutical, and cosmetic sectors. This task builds on the results achieved in Sub-Task 2.2.1, moving from laboratory-scale extractions toward pilot-scale processes in order to complete characterization and assess the feasibility of scaling up the most promising technologies. In particular, the valorization of apple pomace and berry by-products has been considered for the recovery of polyphenols and pigments such as phloretin and anthocyanins. During the last reporting period, different pre-treatments and extraction methods were applied to improve the recovery of total polyphenols from apple pomace. The activities focused on thermal-stirred extraction (TSE), ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE), all performed using water as the extraction solvent. A kinetic study was carried out to determine the optimal extraction conditions, including solid-to-solvent ratio, temperature, extraction time, and applied power. These optimal parameters were then tested at pilot scale, and their environmental impact was evaluated through life cycle assessment. Among the investigated methods, microwave-assisted extraction proved to be the most effective, providing the highest yields while also showing favorable environmental performance. 16 Ultrasound-assisted extraction also gave promising results, and for this reason, it was considered for scale-up. In the ultrasound trials, a fixed frequency of 20 kHz was applied at constant temperature (60 °C), with a solid-to-liquid ratio of 1:30 and an extraction time of five minutes. Scale-up experiments were conducted stepwise, moving from 50 mL to 500 mL, 1000 mL, and finally 5000 mL. The first three volumes were processed using the Sonics Materials VC 750 Ultrasonic Processor, equipped with different probes according to the working volume, while the 5 L trial was carried out with the Hielscher UIP1000hdT-230 device (Fig.1). Figure 7. Apple pomace US processing (Hielscher UIP1000hdT-230) Total polyphenol content was determined using the Folin–Ciocalteu method, and all experiments were performed in triplicate and statistically analyzed with one-way ANOVA and Tukey test (Fig. 17 Tab. 7. Total polyphenol content (mg GAE/g dry basis) obtained at different extraction volumes (50, 500, 1000, and 5000 mL). Data are expressed as mean ± standard deviation (n = 3). Different letters above the bars indicate statistically significant differences among treatments at the 95% confidence level (one-way ANOVA, Tukey test). The results showed that the polyphenol recovery was comparable across the extractions at 50, 500, and 1000 mL, whereas a significant decrease in polyphenol concentration was observed at the 5 L scale. This reduction can likely be attributed to several factors. Beyond the lack of agitation and the limited penetration of ultrasonic waves at larger volumes, the dissipation of acoustic energy plays a crucial role: as the working volume increases, the ultrasonic energy is distributed across a larger mass, reducing the intensity of cavitation in certain regions. Moreover, non-uniform heating at higher volumes can generate localized hot or cold spots; in hot spots, thermal degradation of polyphenols may occur, while in colder regions the extraction kinetics may be reduced. Another aspect concerns the effective surface-to-volume ratio, as particles in suspension may sediment without stirring, thereby reducing solvent accessibility. In addition, the propagation of cavitation is less efficient in larger volumes, with weaker acoustic fields away from the probe tip. Future work will therefore focus on addressing these limitations. New configurations with improved mixing and energy distribution will be tested to enhance the homogeneity of the process at pilot scale. Adjustments in extraction time and energy input will also be explored to minimize degradation phenomena while maximizing yield. Alternative ultrasound frequencies or multi-probe systems may be considered to improve cavitation distribution in large volumes. In parallel, coupling ultrasound with mild mechanical agitation or flow-through systems could provide more reproducible and scalable results. These efforts will contribute to the optimization of the process, ensuring that the promising results obtained at laboratory scale can be translated effectively into pilot-scale production. Final considerations and perspectives The activity here described was primarily directed to characterize the material upcycled and valorized (or extracted/processed) using “green” methods, as well as to complete the description of the best performing material to be selected for the formulation of “final model products” within the pharmaceutical, nutraceutical, cosmetic or fine chemical applications. These products were obtained – also in close collaboration with some Companies interested to develop new processes and new products, as described in the Project - applying the best performed methods/combined processes selected after the comparison of 10 processes (as 18 reported in D2.2.2). The main material characterized (source of bioactive ingredients) were: - Fiber from cocoa bean husks - Extract rich in polyphenols and caffeine/theobromine from cocoa bean husks - Bioactive polyphenols from apple pomace - Bioactive polyphenols and pigments from blueberry pomace - Oils from fruit kernels More materials characterized in RM2 project (more particularly rice husks, soy, cow whey and biomass from Elodea nuttalii) are described in D2.4.2. 19