Preliminary evaluation of pressurized hot water extraction for the solubilization of valuable components from hospital kitchen wastes

Moisture and ash content were determined gravimetrically. For moisture content determination, samples were placed in an air convective oven at 105 °C for 48 h, whereas for the ash content analysis, a muffle was used at 575 °C for 6 h. Protein content was estimated from the total nitrogen content value (Kjeldahl method) using the universal factor 6.25. Soluble protein was measured incubating samples with Bradford reagent (Sigma, Spain) for 20 min at room temperature and reading absorbance at 595 nm in relation to a standard curve prepared with bovine serum albumin (BSA) (Sigma, Spain). Mineral content was determined on the microwave (Marsxpress, CEM, USA)-assisted acid digestion of ashes (HNO₃ and H₂O₂ at 1600 W, 15 min and 200 °C, 10 min). The contents of Na and K were obtained by atomic emission spectrophotometry, whereas atomic absorption spectroscopy was used for Ca, Fe, B, Cu, and Mg employing a 220 Fast sequential spectrophotometer (Varian, Palo Alto, CA). Cd and Pb contents were determined by inductively coupled plasma mass spectrometry (ICP-MS) (X Series, Thermo Scientific, USA). Hg was obtained by cold vapor atomic absoprtion spectroscopy (CVAAS).

The saccharidic composition of the samples was determined after an acid digestion (72% sulfuric acid, 30 °C, 60 min). The sample hydrolysis continued in an autoclave with sulfuric acid at 4% (121 °C, 60 min). The liquid phase was separated by filtration (0.45 μm) and analyzed by high performed liquid chromatography (1100 series, Agilent, Germany) in an Aminex HPX-87H (300 × 7.8 mm, BioRad, Hercules, CA, USA) column at 60 °C with 0.003 M H₂SO₄ at 0.6 mL/min as mobile phase.

The total lipid content of the KW samples was performed following the Folch method [15]. Briefly, the mixtures were homogenized with 20 parts of chloroform:methanol (2:1), for one part of sample and centrifuged for 10 min at 3000 rpm and 15 °C. After filtering, 5 mL of H₂O was added, forming a biphase that was separated by centrifugation. The lipid content was gravimetrically determined once the chloroform was removed by rotary evaporation (320 mbar at 40 °C).

The total phenolic content was determined using the Folin–Ciocalteau reagent (1 N) and sodium carbonate at 20%. Samples were incubated in darkness at room temperature for 60 min, and the absorbance was measured at 730 nm against a distilled water blank and a standard curve with gallic acid (Sigma, Spain).

Total starch content and amylose/amylopectin ratio were calculated using two enzymatic kits (Megazyme, Co., Wicklow, Ireland) following standard procedures [1].

A representative radical scavenging capacity assay was selected, with the ABTS [2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonate)] radical cation (ABTS^·+) [31]. After incubation of samples with the ABTS^·+ solution for 6 min, the absorbance was read at 734 nm, and values were expressed as TEAC value (Trolox equivalent antioxidant capacity).

Isolated starch (moisture content: 10.3%) was used to formulate anti-freezing hydrogels based on the recommendations previously reported [48]. Starch was gelatinized at room temperature using the soluble extract fractions obtained after hydrothermal treatment. The biopolymer (15%, w/w) was dispersed in the hydrothermal liquid phase. The mixture was gelatinized under gentle stirring for 60 min at 25 °C. Starch solutions were incubated in a freezer at − 18 °C for 24 h to develop the anti-freezing hydrogels.

To illustrate the possibility of obtaining printable anti-freezing hydrogels, 3D printing trials were conducted in a Foodini 3D printer (Natural Machines, Barcelona, Spain). Two printed models with cylindrical study (15-mm diameter × 30-mm height) and spherical study (20-mm diameter) shapes were designed using Foodini creator software. The above starch/hydrothermal liquid phase solutions feed were dosed using a syringe extruder with 0.8-mm nozzle after being gellified into the printing tube at 25 °C. The printing settings were directly assigned as a new material using as the main parameters, the printing speed (1800 mm/min), ingredient flow (0.6), and gap between layers (2 mm). Several preliminary tests were performed to optimize the printing conditions taking into account the subsequent rheological features, according to the recommendations given in previous studies [10, 48]. The printed systems were placed at − 18 °C for 24 h to ensure full hydrogel maturation previously to further testing.

The water retention ability test of prepared hydrogels was made according to the method previously reported [39]. Briefly, starch hydrogels were cut into cylinders (15.0-mm diameter, 10.0-mm height) and placed at room temperature. The corresponding dehydration kinetics of the hydrogels were monitored for 2 weeks, following the weight changes to calculate the water content (%) = (initial wet mass − dry mass) / initial wet mass × 100.

Rheological testing of the starch hydrogels, in terms of elastic (G′) and viscous (G″) moduli, was conducted on a controlled-stress rheometer (MCR302, Anton Paar, Austria) using a sand-blasted parallel plate (25-mm diameter, 1-mm gap) equipped with a Peltier temperature control (− 20 to 200 °C). Frequency sweeps were performed at 10 Pa within the linear viscoelastic range (< 25 Pa) at − 18 and 25 °C.

The flow diagram of the proposed valorization of selected pre- and post-consumption hospital kitchen wastes for a representative rich-starch fractions, containing the highest values of this biopolymer (sample 8, processed up to 140 °C), is shown in Fig. 2. The starch present in this D + B waste material was efficiently recovered with the proposed treatment. Biopolymers derived from renewable resources are an emerging class of advanced materials useful as adhesives, gums, binders, and emulsions. Among these biopolymers are starch and proteins with ability to tailor materials with desirable properties [19]. Starch blends are considered new economy bio-based plastics [38]. Hydrogels are attracting extensive attention due to their biocompatibility, flexibility, multifunctionality, and porosity, for food, bioengineering, soft electronics, and agricultural applications. Starch is one of the most economical and available materials for preparing hydrogels, but becoming fragile and losing elasticity at subzero temperatures. One approach to develop anti-freezing starch hydrogels that can maintain their strength and elasticity even at subzero temperatures is the formulation of water/glycerol mixtures, and also the presence of salts in the water could lower the freezing point. Anti-freezing hydrogels with the addition of CaCl₂ could be formed into different 3D structures and could broaden the application of starch in various fields. Anti-freezing starch bio-based hydrogels have been attracting extensive attention for their excellent water retention ability and for the formulation of food, adhesives, medical materials, and intelligent materials [48]. Commonly, glycerol and CaCl₂ were concurrently introduced to improve its mechanical, thermal, and conductive properties. These materials are highly demanded as high performing, cost effective, and safe for wearable electronic devices [24] and power sources [51, 52] with potential as substrates for transient electronics. Starch could lower the electronic waste caused by non-biodegradable and non-reusable electronic components in flexible electronic devices, due to its advantages of biodegradability, cost effectiveness, abundance, and renewability.

In this context, a selected starch-rich HKW samples was evaluated for printable anti-freezing hydrogels using the corresponding soluble extract fractions obtained after hydrothermal treatment. The rheological features of formulated and printed anti-freezing hydrogels measured at both subzero (− 18 °C) and room temperature (25 °C) have been given in Fig. 3a. All the samples exhibited predominant elastic behavior, with G′ > G″, over the tested frequency range. Both elastic and viscous moduli were almost frequency invariant, indicating a typical gel-like behavior [41]. Slight frequency dependence was identified for G″ modulus above 2.5 Hz. At fixed angular frequency, G′ and G″ moduli values were slightly higher for samples measured at subzero temperature, which is consistent with the gel network reinforcement expected at lower temperatures. This suggests that proposed KW starch-based hydrogels can maintain adequate gel mechanical performance at subzero temperatures. Note here that all printed hydrogels exhibited similar mechanical profiles, with slightly lower viscoelastic properties (Fig. 3b). Anyway, the magnitude of G′ and G″ moduli indicated intermediate strength gels, with mechanical behavior at both tested temperatures with better compressive than those obtained by the traditional hydrothermal method [48]. These results suggest that the components present in the hydrothermal liquid phases (minerals as calcium, lipids, or proteins) used here for the anti-freezing hydrogel development favor the hydrophilic cross-linking, without the extra-addition of CaCl₂ or glycerol [17].

The ability of the proposed anti-freezing hydrogels to hold water in the environment was also assessed. For both formulated and printed starchy hydrogels, the water content drop from 100% at the initial stage to 75% (1 week), and then decreased slowly to 60% (2 weeks). The observed water-hold capacities were higher than those previously reported for novel anti-freezing hydrogels from potato and corn starches incorporated with CaCl₂ formulated at subzero temperatures, where incubation times of 1 week at room temperature involved 55% of water and 17–25% for those control hydrogels placed in water with the same incubation time [48]. Later, the authors explained that smaller pores of the network formed at subzero temperature, which trapped water inside the hydro gel network, can help to rise the water holding ability. Ions as Ca²⁺ present in the hydrothermal liquid phase used as solvent have a high charge density and can form strong electrostatic interactions with water molecules, which could have reduced the free water content in the hydrogel [41].

Overall, the outcomes demonstrated that it could be possible to design anti-freezing printable hydrogels into different 3D structures as spheres or cylinders with adequate rheological performance (Fig. 3b). It should be remarked that developed hydrogels were transparent, smooth, and stereo, which is indicative of the quick prototyping ability of anti-freezing starch-based hydrogels.

The flow diagram corresponding to processing a selected protein-rich HKW (sample 9, processed up to 160 °C) has been presented in Fig. 4. The corresponding soluble extracts were further concentrated in a 5-kDa cut-off ultrafiltration membrane operating in concentration mode to a final volume concentration ratio 3. Both retentate and permeate were collected and analyzed. Under these conditions permeate with 71% peptides under 5 kDa was obtained, and the carbohydrate content was 13%. The retentate contained 51% protein and 26% carbohydrates.

Protein hydrolysates obtained from recycling wastes of plant or animal origin are good candidates to be used as plant biostimulants because of their high amino acids and soluble peptide concentrations. Biostimulants are at the moment one of the most eco-friendly sustainable strategy to increase crop production reducing the use of chemicals. Vegetable- and animal-derived protein hydrolysates are interesting biostimulants for their ability to enhance plant growth and mitigate the effects of environmental stresses [37]. Their composition is influenced by the source of proteins and the hydrolytic processes (chemical-thermal and/or enzymatic hydrolysis) utilized. Whereas enzymatically produced vegetable protein hydrolysates are mainly characterized by signaling peptides, the free amino acids are the main bioactive molecules in those from animal origin chemically produced, which are more effective at low doses, because at high doses plants need to counteract the negative effects of Na and Cl at toxic levels [33]. The enzymatically hydrolyzed animal protein-based biostimulants, represent a cost-effective approach to alleviate the negative effects of several stresses in horticultural crops, by stimulating the biosynthesis of specific hormonal pathways [9]. Conventional extraction of protein from biomass relies on acid or alkaline extraction and the further production of protein hydrolysates, composed of large peptides, small peptides, and free aminoacids, can be addressed by chemical, enzymatic, or microbial hydrolysis. The enzymatic hydrolysis offers advantages to be performed under mild conditions with precise control of the degree of hydrolysis, minimizing side reactions and the presence of toxic chemicals in the products. The development of efficient and scalable methods avoiding chemicals, enzymes, and prolonged times and could be addressed using water under subcritical conditions [27]. This is a technically efficient, inexpensive, scalable and environmentally friendly technology although less selective [2].

In this work, high amounts of protein have been identified < 5 kDa (0.250-kg protein). This could make lunch + snack HKW attractive candidates for biostimulants, since it was reported that those samples with higher protein and peptide content under 3 kDa exhibited higher impact on the soil biological properties [40]. This was related to the easier assimilation of low molecular weight proteins by soil microorganisms promoting the vegetation establishment, which can protect the soil erosion. Further studies to optimize the potential of these applications and also to advance in the utilization of the insoluble solid phase are ongoing.