Vegetable lignocellulosic residues and chitosan as valuable resources in the superabsorbent bio-aerogels development for food conservation

doi:10.21203/rs.3.rs-2740851/v1

Superabsorbent bio-aerogels were developed from TEMPO-oxidized cellulose nanofibers (isolated from vegetables lignocellulosic residues) and two different commercial chitosan reactants (low and high molecular weight) by polyelectrolyte complex formation at room temperature. Three different thickness values were considered, and two different aging times were employed, thus leading to twelve different bio-aerogels samples. Remarkably, no crosslink agents nor organic solvents were used. All the developed materials were characterized and deeply studied. The bio-aerogels components were strongly interacting through hydrogen bonding, leading to highly porous and stable materials able to absorb high amounts of water and soybean oil. Then, the specific tests to visualize the material’s potential use as food absorbent pads showed: i) they were able to absorb simulated food exudates under a specific pressure in higher quantities (≈ 4000%) than some commercial pads (≈ 1600%); ii) the materials have low to moderate antibacterial activities exposing better reduction effectiveness for Listeria monocytogenes than for Salmonella typhimurium; and iii) the antioxidant properties were in general low. In the light of all the results achieved, the bio-aerogels prepared from H-CH with an aging time of 24 h and whose thickness was around 2.1 mm (H-10g-24) were selected as the most appropriate for the desired application.

Cellulose Nanofibers

Chitosan

Bio-aerogels

Absorbent Pads

Green Engineering

Consumers criteria to buy a certain product have evolved over time. People have different ways to satisfy the same need since their intrinsic history and specific consumption behavior, as well as the social influences under which they are immersed, determine their wants. In this sense, buying a food product in many cases not only implies paying attention to the good’s both price and quality, but it is also necessary to take notice about the product’s precedence, freshness, safety, packaging, and environmental impact characteristics, among many others. Several of the above-mentioned characteristics have a close relation one with each other and, from some decades up to now, have been attracting increasing attention (Li et al. 2022). For example, meat products have incorporated absorption pads as part of their packaging to entrap their possible exudates and, thus, to avoid visible drip or blood as well as undesired bad odors. Furthermore, some pads contain active compounds able to delay microbial growth and, at the same time, to extend the food shelf life (Yao et al. 2021). However, these pads are usually made of synthetic polymers making them harmful to the environment due to their non-biodegradable nature. Then, the moving towards equally useful, bio-based, and environmentally friendly food packaging materials is highly desired.

The extraction of biopolymers from residue involves various positive ecological impacts, such as the reduction of i) residues accumulation due to its exploitation; ii) the use of non-renewable resources; and iii) non-biodegradable packaging waste. It is important to remark that this practice is not competing with the food chain, and it is in line with the principles of the circular economy. On the one hand, the main green material that can be isolated from agricultural, forestry and food residues is cellulose, the most widespread biopolymer on the Earth. In general, cellulose and its micro- and nanoscale derivatives are biodegradable and suitable to develop a variety of materials, including aerogel structures. Cellulose-derived aerogels, being light-weight, meso- and macroporous materials, can be considered as suitable to be used as food absorbent pads (Plazzotta et al. 2018). However, their poor mechanical properties as well as their fragile nature are some limitations that need to be addressed (Luo et al. 2021). On the other hand, from marine residue resources (crustacean’s exoskeletons) it can be obtained the second more abundant biopolymer chitin and, after its deacetylation, chitosan. This polysaccharide not only can be considered as a useful building block to produce engineered materials, but it also displays intrinsic antimicrobial activity (Costa et al. 2021a). Nevertheless, chitosan-based aerogels have a key limitation as absorbents: they present a low absorption capability in neutral media (Ko and Kim 2020). To successfully take advantage of the intrinsic properties of both cellulose and chitosan, as well as to overcome some of their limitations, these two biopolymers are usually combined to generate new bio-aerogel materials. For example, Fan and coworkers prepared Nanocellulose/Chitosan aerogels with enhanced properties by exploiting the opposite charge of the starting materials to generate polyelectrolyte complexes (PECs) (Fan et al. 2020). The electrostatic interactions between the starting materials allow the formation of stable structures thus avoiding the employment of auxiliary substances, like crosslinkers.

In the present research work the main objective was to develop, through eco-friendly procedures, low-density and highly absorbent bio-aerogels with antimicrobial properties potentially useful as food pads. For this, TEMPO-oxidized cellulose nanofibers (CNF) isolated from vegetables lignocellulosic residues and two different molecular weight (Mw) chitosan powders (low and high Mw, L-CH and H-CH, respectively) were employed. Remarkably, no crosslink agents nor organic solvents were used. Even though both biopolymers have been previously considered as starting materials for the development of different food packaging products, to the best of our knowledge the here presented specific reactants combined and treated by this preparation procedure have not been previously considered (Abdullah et al. 2022; da Silva et al. 2020; Zhou et al. 2022).

2.1- Materials

Vegetable lignocellulosic residues were kindly donated by an independent farmer from Córdoba, Spain. The reagents used for cellulose nanofibers production were: Sodium Hydroxide (NaOH, ≥ 97%), Sodium Chlorite (ClNaO₂, > 80%), Acetone, Acetic Acid (ACS reagent, ≥ 99.7%); 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO, free radical, 98%) from Sigma Aldrich; Sodium Bromide (NaBr, 99%) and Sodium Hypochlorite (NaClO, 80%) supplied from Honeywell, Fluka™. Furthermore, the reagents used for aerogels preparation and characterization were: L-CH and H-CH supplied by Sigma-Aldrich; Soybean Oil supplied by Guinama S.L.U.; Sodium Chloride (NaCl, > 99.8%) from Labbox; Mueller-Hilton Agar was supplied by Oxoid Ltd; Methanol (> 99,9%) from PanReac AppliChem ITW Reagents and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) supplied by Sigma-Aldrich.

2.2- Methods

Scheme 1 shows the workflow followed during the present study. Briefly, cellulose nanofibers have been obtained from vegetable lignocellulosic residues and used for the formulation of aerogeles together with chitosan. The detailed methodology for these procedures as well as the characterization performed for all the fractions are described in this section.

2.2.1- Nanocellulose preparation and characterization

Cellulose nanofibers were isolated from cellulose pulp obtained from a mixture of eggplant, tomato and bell pepper pruning in identical proportions (33%). The pruning mixture was subjected to an environmentally friendly alkaline process using a 15 L rotary reactor (Metrotec S.A.) under the following conditions: 7% NaOH on dry matter, 100°C, 150 min and a liquid/solid ratio of 10/1. Once chemically treated, the pruning mixture was passed through a disintegrator (Metrotec S.A.) and subsequently processed in a Sprout-Bauer defibrillator (Combustion Engineering) to achieve the fibers fibrillation. The resulting suspension was sieved using a 0.14 mm mesh to obtain cellulose pulp (Eduardo Espinosa et al. 2019).

The cellulose pulp was bleached by successive treatments with chlorine dioxide produced from sodium chlorite and concentrated acetic acid. Three bleaching treatments were carried out by adding 15 g of sodium chlorite and 5 mL of concentrated acetic acid to a suspension of 50 g of dry pulp in 1600 mL of water. This mixture was kept at 75–80°C in a water bath. This specific treatment was applied every hour until the bleached pulp was obtained.

Regarding the chemical characterization of the raw materials on their ethanol extractables, ash, holocellulose, lignin, and α-cellulose, it was done according to TAPPI standardization standards T-204, T-211, T-222, T-203os61 and T-9m54, respectively.

CNF were obtained using an oxidation pretreatment with TEMPO catalyst by adding as oxidizing agent 5 mmol/g of NaClO at 10% maintaining a medium pH = 10.2. Once the addition of the oxidant was finished, 0.5 M NaOH was added to maintain the pH constant until the end of the process. To stop the reaction, 100 mL of ethanol was added and then washed with distilled water (Besbes et al. 2011). The suspension obtained at a concentration of 1% was subjected to a high-pressure homogenization process in a homogenizer (Panda GEA 2K Niro). This process was carried out in 4 cycles at 300 bar, 3 cycles at 600 bar and 3 cycles at 900 bar pressure (Eduardo Espinosa et al. 2019).

For the characterization of CNF, several tests described in the literature were performed. The nanofibrillation yield was determined by centrifuging a 0.1% CNF suspension at 10,000 rpm for 20 min (Eduardo Espinosa et al. 2019). The optical transmittance was measured from 400 to 800 nm at 0.1% suspension on a Perkin Elmer Lambda 25 UV spectrometer. Cationic demand (CD) was determined following the methodology described in the literature using a Particle Charge Detector (Mütek PCD 05) (Carrasco et al. 1998). The carboxyl content (CC) was measured by conductimetric titration according to the methodology proposed by Besbes et. al (Besbes et al. 2011). The intrinsic viscosity (η_s) of the CNF was determined according to ISO 5351:2010. The intrinsic viscosity value is related to the degree of polymerization (DP) of the CNF based on the following equation (Eq. 1) (Marx-Figini 1987):

\(DP \left(<950\right):DP=\frac{{{\eta }}_{s}}{\text{0,42}}\) (Eq. 1)

Then, the DP is related to the length of the nanofibers according to the equation described by Shinoda et. al (Eq. 2) (Shinoda et al. 2012):

\(Length \left(nm\right)=4.286·DP-757\) (Eq. 2)

The calculation of the theoretical specific surface area (\({\sigma }_{CNF})\) was obtained from the known surface area of the cation used in the determination of the cation demand, polydiallyldimethy- lammonium chloride (Poly-DADMAC) by employing the Eq. 3 (E. Espinosa et al. 2016). For this purpose, the net cationic demand of the nanofiber (CD - CC) was determined, and assuming the monolayer interaction of the poly-DADMAC on the fiber surface, the specific surface area of the nanofiber was determined.

\({\sigma }_{CNF}=\left(CD-CC\right)· {\sigma }_{Poly-DADMAC}\) (Eq. 3)

where \({\sigma }_{Poly-DADMAC}\) corresponds to the surface area of a single poly-DADMAC molecule.

Finally, TEMPO oxidized CNF were also prepared from unbleached pulp (L-CNF) to compare the obtained results and to better study the prepared materials.

2.2.2- Bio-aerogels preparation

Firstly, two different CH 0.5 wt% solutions were prepared: one employing the high Mw reactant whereas the other one contained the low Mw polymeric material, both being dissolved in acetic acid 1 v/v %. Then, an aqueous suspension of CNF (0.5 wt%) was also prepared. For each bio-aerogel, equal mass quantities of a CH solution and the CNF suspension were mixed during 3 minutes at 10,000 RPM in an ultraturrax homogenizer (IKA T18 digital Ultra Turrax). After this, 5, 10 and 15 g of each suspension were poured into Petri dishes in order to finally have different bio-aerogel thickness values. Then, to determine if the aging time influences the biopolymers possible spatial rearrangement and the further formation of intra/inter PEC complexes, one half of the Petri dishes were directly freeze and lyophilized, whereas the other half was left at room temperature during 24h before this final freeze-drying treatment. Thus, 12 different bio-aerogel materials were developed (Table 1). Additionally, blank CNF and CH bio-aerogel materials were prepared using 10 g of each corresponding solution/ suspension, following an analogous procedure.

Table 1

Bio-aerogel formulations prepared.
Number	Chitosan	Mass (g)	Aging (h)	Sample	Thickness (mm)
1	Low Mw	5	0	L-5g-0	0.650 ± 0.066
2		5	24	L-5g-24	0.819 ± 0.080
3		10	0	L-10g-0	2.218 ± 0.066
4		10	24	L-10g-24	1.83 ± 0.296
5		15	0	L-15g-0	3.807 ± 0.327
6		15	24	L-15g-24	3.069 ± 0.902
7	High Mw	5	0	H-5g-0	0.858 ± 0.202
8		5	24	H-5g-24	0.777 ± 0.136
9		10	0	H-10g-0	2.189 ± 0.382
10		10	24	H-10g-24	2.168 ± 0.381
11		15	0	H-15g-0	3.890 ± 0.341
12		15	24	H-15g-24	3.681 ± 0.186

The pH of the bio-aerogel-forming solutions and suspensions were all determined in triplicate. The zeta potential of these same suspensions was measured using a Zetasizer (ZSP, Malvern Instrument) at 25°C. A concentration of 10^− 3 g/mL was prepared for all measured samples using distilled water as solvent and an inert electrolyte. Previously, the suspensions were homogenized using the ultraturrax homogenizer for a period of 60 s. The analysis was realized by triplicate and the corresponding average values and standard deviation were calculated.

2.2.3- Bio-aerogels characterization

The obtained biomaterials were characterized through the employment of different techniques. The aerogels thickness was measured on triplicate using a Digital Micrometer IP65 0–1” (Digimatic, Mitutoyo). The Swelling Degree (SD, %) of each aerogel (square samples with a surface area of 4 cm²) at t time in both distilled water and soybean oil at 25°C was obtained by using the following equation (Eq. 4):

\(\text{S}\text{D} \left(\text{%}\right)=\frac{{\text{W}}_{\text{t}}-{\text{W}}_{\text{i}}}{{\text{W}}_{\text{i}}} \times 100\) (Eq. 4)

where W_i is the weight of the samples before immersion, and W_t is the weight of the sample at t time. The water/oil retention at 120 minutes is also expressed as mass of fluid (g) per gram of material. Measurements were run on triplicate.

After analyzing the swelling results, the following characterizations were conducted by using some selected samples. The chemical interactions between the CNF and the CH were explored through Fourier Transform Infrared Spectroscopy analysis (FTIR). Thus, the FTIR Spectra of the aerogels were recorded on a FTIR-ATR Perkin-Elmer Spectrum Two equipment, in the range of 400–4000 cm^− 1 and employing a resolution of 4 cm^− 1 (40 scans for each sample).

The materials morphology was studied by Scanning Electron Microscopy (SEM) in a ZEISS DSM 960A instrument. All the samples had been previously swollen in a water medium, frozen, lyophilized, cryo-fractured after their immersion in liquid N₂, and sputtered with a thin gold layer. The Image J Software was employed to measure the samples pores diameters.

Regarding the thermal properties of the bio-aerogels, they were explored by Thermogravimetric Analysis (TGA). The measurements were conducted in Netzsch TG 209F3 equipment from room temperature to 900°C at a heating rate of 10°C/min, and under nitrogen atmosphere. Additionally, X-Ray Diffraction (XRD) determinations were carried out in a Bruker D8 Discover Instrument for 2θ values from 10 to 65 degrees (rate of 2°/min), employing monochromatic Cu-Kα radiation.

Finally, to explore and obtain information about the soluble fraction characteristics of the bio-aerogel materials, a fixed mass of them was immersed into distilled water at 25°C for a time period of 4 days. Then, each sample was removed from distilled water, and dried until constant weight was reached. Therefore, the corresponding Soluble Fraction (SF %) values were calculated as follows (Eq. 5):

\(\text{S}\text{F} \left(\text{%}\right)=\frac{{\text{W}}_{\text{i}}-{\text{W}}_{\text{f}}}{{\text{W}}_{\text{i}}} \times 100\) (Eq. 5)

where W_i and W_f are the weights of the dried bio-aerogels before and after immersion, respectively. Measurements were run on triplicate.

2.2.4- Bio-aerogels performance as food absorbent pads

To determine the capability of the selected bio-aerogels to act as food absorbent pads, three different specific tests were conducted. The first set was the material’s capacity to absorb a simulated exudate fluid, while the second and third ones were oriented to better know their antimicrobial and antioxidant capacity, respectively.

The exudates absorption capability was tested on triplicate in accordance to a bibliographic methodology (Costa et al. 2021b). Briefly, previously weighted discs of aerogels (5.6 cm of diameter) were immersed in NaCl 0.9 wt% (pH 5) and submitted to 80 g of pressure for 2 minutes. After this, the samples were weighted again. The results were processed by using an equation analogous to Eq. 5, and they were finally expressed in percentage relative to the initial aerogel mass.

The antimicrobial activity of the different bio-aerogels was performed by using the immersion method. Two different pathogenic bacteria commonly found in meat products were assayed, the Gram-negative bacteria Salmonella typhimurium (CECT 704) and the Gram-positive bacteria Listeria monocytogenes (CECT 4032). Concisely, bacterial inoculate were prepared by taking an aliquot of a fresh culture to saline solution (0.85% w/v) until an absorbance between 0.08–0.1 at 625 nm (approximately 10⁸ cfu/mL) was reached. A 20-fold dilution was made to obtain 5 x 10⁶ cfu/mL in the test tube. The different materials were weighted and immersed in the respective tubes. These were incubated for 24 h at 37 ± 1°C, taking samples at 0, 1, 3, 5, 7 and 24 hours, determining the number of CFU by plating serial dilutions. Control samples were also performed for both bacteria. The antimicrobial activity of the aerogels was expressed as a logarithmic reduction calculated according to Eq. 6, where R stands for the logarithmic reduction, C_t0 is the mean number of viable cells of bacteria in the test tube at the moment of contact with the material, C_tn is the mean number of viable cells of bacteria in the test tube at a given time and W the weight of the aerogel in milligrams.

\(\text{R}=\frac{\text{log}\frac{\text{C}\text{t}0}{\text{C}\text{t}\text{n}}}{W}\) (Eq. 6)

Finally, the antioxidant activity of the prepared materials was evaluated according to the free radical 2,2-Diphenyl-1-picrylhydrazyl (DPPH) methodology, following the protocol proposed by de Oliveira et al. (Brand-Williams et al. 1995; Marcucci et al. 2020). Briefly, 1 mg of aerogel samples were mixed with 3.9 mL of previously prepared 0.02 mM DPPH solution in methanol. The samples were allowed to stand out from the light during 2h 30min and the absorbance was measured at 517 nm in a Perkin Elmer UV/Vis spectrometer Lambda 25. The ability to sequester the radical, expressed as a percentage, was calculated following the Eq. 7:

\(\text{I}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n} \left(\text{%}\right)=\frac{{\text{A}\text{B}\text{S}}_{\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}-{\text{A}\text{B}\text{S}}_{\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}}{{\text{A}\text{B}\text{S}}_{\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}}\text{x} 100\) (Eq. 7)

3.1- Nanocellulose

During the pulping process a yield of 22.3 ± 3.31% was obtained. The data obtained from the chemical characterization of the raw material as well as both the bleached and unbleached pulp are detailed in the Table 2. It can be seen how, comparing the data between the raw material and the unbleached pulp, the treatment affects the fiber by reducing the content of inorganic compounds (ash), obtaining values of 14.86% for the raw material and 4.68% for the unbleached pulp. Furthermore, there was a 6.1% decrease in extractables and an increase in the amount of α-cellulose from 32.10% of the raw material to 50.45% of the unbleached pulp. This increase is due to the total decrease in mass during the pulping process, leaving the cellulose intact. The bleached pulp did not show significant differences with respect to the unbleached pulp in terms of ash and extractable compounds. There was a decrease to 6.39% of lignin with respect to the unbleached pulp (16%), which demonstrates the effectiveness of the chlorine oxide treatment. There were no significant differences in the rest of the parameters except for an increase in the amount of α-cellulose produced by decreasing the total mass by removing most of the lignin.

Table 2

Chemical characterization of the raw material as well as both the bleached and unbleached pulp.
	Horticultural plants mixture (%)	Bleached pulp (%)	Unbleached Pulp (%)
Ashes	14.86 ± 1.35	3.20 ± 0.35	4.68 ± 0.02
Extractables	17.12 ± 1.55	9.11 ± 0.41	11.02 ± 0.86
Lignin	15.03 ± 1.36	7.31 ± 2.37	16.00 ± 0.63
Hemicellulose	20.89 ± 1.90	16.23 ± 0.44	18.17 ± 1.21
α-Cellulose	32.10 ± 2.91	64.42 ± 5.91	50.45 ± 2.81

Then, the prepared CNF and L-CNF were fully characterized (Table 3). The nanofibrillation yield obtained from the considered raw material was significantly lower than that obtained in other studies with other raw materials, where values above 90% have been achieved (Eduardo Espinosa et al. 2019). The value obtained in the optical transmittance test was also lower in the data reflected regarding other studies, something that agrees with the yield obtained since the samples with lower nanofibrillation usually present lower transmittance due to the lower light dissipation of the nanofibers that are inactive from the optical point of view (Eduardo Espinosa et al. 2019). A relatively high value is observed in the CD, a measure that provides information about the interaction of the anions on the fiber surface with the surrounding area and therefore it is possible to estimate the specific surface area of the nanofibers (E. Espinosa et al. 2016). The CD values obtained for CNF and L-CNF are 1043.54 µeq/g and 621.55 µeq/g, respectively. This difference in cation demand values shows how the effectiveness of the TEMPO-mediated oxidation pretreatment varies in introducing carboxyl groups into the cellulose when the lignin has not been removed from the cellulose fibers, and improving the nanofibrillation efficiency that results in higher values for the specific surface area of each sample (436 m²/g for CNF and 262 m²/g for L-CNF). The effectiveness of the TEMPO pretreatment when decreasing the amount of lignin is also evident in the determination of the CC, where a higher value is seen for the CNF sample (148.12 µeq/g) than for the L-CNF sample (83.83 µeq/g). With respect to morphology, the smaller diameter of the bleached sample is indicative of a larger specific surface area, characteristic that allows a greater interaction of the nanofibers with nearby compounds (Delgado-Aguilar et al. 2015). Additionally, it can be observed that there is a decrease in the DP, a parameter that is related to the length of the fibers (Shinoda et al. 2012). As the number of monomers forming the polymer chain decreases the fiber length also does it. This parameter does not significantly affect the formation of the material in question since it depends to a greater extent on the number of interactions that can occur with the fiber and, therefore, on the specific surface area (Eduardo Espinosa et al. 2019). Based on the results obtained, and the use of nanofibers as co-polymer in the formulation of bio-aerogels together with chitosan (negatively charged), CNF samples were selected for the formulation of these bio-aerogels due to their higher nanofibrillation performance and cation demand that will allow a higher electrostatic interaction with the chitosan chains.

Table 3

CNF and LCNF TEMPO main characterization results.
Sample	Yield (%)	T 800 (%)	CD (µeq/g)	CC (µeq/g)	Length (nm)	σ_especific (m²/g)	Diameter (nm)
CNF	63.44 ± 4.52	74.7	1043.54 ± 18.2	148.12 ± 5.26	614.26	436	6
L-CNF	49.52 ± 2.95	44.3	621.55 ± 9.45	83.83 ± 4.28	812.96	262	10

Obtaining cellulose nanofibers from a mixture of agricultural residues increases the plasticity of the process since it does not depend solely on a raw material that can be obtained seasonally, thus guaranteeing to a certain extent the availability of resources for the process. The results obtained from one mixture can be extrapolated to other tests, which is difficult when only one raw material is involved. In addition, the results show a smaller fiber diameter, increasing the surface area for hydrogen bridging between nearby fibers and thus improving the properties of the material.

3.2- Bio-aerogels

Twelve different bio-aerogel materials were successfully obtained through the electrostatic interaction of poly-anionic (CNF) and poly-cationic (CH) species, which were able to form PECs. Among the variables that usually have a strong influence on the PECs characteristics are the poly-ion species relative Mw, their functional groups strength, their concentration and stoichiometric ratio, the medium’s pH, the order of addition of each component and the mixing protocol (Kulkarni et al. 2016; Sæther et al. 2008). Taking into account the desired materials ability to control the microbial stability in food, and since having positive charges in the chitosan’s structure is a pre-requisite for having antibacterial activity towards both Gram positive and Gram negative bacteria, an excess of positive unreacted charges was looked-for in the preparation of the bio-aerogels (Raafat and Sahl 2009; Sahariah et al. 2014).

In this research, some preliminary bio-aerogels were prepared with higher amounts of the CNF suspension (CH:CNF ratios 1:3; 1:6 and 1:9). To preserve the charges imbalance, Ca²⁺ was also incorporated to the formulations to crosslink the CNF chains without the use of all the available –NH₃⁺ species. The so-obtained materials were discarded due to their poor mechanical and water-resistance properties. Then, only the materials prepared from a 1:1 ratio were considered without the use of crosslinkers or auxiliary substances, and their precursor CH solutions, the CNF suspension and the two bio-aerogel forming suspensions both pH and Zeta potential were studied (Table 4).

Table 4

pH and Zeta potential of the precursor CH solutions, the CNF suspension and the two bio-aerogel forming suspensions.
Sample	pH	Zeta potential
L-CH 0.5 wt% solution	3.65 ± 0.01	45.7 ± 2.69
H-CH 0.5 wt% solution	3.59 ± 0.07	37.85 ± 2.62
CNF 0.5 wt% suspension	5.61 ± 0.17	-11.5 ± 1.48
L-bio-aerogel-forming suspension	3.82 ± 0.03	20.97 ± 1.25
H-bio-aerogel-forming suspensions	3.86 ± 0.06	17.03 ± 1.93

At the working pH conditions, the precursor CH solutions and the CNF suspension have enough opposite charges to be attracted and mutually complexed. Despite having PEC stable complexes, the resulting forming suspensions showed the wanted excess of positive charges. Then, in order to determine the swelling properties of the prepared materials, several tests were conducted in both water and soybean oil medium (Fig. 1).

It is widely known that both the hydrophilicity and the PEC stability in a liquid medium depend on a specific combination of factors. The obtained results showed that almost all materials have excellent both water and oil uptake properties, being them significantly higher than the offered by other bio-based absorbent pads (Ebadi et al. 2021), and from similar to higher regarding some superabsorbent bio-aerogels designed for food packaging purposes (Fontes-Candia et al. 2019). In general, the 5 g samples showed lower swelling capabilities than the 10 g and 15 g ones, being the SD of these last materials quite similar. Then, the bio-aerogels thickness characteristics (Table 1) might not be considered as determinant in the swelling behavior of the bio-aerogels. Otherwise, in the water swelling phenomenon, the CH’s Mw plays some important role since all the samples prepared with the higher Mw presented higher SD. On the contrary, although the differences are not so noticeable, in the oil swelling tests the best performance was achieved by the samples containing the CH with lower Mw. These observations might be explained by considering the PEC formation mechanism and their further aggregation (Fig. 2) (Kulkarni et al. 2016). Thus, the lower CH-Mw bio-aerogels might contain a higher amount of hydrophobic well-ordered secondary structures. Their formation requires the presence of new interactions and/or the correction of the possible existing distortions of the polymer chains to define that new conformation of the PEC complex, so the process may be favored by the employment of shorter starting materials. Additionally, since the aging time determines not only the intra-complex formation but also the inter-complex one, it can be expected to observe higher oil swelling affinities by the 24h-samples. This fact was experimentally confirmed, chiefly in the lower CH-Mw samples, thus reinforcing the previously stated hypothesis.

In general, the bio-aerogels swelling in soybean oil reached the equilibrium more quickly than when the analogous tests were conducted in water. This kind of aerogel’s sorption behavior dependence on the liquid medium was also observed by other authors (Chen et al. 2022). On the one hand, in the water medium the sorption process takes place by both simple capillary suction and by the pressure exerted by the liquid. During this, there are also charge interactions between the water, the motionless polymers integrating the aerogel network and their corresponding mobile counterions (Gorshkova et al. 2021). Thus, reaching the equilibrium in this polar fluid takes more time than in non-polar ones, like the soybean oil. The viscosity of the soybean oil (48.25 ± 0.05 MPa.s at 20°C) is higher than the corresponding to the water (1.00 MPa.s at 20°C), leading to a more easy adhesion to the near surface and inside the aerogels, but not to an easy diffusion and further penetration to the aerogel’s interior (Korson et al. 1969; Wu et al. 2021).

The corresponding swelling behaviors of the three blank CNF and CH bio-aerogel materials were also evaluated. It was found that the pure CH materials were completely unable to retain its structural integrity when they were submerged in water and, thus, these materials were discarded. Regarding the CNF bio-aerogels, their water and soybean oil absorption capacities at 120 min were 63.5 ± 9.78 g/g and 39.41 ± 21.87 g/g, respectively. As it could be expected, the water sorption properties were similar or not-remarkably lower than some of the PEC bio-aerogel samples (especially in the case of L-CH based materials), whereas when this characteristic is compared to the one corresponding to the H-CH based materials a great difference is observed. As to the soybean oil uptake, in almost all cases an improvement was achieved in the case of PEC bio-aerogel samples regarding the blank CNF ones. So, after a deep analysis of the swelling results and taking into account the bio-aerogel’s ability to preserve their structural integrity after the essays, two different materials samples were selected to keep doing the characterization tests: L-10g-24 and H-10g-24. However, in several cases the samples L-10g-0 and H-10g-0 were employed as blank samples in order to better study the bio-aerogel materials developed.

The FTIR spectra not only of the aerogels but also of the starting materials were analyzed (Fig. 3). The typical main bands were observed for the CNF sample: at 3200–3400 cm^− 1corresponding to the -OH groups, near to 2900 cm^− 1 due to -CH stretching and at 1600 cm^− 1 being related to the -C = O functional groups present as result of the TEMPO oxidation treatment (Eduardo Espinosa et al. 2017). On the other hand, both CH materials showed a broad band centered at 3300 cm^− 1 corresponding to -OH and -NH groups, another one at 2870 cm^− 1 because of the -CH groups, whereas the ones at 1659 cm^− 1 and 1555 cm^− 1 were indicating the Amide I and Amide II bands (Costa et al. 2021a). Then, as it was expected, aerogels have a mixture of bands from both components, and no covalent linkages formation were confirmed. However, there is evidence about ionic interactions in the PECs formed: the absence of the CNF band at 1600 cm^− 1and of the CH band from 1659 cm^− 1, the appearance of the 1638 cm^− 1 band indicating the electrostatic interaction between the two biopolymers, as well as the carboxylate shift from 1427 to 1411 cm^− 1. These bands were also observed by other authors in similar PEC materials (Ostrowska-Czubenko and Gierszewska-Druzyńska 2009). No significant differences were observed by comparing the 0 and the 24 h samples.

Regarding the morphology of the prepared bio-aerogels, the SEM micrographs showed the highly porous nature of the materials (Fig. 4). The pores sizes diameters were higher in the L-10g-24 samples than in the H-10g-24 materials, whereas the pores size distribution was also broader in the case of L-10g-24 bio-aerogels. Since the precursors concentrations and freezing conditions before and during lyophilization were identical, the only intervening factor is related to CH’s Mw. During the freezing stage, ice crystals nucleates and propagates, leading to water-rich and water-poor zones, influencing the pore walls formation (Sanchez et al. 2019; Sanchez and Alvarez 2019). During this process, the polymer chains length and mobility can limit the ice crystals growth: the longer the CH chain the smaller the ice crystals growth and, thus, the obtained pores sizes decrease (Liao et al. 2019). Moreover, from the obtained results, it is expectable that the H-10g-24 bio-aerogels exert a higher capillary pressure (Young-Laplace Equation, Eq. 8) during swelling tests and, thus, to offer higher oil-swelling capabilities than the L-10g-24 materials.

\({\Delta }\text{P}=\frac{2{\gamma }\text{c}\text{o}\text{s}{\theta }}{\text{r}}\) (Eq. 8)

where \({\Delta }\)P is the capillary pressure, θ is the contact angle of the air/fluid interface formed with the pores wall surface, γ refers to the surface tension of the liquid and r corresponds to the pore size (Ding and Li 2022). Even though the obtained results have shown a contrary trend, that is, the H-10g-24 materials presented the lower oil-swelling capabilities, it is important to mention that some other factors are also of key importance in the oil sorption phenomenon. In this sense, to overcome the minimum required energy for the oil to be absorbed it is necessary to have chemical compatibility between the bio-aerogel surface and the fluid at issue. Furthermore, the total porosity of the sample as well as the surfaces roughness also represent key factors (Rafieian et al. 2018). Then, in this case it might be happening that the other intervening variables result more defining than the pores sizes by themselves.

The thermal properties studies through TGA and DTGA showed the main thermal event in CH samples at 287°C, near to the already reported data (300°C), whereas the CNF sample presented two remarkable events at 234 and 291°C, also similar to those observed by other authors (Costa et al. 2021a; Ostrowska-Czubenko and Gierszewska-Druzyńska 2009; Tang et al. 2018). On the other hand, both bio-aerogels presented a maximum at 263°C, which represents an improvement in thermal stability of the materials regarding the CNF starting material, similarly to previous reported results (Tang et al. 2018). Finally, all the thermal events occurring at temperatures lower than 150°C are related to the loss of both free and bonded water molecules.

The diffraction patterns obtained by XRD for both CH samples and the CNF showed the expected diffraction patterns (Figure S1, 20° for CH and 22.4° for CNF), whereas the bio-aerogels presented broader peaks as a result of the electrostatic interactions leading to have more amorphous and less crystalline zones all along the materials (Hermanto et al. 2019; Morcillo-Martín et al. 2022; Ostrowska-Czubenko and Gierszewska-Druzyńska 2009). As to the SF determinations (Figure S2), the results have showed that in both cases less of half the materials weight is loss after 4 days immersion tests, condition that is far much more destructive that the ones usually present during the employment of the materials as food absorbent pads. Thus, these can be considered as encouraging results.

As the first test regarding the possible in-service specific properties of the developed materials, their ability to absorb simulated food exudates under a specific pressure was evaluated (Table 5). The obtained performances were much higher than other previously observed including not only new materials potentially useful as food pads (70 and 120%), but also commercial pads (1600%) (Costa et al. 2021a). Furthermore, the blank CNF bio-aerogel material was also subjected to this evaluation, and the model exudates absorption capability was found to be smaller than the ones achieved by the PEC bio-aerogel materials. Then, the evaluation of these bio-aerogel’s antimicrobial activity was also conducted (Table 6). Clear differences were observed between the two microorganisms tested. Overall, the antimicrobial activity of the aerogels is moderate, having slight changes along time, and reaching in most cases the maximum antimicrobial activity in the first hour of contact for the two considered microorganisms. When the reduction effectiveness for both pathogenic bacteria is compared, it is possible to see that Salmonella typhimurium was practically unaffected by the presence of the aerogel in the medium (samples S-H-10g-24 and S-L-10g-24). However, for Listeria monocytogenes higher reductions were observed, especially when aerogels made with high CH-Mw were employed: 0.36 logarithmic reductions per mg of material were reached in the first hour (L-H-10g-24). These observations are in agreement with previously reported observations regarding the inhibitory effects of unmodified chitosan towards both Gram positive and Gram negative bacteria due to the intrinsic nature of each considered microorganisms group (Ardean et al. 2021; Jeon et al. 2001; Sahariah et al. 2014). First, Salmonella typhimurium is a Gram-negative bacterium; it possesses an outer membrane that protects it from the hydrolysis of the peptidoglycans caused by the electrostatic interactions between protonated NH₃⁺ and the negative residues present in cell membrane, which usually leads to the leakage of intracellular electrolytes such as potassium ions and other low molecular weight proteinaceous constituents (Atay 2020). In contrast, Listeria monocytogene is a Gram-positive bacterium lacking the protection offered by this outer membrane. Therefore, Listeria monocytogene has greater vulnerability to the bio-aerogel’s components.

Table 5

Exudates absorption performance.
Absorbent	Model exudate absorption (%)
L-10g-24	4064.0 ± 252.9
H-10g-24	3949.3 ± 532.6
Blank CNF	2230.5 ± 90.69
Commercial	1600 (Costa et al. 2021a)

Table 6

Value of antimicrobial activity of the different bio-aerogels expressed as a logarithmic reduction per mg of material.
Time (h)	Salmonella typhimurium Control	Listeria monocytogene Control	S-L-10g-24	S-H-10g-24	L-L-10g-24	L-H-10g-24
1	0.12	0.00	0.00	0.02	0.06	0.36
3	0.11	0.09	0.02	0.01	0.03	0.33
5	0.14	0.18	0.02	0.00	0.04	0.35
7	0.10	0.12	0.02	0.00	0.04	0.33
24	0.16	0.03	0.02	0.00	0.04	0.32

In the present study, it was found that as CH’s Mw in the bio-aerogel increases the antimicrobial capacity against gram-positive bacteria also does it. A priori, these results could be considered as contradictory when considering the bio-aerogel forming suspensions zeta-potentials and that the CH’s antimicrobial activity is a result of its cationic property (Han et al. 2010). However, the presence of a certain number of positive charges in the materials is not the only determining factor: the positioning and availability of the cationic charges also play a key role (Ardean et al. 2021; Sahariah et al. 2014). It is possible to connect the hypothesis previously exposed about the biopolymers interaction to form the PEC complexes with this observation. As it was stated some paragraphs ago, the lower CH-Mw bio-aerogels might contain a higher amount of hydrophobic well-ordered secondary structures. Then, the biopolymer’s positive charges are expected not to be available for the cell disruption of the bacteria by stablishing the expected electrostatic interactions with the anionic components of its membrane, thus leading to a lower antimicrobial activity than the offered by the higher CH-Mw bio-aerogels. Although the achieved antimicrobial activities might be considered as low, it is of key importance to mention that in most of the articles focused on the manufacture of aerogels for meat products, the materials were functionalized with some antimicrobial agent such as essential oils, acids, metals, among others (Agrimonti et al. 2019; Fontes-Candia et al. 2019; Kim et al. 2021). Additionally, establishing comparisons with other authors who developed similar materials is sometimes difficult since the antimicrobial properties are not reported by mass of material, which makes the results imprecise. Nonetheless, other authors have produced similar aerogels showing a lower antimicrobial capacity as the chitosan: cellulose nanofiber and alginate: chitosan ratio was close to 1:1 (Costa et al. 2021a; Pan et al. 2021).

Finally, since preventing meat both color changes and the appearance of disgusting odors by simple avoiding the oxidation processes is highly looked-for in the meat packaging field, the antioxidant properties of the bio-aerogels were tested (Fig. 6). The results showed that the inhibition percentages were in general very low. Interestingly, the bio-aerogel L-10g-24 presented the lower inhibition percentage. Although at present the action mechanism is not completely known, various researchers agree that the CH antioxidant activity has a close bond to the –OH and –NH₂ functional groups (Sun et al. 2007; Xie et al. 2001). Then, to offer scavenging activities against radicals it is necessary to have the hydroxyl and amino groups exposed and available, which means they are needed not to be sterically impeded nor forming hydrogen bonds. In accordance to the present and previously discussed results, the L-10g-24 material do not have this required characteristics. The stated hypothesis is, once again, reinforced.

Twelve different bio-aerogels were prepared by considering a certain set of variables: two CH’s Mw (low and high Mw), three thickness values (from 0.65 to 3.89 mm) and two aging times (0 and 24 h). Then, the materials were fully characterized not only in general terms but also taking into account the specific properties needed to have bioactive superabsorbent food pads. In all cases the polymer chains were interacting via hydrogen bonding, forming stable PEC complexes able to retain high quantities of both water and soybean oil. Although there was some trend of L-CH based materials to be better absorbents of soybean oil than H-CH ones, and worse at absorbing water, this property was not influenced by the bio-aerogels thickness. Furthermore, the pores sizes diameters were smaller in the H-10g-24 samples than in the L-10g-24 materials, whereas the pores size distribution was less wide in the case of H-10g-24 bio-aerogels. Moreover, the thermal analysis showed a little improvement in the bio-aerogel samples characteristics regarding the CNF taking into account its main thermal degradation event. It is of key importance to note that the L-10g-24 and H-10g-24 bio-aerogels were able to absorb simulated food exudates under a specific pressure in higher quantities (≈ 4000%) than some commercial pads (≈ 1600%). The two considered materials offered low to moderate antibacterial activities, exposing better reduction effectiveness for Listeria monocytogenes (0.36 logarithmic reductions per mg of material were reached in the first hour when using H-10g-24) than for Salmonella typhimurium. Finally, the antioxidant properties were in general low. However, since the other bio-aerogel properties make these materials suitable to be considered as food absorbent pads (specially the H-10g-24 material) and taking into account their possible compatibility with both natural extracts and essential oils (rosemary, eugenol, clove) having antioxidant activities, the authors consider that incorporating them to the here presented bio-aerogels in further studies might lead to active materials even more suitable for the desired application.

Declarations

Conflict of Interest

Funding

Dr. Laura M. Sanchez acknowledge the financial support of CONICET, ANPCyT (PICT-2018-0711) and UNMdP, and she was benefited from conversations during her research stays supported by Fundación Carolina, Fundación Bunge y Born and Fulbright Argentina - Ministerio de Educación. Esther Rincón acknowledge the support from Universidad de Córdoba-Predoctoral Grant 2019. The authors acknowledge the financial support from the Department of Economic Transformation, Industry, Knowledge and Universities (Regional Government of Andalusia). This research is part of the project P18-RT-4064. The authors also thank the staff of the Central Research Support Service (SCAI) of the Universidad de Córdoba. The graphical abstract was created in BioRender.com.

Author contributions

The experimental work was carried out by Dr. Laura M. Sanchez, Esther Rincón, Jorge de Haro Niza and Ramón Morcillo Martín; data analysis was performed by Dr. Laura M. Sanchez, Esther Rincón, Jorge de Haro Niza and Ramón Morcillo Martín; the article was written by Dr. Laura M. Sanchez, Jorge de Haro Niza and Ramón Morcillo Martín; the article was reviewed by Dr. Laura M. Sanchez, Esther Rincón, Jorge de Haro Niza and Ramón Morcillo Martín, Dr. Eduardo Espinosa and Dr. Alejandro Rodríguez; the article was supervised by Dr. Laura M. Sanchez, Dr. Eduardo Espinosa and Dr. Alejandro Rodríguez. All authors have read and agreed to the published version of the manuscript.

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Scheme 1 is available in the Supplementary Files section.

No competing interests reported.

SI140323.docx
Scheme1.tif
Scheme 1. Workflow for the preparation and study of bio-aerogels as food conservation systems.

Vegetable lignocellulosic residues and chitosan as valuable resources in the superabsorbent bio-aerogels development for food conservation

Status:

Journal Publication

Version 1

Abstract

Figures

1.0- Introduction

2.0- Materials And Methods

2.1- Materials

2.2- Methods

2.2.1- Nanocellulose preparation and characterization

2.2.2- Bio-aerogels preparation

2.2.3- Bio-aerogels characterization

2.2.4- Bio-aerogels performance as food absorbent pads

3.0- Results And Discussion

3.1- Nanocellulose

3.2- Bio-aerogels

4.0- Conclusions

Declarations

Declarations

Funding

Author contributions

References

Scheme

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1