Dried Porous Biomaterials from Mealworm Protein Gels: Proof of Concept and Impact of Drying Method on Structural Properties and Zinc Retention [original]

Citation: Klost, M.; Keil, C.; Gurikov,

P. Dried Porous Biomaterials from

Mealworm Protein Gels: Proof of

Concept and Impact of Drying

Method on Structural Properties and

Zinc Retention. Gels 2024,10, 275.

https://doi.org/10.3390/

gels10040275

Academic Editor: Song He

Received: 26 February 2024

Revised: 4 April 2024

Accepted: 10 April 2024

Published: 18 April 2024

Licensee MDPI, Basel, Switzerland.

This article is an open access article

distributed under the terms and

conditions of the Creative Commons

Attribution (CC BY) license (https://

creativecommons.org/licenses/by/

4.0/).

gels

Communication

Dried Porous Biomaterials from Mealworm Protein Gels: Proof

of Concept and Impact of Drying Method on Structural

Properties and Zinc Retention

Martina Klost 1, Claudia Keil 2and Pavel Gurikov 3,4,*

1Faculty III Process Sciences, Institute for Food Technology and Food Chemistry, Department of Food

Technology and Food Material Science, Technische Universität Berlin, Straße des 17. Juni 135,

10623 Berlin, Germany; [email protected]

2Faculty III Process Sciences, Institute of Food Technology and Food Chemistry, Department of Food

Chemistry and Toxicology, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany;

[email protected]

3Laboratory for Development and Modelling of Novel Nanoporous Materials, Hamburg University of

Technology, Eißendorfer Straße 38, 21073 Hamburg, Germany

4aerogel-it GmbH, Albert-Einstein-Str. 1, 49076 Osnabrück, Germany

*Correspondence: [email protected]; Tel.: +49-40-42878-4275

Abstract: Dried porous materials can be found in a wide range of applications. So far, they are

mostly prepared from inorganic or indigestible raw materials. The aim of the presented study was to

provide a proof of concept for (a) the suitability of mealworm protein gels to be turned into dried

porous biomaterials by either a combination of solvent exchange and supercritical drying to obtain

aerogels or by lyophilization to obtain lyophilized hydrogels and (b) the suitability of either drying

method to retain trace elements such as zinc in the gels throughout the drying process. Hydrogels

were prepared from mealworm protein, subsequently dried using either method, and characterized

via FT-IR, BET volume, and high-resolution scanning electron microscopy. Retention of zinc was

evaluated via energy-dispersive X-ray spectroscopy. Results showed that both drying methods were

suitable for obtaining dried porous biomaterials and that the drying method mainly influenced the

overall surface area and pore hydrophobicity but not the secondary structure of the proteins in the

gels or their zinc content after drying. Therefore, a first proof of concept for utilizing mealworm

protein hydrogels as a base for dried porous biomaterials was successful and elucidated the potential

of these materials as future sustainable alternatives to more conventional dried porous materials.

Keywords: aerogel; lyophilized hydrogel; biobased; Tenebrio molitor; zinc

1. Introduction

Dried porous materials such as aerogels can be found in applications ranging from

aerospace and construction to various life and food science branches. With porosity (at

the micro (<2 nm), meso- (2–50 nm), and macrolevels (>50 nm), high porosity (up to

99.98%) and large surface areas (up to 1500 m

/g) aerogels can be applied in various

areas [

]. Bio-based aerogels made from either biopolymer materials (such as cellulose,

chitosan, and alginate) [

] or from renewable side streams (banana peel, apple pomace,

and coffee powders) [

] are highly valued in life- and food science applications. They may

serve as carriers for bioactive compounds, functional ingredients, or micronutrients. For

example, they can be used as carrier systems for trace elements requiring gastrointestinal

compatibility [

–

]. One example for such trace elements is zinc of which about 17% of the

world’s population has a deficiency. As a consequence, it is critical to find a way to provide

zinc in a food or supplement format that is easily accessible [10,11].

Research efforts towards the production of tailor-made aerogels from bio-based raw

materials have vastly increased over the past decade [

]. While our estimations show

Gels 2024,10, 275. https://doi.org/10.3390/gels10040275 https://www.mdpi.com/journal/gels

Gels 2024,10, 275 2 of 9

that over a thousand peer-reviewed publications appear annually on biopolymer aerogels,

most of these efforts focus on cellulose and other enzymatically inaccessible carbohydrates

as building blocks for porous gels. However, the indigestibility of polysaccharide-based

aerogels may not be appropriate in all applications. This has led to a growing number

of studies focusing on protein aerogels derived from various sources such as milk, eggs,

and different plants [

] (and references therein). However, this research branch still

only accounts for approximately 3.5% of all research on biopolymer-based aerogels. When

considering protein sources, not only should their techno-functional properties be con-

sidered, but their environmental impact should also be considered [

]. In this context,

insects such as mealworms (Tenebrio molitor) are considered to be promising because they

require only a little water and can be reared on organic waste [

]. Moreover, mealworm

is an EFSA-approved raw material for food applications [

]. However, research on

the gelation of mealworm protein is only just emerging [

], and, to our knowledge, so

far, there are no reports on dried porous biomaterials prepared from mealworm protein

gels. Therefore, the aim of the presented study was to provide a first proof of concept for

the suitability of mealworm protein hydrogels to be turned into dried porous biomaterials

and to investigate whether these gels could retain added zinc as a model trace element

throughout the fabrication process.

2. Results and Discussion

2.1. Characterization of Aerogels and Lyophilized Hydrogels

In this section, we will mainly focus on the characterization and comparison of dried

porous biomaterials such as aerogels (AG) and lyophilized hydrogels (LHG) from meal-

worm protein hydrogels (HG) prepared in distilled water. Discussion on the coordination

of zinc with protein will additionally compare results from samples prepared in distilled

water to those from samples prepared with zinc. As previously shown by Klost et al.,

mealworm protein could form heat-induced hydrogels (HG) with and without the addition

of ZnSO

[

]. At the selected protein concentration (13.4%) and pH values (3.5, 5.5, and

7.5) in the present study, the hydrogels were self-supporting—more so at pH 5.5 and 7.5

compared to pH 3.5—and could be turned into dried gels by both solvent exchange or

lyophilization, leading to aerogels (AG) and lyophilized hydrogels (LHG) respectively

(Figure 1). The brittleness of some of the dried gels (as shown in Figure 1) may be related to

the nature of the interactions stabilizing the hydrogels (i.e., hydrogen bonds, hydrophobic

interactions, and some ionic or covalent bonds, especially if the gels contain zinc [

]) as

well as the distribution of these interactions within and between aggregates in the gel.

Gels 2024, 10, 275 2 of 9

Research eﬀorts towards the production of tailor-made aerogels from bio-based raw

materials have vastly increased over the past decade [12]. While our estimations show that

over a thousand peer-reviewed publications appear annually on biopolymer aerogels,

most of these eﬀorts focus on cellulose and other enzymatically inaccessible carbohydrates

as building blocks for porous gels. However, the indigestibility of polysaccharide-based

aerogels may not be appropriate in all applications. This has led to a growing number of

studies focusing on protein aerogels derived from various sources such as milk, eggs, and

diﬀerent plants [13,14] (and references therein). However, this research branch still only

accounts for approximately 3.5% of all research on biopolymer-based aerogels. When con-

sidering protein sources, not only should their techno-functional properties be consid-

ered, but their environmental impact should also be considered [6,15]. In this context, in-

sects such as mealworms (Tenebrio molitor) are considered to be promising because they

require only a little water and can be reared on organic waste [16]. Moreover, mealworm

is an EFSA-approved raw material for food applications [17,18]. However, research on the

gelation of mealworm protein is only just emerging [19], and, to our knowledge, so far,

there are no reports on dried porous biomaterials prepared from mealworm protein gels.

Therefore, the aim of the presented study was to provide a first proof of concept for the

suitability of mealworm protein hydrogels to be turned into dried porous biomaterials

and to investigate whether these gels could retain added zinc as a model trace element

throughout the fabrication process.

2. Results and Discussion

2.1. Characterization of Aerogels and Lyophilized Hydrogels

In this section, we will mainly focus on the characterization and comparison of dried

porous biomaterials such as aerogels (AG) and lyophilized hydrogels (LHG) from meal-

worm protein hydrogels (HG) prepared in distilled water. Discussion on the coordination

of zinc with protein will additionally compare results from samples prepared in distilled

water to those from samples prepared with zinc. As previously shown by Klost et al.,

mealworm protein could form heat-induced hydrogels (HG) with and without the addi-

tion of ZnSO4 [19]. At the selected protein concentration (13.4%) and pH values (3.5, 5.5,

and 7.5) in the present study, the hydrogels were self-supporting—more so at pH 5.5 and

7.5 compared to pH 3.5—and could be turned into dried gels by both solvent exchange or

lyophilization, leading to aerogels (AG) and lyophilized hydrogels (LHG) respectively

(Figure 1). The brittleness of some of the dried gels (as shown in Figure 1) may be related

to the nature of the interactions stabilizing the hydrogels (i.e., hydrogen bonds, hydro-

phobic interactions, and some ionic or covalent bonds, especially if the gels contain zinc

[19]) as well as the distribution of these interactions within and between aggregates in the

gel.

Figure 1. Photographs of HG (left), LHG (center), and AG (right) from mealworm protein gels pre-

pared in distilled water at pH 3.5 (a), 5.5 (b), 7.5 (c), and 0.3 M ZnSO4 solution at pH 3.5 (d), 5.5 (e),

and 7.5 (f).

Figure 1. Photographs of HG (left), LHG (center), and AG (right) from mealworm protein gels

prepared in distilled water at pH 3.5 (a), 5.5 (b), 7.5 (c), and 0.3 M ZnSO

solution at pH 3.5 (d),

5.5 (e), and 7.5 (f).

The microstructure is of utmost relevance to future applications in dried porous

materials. Therefore, the AG and LHG were characterized and compared with regard to

the impact of the drying method on the molecular and microscopic levels.

Gels 2024,10, 275 3 of 9

Characterization of AG and LHG at the molecular level and comparison to the HG

was carried out via FT-IR (Figure 2). FT-IR spectra of HG are dominated by intermolec-

ular

-sheet structures (approx. 1620 cm

−1

) that formed during the heating process [

Drying leads to a shift in the wavenumber of intermolecular

-sheets from approximately

1620 cm

−1

(Figure 2a,d) toward approximately 1624 cm

−1

(Figure 2b,c,e,f), indicating a

slight weakening of the involved intermolecular hydrogen bonds [

] independent of dry-

ing method and addition of zinc. Observed differences in peak intensity, e.g., depending

on pH value or drying method in Figure 2, cannot be attributed to differences between

samples because Figure 2shows the second derivative of the spectrum and, therefore, only

allows for qualitative evaluation.

Gels 2024, 10, 275 3 of 9

The microstructure is of utmost relevance to future applications in dried porous ma-

terials. Therefore, the AG and LHG were characterized and compared with regard to the

impact of the drying method on the molecular and microscopic levels.

Characterization of AG and LHG at the molecular level and comparison to the HG

was carried out via FT-IR (Figure 2). FT-IR spectra of HG are dominated by intermolecular

β-sheet structures (approx. 1620 cm−1) that formed during the heating process [20]. Drying

leads to a shift in the wavenumber of intermolecular β-sheets from approximately 1620

cm−1 (Figure 2a,d) toward approximately 1624 cm−1 (Figure 2b,c,e,f), indicating a slight

weakening of the involved intermolecular hydrogen bonds [21] independent of drying

method and addition of zinc. Observed diﬀerences in peak intensity, e.g., depending on

pH value or drying method in Figure 2, cannot be attributed to diﬀerences between sam-

ples because Figure 2 shows the second derivative of the spectrum and, therefore, only

allows for qualitative evaluation.

Figure 2. Second derivate spectrum from FT-IR measurements at wavenumbers representing the

amide I band. (a) HG (b) AG (c) LHG prepared in distilled water; (d) HG (e) AG (f) LHG prepared

in 0.3 M ZnSO4 solution. Black lines: pH 3.5, red lines: pH 5.5, and blue lines: pH 7.5. Peaks in zone

1 represent antiparallel intramolecular β-sheets, peaks in zone 2 represent α-helices, peaks in zone

3 represent parallel intramolecular β-sheets and peaks in zone 4 represent intermolecular β-sheets.

Error bars represent deviations between individual measurements of the same sample.

Besides obtaining information on secondary structures of the protein, FT-IR spectros-

copy can give some information on the coordination of metal ions with proteins. The lit-

erature implies that oxygen, nitrogen, and sulfur donors from amino acid side chains limit

the zinc coordination environment in proteins under physiological conditions. Changes

in the number of ligands available for the metal ions, the ideal geometry of the protein

ligands, or the number of interactions between the secondary coordination sphere and the

global protein structure can change zinc’s aﬃnity to the protein [22,23]. Since we are look-

ing at heat-treated and, therefore, non-native proteins in our study, the physiological af-

finity of the proteins to the zinc will most likely be altered. With regard to the coordination

of metal ions with various proteins, the literature indicates an impact of these cations on

the νCOO− symmetric (1430–1360 cm−1) and asymmetric (1580–1560 cm−1) stretching vi-

brations, e.g., [24,25]. Shifts in the frequencies of the respective bands and their relation to

each other may, in turn, allow for the interpretation of the coordination mode of the metal

ions [24,26]. Since the νCOO−asym band overlaps with the amid II band, it cannot easily be

distinguished in our study. For hydrogels, we found the wavenumbers of the νCOO−sym

band to increase slightly with the addition of ZnSO4 at pH 5.5 and 7.5 and to decrease

slightly at pH 3.5. Compared to shifts shown in the literature for various metal ions with

Figure 2. Second derivate spectrum from FT-IR measurements at wavenumbers representing the

amide I band. (a) HG (b) AG (c) LHG prepared in distilled water; (d) HG (e) AG (f) LHG prepared in

0.3 M ZnSO

solution. Black lines: pH 3.5, red lines: pH 5.5, and blue lines: pH 7.5. Peaks in zone

1 represent antiparallel intramolecular

-sheets, peaks in zone 2 represent

-helices, peaks in zone

3 represent parallel intramolecular

-sheets and peaks in zone 4 represent intermolecular

-sheets.

Error bars represent deviations between individual measurements of the same sample.

Besides obtaining information on secondary structures of the protein, FT-IR spec-

troscopy can give some information on the coordination of metal ions with proteins. The

literature implies that oxygen, nitrogen, and sulfur donors from amino acid side chains limit

the zinc coordination environment in proteins under physiological conditions. Changes in

the number of ligands available for the metal ions, the ideal geometry of the protein ligands,

or the number of interactions between the secondary coordination sphere and the global

protein structure can change zinc’s affinity to the protein [

]. Since we are looking

at heat-treated and, therefore, non-native proteins in our study, the physiological affinity

of the proteins to the zinc will most likely be altered. With regard to the coordination of

metal ions with various proteins, the literature indicates an impact of these cations on

the

COO

−

symmetric (1430–1360 cm

−1

) and asymmetric (1580–1560 cm

−1

) stretching

vibrations, e.g., [

]. Shifts in the frequencies of the respective bands and their relation to

each other may, in turn, allow for the interpretation of the coordination mode of the metal

ions [

]. Since the

COO

−asym

band overlaps with the amid II band, it cannot easily be

distinguished in our study. For hydrogels, we found the wavenumbers of the

COO

−sym

band to increase slightly with the addition of ZnSO

at pH 5.5 and 7.5 and to decrease

slightly at pH 3.5. Compared to shifts shown in the literature for various metal ions with

Lactobacillus kefir S-layers [

], these shifts are very minor. Moreover, without information

on the wavenumber of the

COO

−asym

band, they cannot be related to any coordination

mechanism. Consequently, future research should be conducted under conditions where

Gels 2024,10, 275 4 of 9

exchangeable protons in the mealworm protein are completely deuterated [

] so that the

νCOO−asym band can be evaluated more easily.

Regarding the coordination of zinc with sulfur donors, in our study, we did not

consider bands corresponding to sulfur groups as a distinction between Zn-sidechain

interactions and the effect of the utilized salt could not be sufficiently distinguished.

Determination of the BET surface area was used to investigate structural properties

at the microscopic level (Figure 3; see Supplementary Figure S2 for adsorption isotherms).

With regard to the AG, we found an increase in the surface area with decreasing pH value

(cf. 133, 53, and 42 m

/g for AG prepared at pH 3.5, 5.5. and 7.5, respectively; Figure 3a–c).

This indicates a higher porosity of the corresponding samples, at least in the mesoporous

range [

]. A similar behavior of the specific surface area with respect to the pH increase has

been reported by Andlinger et al. [

] for potato protein aerogels. Differences in the protein

conformation conventionally explain the drop in the surface area: compacted globular

structures are present in the gelling solution at a pH close to the isoelectric point. The

resulting gels and corresponding aerogels thus demonstrate a more granular structure with

a lower specific surface compared to the gels obtained under acid or basic conditions. LHG

did not show a distinct influence of pH (24, 37, and 20 m

/g for LHG prepared at 3.5, 5.5,

and 7.5, respectively, Figure 3d–f).

Gels 2024, 10, 275 4 of 9

Lactobacillus kefir S-layers [25], these shifts are very minor. Moreover, without infor-

mation on the wavenumber of the νCOO−asym band, they cannot be related to any coordi-

nation mechanism. Consequently, future research should be conducted under conditions

where exchangeable protons in the mealworm protein are completely deuterated [24] so

that the νCOO−asym band can be evaluated more easily.

Regarding the coordination of zinc with sulfur donors, in our study, we did not con-

sider bands corresponding to sulfur groups as a distinction between Zn-sidechain inter-

actions and the eﬀect of the utilized salt could not be suﬃciently distinguished.

Determination of the BET surface area was used to investigate structural properties

at the microscopic level (Figure 3; see Supplementary Figure S2 for adsorption isotherms).

With regard to the AG, we found an increase in the surface area with decreasing pH value

(cf. 133, 53, and 42 m2/g for AG prepared at pH 3.5, 5.5. and 7.5, respectively; Figure 3a–c).

This indicates a higher porosity of the corresponding samples, at least in the mesoporous

range [27]. A similar behavior of the specific surface area with respect to the pH increase

has been reported by Andlinger et al. [13] for potato protein aerogels. Diﬀerences in the

protein conformation conventionally explain the drop in the surface area: compacted

globular structures are present in the gelling solution at a pH close to the isoelectric point.

The resulting gels and corresponding aerogels thus demonstrate a more granular struc-

ture with a lower specific surface compared to the gels obtained under acid or basic con-

ditions. LHG did not show a distinct influence of pH (24, 37, and 20 m2/g for LHG pre-

pared at 3.5, 5.5, and 7.5, respectively, Figure 3d–f).

Figure 3. SEM pictures and surface areas of AG and LHG (both zinc-free) at 20,000

magnification

at pH 3.5, 5.5, and 7.5, respectively. (a) AG-pH 3.5, (b) AG-pH 5.5, (c) AG-pH 7.5, (d) LHG-pH 3.5,

(e) LHG-pH 5.5, and (f) LHG-pH 7.5.

Gels 2024,10, 275 5 of 9

The BET constant C is also distinctly higher for AG than for LHG (81 vs. 8 for AG and

LHG at pH 3.5, respectively). As higher values for C indicate a more polar surface [

we can conclude that solvent exchange-based drying preserves the polar surface of a HG

to a greater extent while freezing in liquid nitrogen followed by lyophilization leads to

more hydrophobic pores in the corresponding LHG. These findings are supported by high-

resolution SEM micrographs recorded at 20,000

magnification. In agreement with results

from surface area measurements, AG (Figure 3a–c) shows finer, more porous structures

compared to the corresponding LHG (Figure 3d–f).

2.2. Retention of Zinc during Drying of Mealworm Protein Hydrogels

With regard to the future utilization of AG and/or LHG as possible delivery systems

for trace elements in biomedical applications, this proof of concept also aimed to determine

if zinc added in the course of HG fabrication would be retained in the samples during

the drying procedure. With regard to a potential loss of Zn

, it can be assumed that the

zinc should be retained in the sample during freeze drying as it is not volatile. However,

with regard to the solvent exchange processes during AG preparation, zinc may be washed

from the samples, as has already been shown for zinc-alginate gels in the past [

]. To

this purpose, we investigated AG and LHG using energy-dispersive X-ray spectroscopy to

determine their elemental composition. Figure 4shows the corresponding EDX spectra

and EDX maps for samples prepared at pH 5.5 as an example.

Gels 2024, 10, 275 5 of 9

Figure 3. SEM pictures and surface areas of AG and LHG (both zinc-free) at 20,000× magnification

at pH 3.5, 5.5, and 7.5, respectively. (a) AG-pH 3.5, (b) AG-pH 5.5, (c) AG-pH 7.5, (d) LHG-pH 3.5,

(e) LHG-pH 5.5, and (f) LHG-pH 7.5.

The BET constant C is also distinctly higher for AG than for LHG (81 vs. 8 for AG and

LHG at pH 3.5, respectively). As higher values for C indicate a more polar surface [28], we

can conclude that solvent exchange-based drying preserves the polar surface of a HG to a

greater extent while freezing in liquid nitrogen followed by lyophilization leads to more

hydrophobic pores in the corresponding LHG. These findings are supported by high-

resolution SEM micrographs recorded at 20,000× magnification. In agreement with results

from surface area measurements, AG (Figure 3a–c) shows finer, more porous structures

compared to the corresponding LHG (Figure 3d–f).

2.2. Retention of Zinc during Drying of Mealworm Protein Hydrogels

With regard to the future utilization of AG and/or LHG as possible delivery systems

for trace elements in biomedical applications, this proof of concept also aimed to

determine if zinc added in the course of HG fabrication would be retained in the samples

during the drying procedure. With regard to a potential loss of Zn

, it can be assumed

that the zinc should be retained in the sample during freeze drying as it is not volatile.

However, with regard to the solvent exchange processes during AG preparation, zinc may

be washed from the samples, as has already been shown for zinc-alginate gels in the past

[29]. To this purpose, we investigated AG and LHG using energy-dispersive X-ray

spectroscopy to determine their elemental composition. Figure 4 shows the corresponding

EDX spectra and EDX maps for samples prepared at pH 5.5 as an example.

Figure 4. EDX spectra of AG (a) and LHG (b) prepared with 0.3 M ZnSO

. The inset table shows the

results of the semi-quantitative standardless EDX analysis. EDX maps (k line of x-ray emission) of

the spatial distributions of Zn, N, O, S, C, and P in the AG (c) and LHG (d). Scale bars = 40 µm.

If AG and LHG were produced without adding ZnSO

, no zinc could be determined

in the corresponding EDX spectra (Supplementary Figure S1). In contrast, all AG and LHG

that had been produced with the addition of ZnSO

retained some of the zinc throughout

either drying process. This is, to some extent, in agreement with previous findings, where

zinc was shown to decrease the gel solubility of mealworm protein hydrogels in various

aqueous solvents, thus indicating an incorporation of zinc into the protein gel network

Figure 4. EDX spectra of AG (a) and LHG (b) prepared with 0.3 M ZnSO

. The inset table shows the

results of the semi-quantitative standardless EDX analysis. EDX maps (k line of x-ray emission) of

the spatial distributions of Zn, N, O, S, C, and P in the AG (c) and LHG (d). Scale bars = 40 µm.

If AG and LHG were produced without adding ZnSO4, no zinc could be determined

in the corresponding EDX spectra (Supplementary Figure S1). In contrast, all AG and LHG

that had been produced with the addition of ZnSO

retained some of the zinc through-

out either drying process. This is, to some extent, in agreement with previous findings,

where zinc was shown to decrease the gel solubility of mealworm protein hydrogels in

various aqueous solvents, thus indicating an incorporation of zinc into the protein gel

network structure. In that study, the decrease in gel solubility was related to the additional

occurrence of some ionic or covalent bonds [19].

Gels 2024,10, 275 6 of 9

3. Conclusions

The presented study showed that mealworm protein hydrogels form dried porous

bio-materials when processed into aerogels by supercritical CO2drying, or lyophilization

to obtain lyophilized hydrogels. With regard to future utilization in biomedical, life science,

and food applications, we could demonstrate that added zinc could be retained throughout

drying by lyophilization or solvent exchange followed by supercritical CO

drying. From a

mechanistic point of view, future research should aim at further investigation of structural

changes on the molecular level and investigation of effects causing the shift towards more

hydrophobic pores in lyophilized hydrogels compared to the corresponding aerogels. In

addition, the influence of this difference in pore hydrophobicity on techno-functional

properties such as water uptake, swelling rate, and solubility should be investigated

with regard to future applications. In principle, zinc-binding protein domains and zinc-

binding sequence motifs are found in all domains of life. As mentioned above, there are

various limiting factors to the coordination environment of zinc in proteins. These may be

related to the availability of ligands as well as to protein conformation [

]. The zinc

proteome accounts for about 9% of the total proteome in eukaryotes. For example, in the

fruit fly Drosophila melanogaster, the zinc proteome represents about 10.2% of the entire

proteome [

]. Metal-protein interactions under cell/tissue homeostasis conditions involve

a dynamic coordination environment that includes mechanisms for metal dissociation and

association over a timescale of seconds to years. However, zinc’s transfer into proteins

differs considerably when administered under chemical/technological conditions, as in

our approach [

]. So, further research is needed to understand the zinc coordination

spheres in the mealworm HG and possible changes in the course of the conversion to

AG/LHG protein structures.

To counteract the brittleness of some of the AG and LHG, further experiments should

additionally focus on increasing the gel-stability. To this regard, crosslinking with transg-

lutaminase or protein modification with sodium triphosphate are potential options. The

former leads to iso-peptide bonds between glutamine and lysine residues, while the latter

promotes the formation of salt bridges by zinc ions [33].

Drying is considered to be the most critical step in the aerogel production process since

it preserves the three-dimensional pore structure that results in unique material properties.

Both the supercritical CO

and freeze-drying methods are widely used to produce aerogels,

e.g., [34–36]. In recent years, ambient pressure, microwave, and vacuum drying have also

gained popularity [

]. The ability to tailor bio-based sustainable aerogel properties

to meet application requirements requires a thorough understanding of how different

processing parameters affect microstructure.

With regard to possible applications of mealworm protein gels in the biomedical,

life-science, and food sectors, future research should additionally focus on the investigation

of the release of retained trace elements such as zinc.

Despite the necessity for future research toward a complete comprehension of these

novel dried porous biomaterials from mealworm gels, the proof of concept in this study

elucidated their potential as sustainable alternatives to more conventional dried

porous materials.

4. Materials and Methods

4.1. Materials

Living mealworms were purchased from ENTAVA (Roggentin, Germany), frozen

in liquid nitrogen, and subsequently lyophilized. Mealworm proteins were extracted

according to Klost et al. [

]. The protein content was 65.4% (derived from nitrogen content

according to Dumas (DUMATHERM DT, C. Gerhardt, Königswinter, Germany), protein

factor 5.60 [

]). Hexane, HCl, NaOH, ethanol, and ZnSO

were purchased from Carl

Roth (Karlsruhe, Germany) or VWR (Darmstadt, Germany) and were of analytical grade.

Denatured ethanol and CO

for drying were obtained from Carl Roth (Karlsruhe, Germany)

and Nippon Gases Deutschland GmbH (Hürth, Germany), respectively.

Gels 2024,10, 275 7 of 9

4.2. Preparation of Mealworm Protein Hydrogels, Lyophilized Hydrogels and Aerogels

Solutions with 13.4% protein content were prepared in distilled water, according to

Klost et al. [

]. pH was adjusted to pH 3.5, 5.5 or 7.5 with NaOH and HCl, respectively.

Gels containing zinc gels were prepared with 0.3 M ZnSO

instead of water. Solutions were

subsequently poured into plastic cylinders (14 mm diameter, 14 mm height), covered with

a Petri dish to avoid evaporation, and heat-induced gelation was induced by placing the

samples into a drying cabinet at 90

◦

C for 30 min. One batch of the hydrogel (HG) samples

was turned into alcogels by treatment with a large excess of 100% ethanol (approx. 1:10

gel:EtOH v/v). After the solvent exchange, the alcogels were dried with supercritical carbon

dioxide at 35

◦

C and 100 bar for 2 h to obtain aerogels (AG). Another batch of HG samples

was produced for characterization of the HG and subsequent freeze drying to obtain the

lyophilized hydrogels (LHG). To this purpose, HG was frozen in liquid nitrogen and

subsequently lyophilized (Beta 1–8 LSCplus, Christ Gefriertrocknungsanlagen, Osterode

am Harz, Germany). As the study was intended only as a proof-of-concept, no replicates

were prepared.

4.3. Fourier Transform Infrared Spectroscopy

FT-IR spectra of AG, HG, and LHG were recorded at room temperature in the range

from 4000 to 800 cm

−1

(Bruker Optic GmbH, Karlsruhe, Germany) equipped with a liquid

nitrogen-cooled mercury-cadmium-telluride detector. Measurements were carried out at

least eight times from the same sample. The second derivative within the amide I band

(1680 to 1600 cm

−1

) was calculated to investigate changes to the secondary structure of

the protein.

4.4. Determination of the Specific Surface Area

Determination of the specific surface areas of AG and LHG was carried out by low-

temperature N

adsorption analysis (NOVA 4000e, Quantachrome Instrument. Anton Paar,

Graz, Austria) using the Brunauer–Emmett–Teller (BET) method [6].

4.5. High-Resolution Scanning Electron Microscopy and X-ray Spectra

Gold sputtering and high-resolution scanning electron microscopy (HR-SEM) analysis

of AG and LHG samples was done at the Center for Electron Microscopy (ZELMI), Tech-

nische Universität Berlin, Germany (Microscope ZEISS GeminiSEM500 Nano VP with an

in-lens detector (Carl Zeiss Microscopy GmbH, Jena, Germany)). Energy-dispersive X-ray

spectra were also recorded at the Center for Electron Microscopy (ZELMI), Technische

Universität Berlin, Germany, using S-2700 scanning electron microscope (Hitachi, Tokyo,

Japan) with SDD-detector with Si3N4-window (remX GmbH (Bruchsal, Germany)).

Supplementary Materials: The following supporting information can be downloaded at: https:

//www.mdpi.com/article/10.3390/gels10040275/s1, Figure S1: EDX spectra of all samples prepared

with and without 0.3 M ZnSO

; Figure S2: Nitrogen adsorption isotherms of AG (a) and LHG (b) at

pH 3.5, 5.5 and 7.5 respectively.

Author Contributions: All authors contributed equally. All authors have read and agreed to the

published version of the manuscript.

Funding: This research was funded by Technische Universität Berlin, TU-internal research funding:

Strategic Call “Pro Nachhaltigkeit”.

Data Availability Statement: The data presented in this study are available upon request from the

corresponding author.

Acknowledgments: We thank Jörg Nissen and Christoph Fahrenson from the Center for Electron

Microscopy (ZELMI), Technische Universität Berlin, for carrying out the SEM investigations. We

also thank Silvia Heim for conducting FT-IR analysis and Elena Köster for protein extraction and

support in the preparation of the gels. Publishing fees supported by Funding Programme Open

Access Publishing of Hamburg University of Technology (TUHH).

Gels 2024,10, 275 8 of 9

Conflicts of Interest: Pavel Gurikov was employed by the company aerogel-it GmbH. The remaining

authors declare that the research was conducted in the absence of any commercial or financial

relationships that could be construed as a potential conflict of interest.

References

Smirnova, I.; Gurikov, P. Aerogels in Chemical Engineering: Strategies Toward Tailor-Made Aerogels. Annu. Rev. Chem. Biomol.

Eng. 2017,7, 307–334. [CrossRef]

Sonu, S.S.; Rai, N.; Chauhan, I. Multifunctional Aerogels: A Comprehensive Review on Types, Synthesis and Applications of

Aerogels. J. Sol-Gel Sci. Technol. 2023,105, 324–336. [CrossRef]

Zhao, S.; Malfait, W.J.; Guerrero-Alburquerque, N.; Koebel, M.M.; Nyström, G. Biopolymer Aerogels and Foams: Chemistry,

Properties, and Applications. Angew. Chem. Int. Ed. 2018,57, 7580–7608. [CrossRef] [PubMed]

Nita, L.E.; Ghilan, A.; Rusu, A.G.; Neamtu, I.; Chiriac, A.P. New Trends in Bio-Based Aerogels. Pharmaceutics 2020,12, 449.

[CrossRef] [PubMed]

Yue, X.; Zhang, T.; Yang, D.; Qiu, F.; Li, Z. Hybrid Aerogels Derived from Banana Peel and Waste Paper for Efficient Oil Absorption

and Emulsion Separation. J. Clean. Prod. 2018,199, 411–419. [CrossRef]

Gaggero, G.; Subrahmanyam, R.P.; Schroeter, B.; Gurikov, P.; Delucchi, M. Organic Bio-Based Aerogel from Food Waste:

Preparation and Hydrophobization. Gels 2022,8, 691. [CrossRef] [PubMed]

Manzocco, L.; Mikkonen, K.S.; García-González, C.A. Aerogels as Porous Structures for Food Applications: Smart Ingredients

and Novel Packaging Materials. Food Struct. 2021,28, 100188. [CrossRef]

Selvasekaran, P.; Chidambaram, R. Bioaerogels as Food Materials: A State-of-the-Art on Production and Application in Micronu-

trient Fortification and Active Packaging of Foods. Food Hydrocoll. 2022,131, 107760. [CrossRef]

Selvasekaran, P.; Chidambaram, R. Food-Grade Aerogels Obtained from Polysaccharides, Proteins, and Seed Mucilages: Role as a

Carrier Matrix of Functional Food IngredientsNo Title. Trends Food Sci. Technol. 2021,112, 455–470. [CrossRef]

10.

Beal, T.; Massiot, E.; Arsenault, J.E.; Smith, M.R.; Hijmans, R.J. Global Trends in Dietary Micronutrient Supplies and Estimated

Prevalence of Inadequate Intakes. PLoS ONE 2017,12, e0175554. [CrossRef]

11.

Roohani, N.; Hurrell, R.; Kelishadi, R.; Schulin, R. Zinc and Its Importance for Human Health: An Integrative Review. J. Res. Med.

Sci. 2013,18, 144–157. [PubMed]

12.

García-González, C.A.; Budtova, T.; Dures, L.; Erkey, C.; Del Gaudio, P.; Gurikov, P.; Koebel, M.; Liebner, F.; Neagu, M.; Smirnova,

I. An Opinion Paper on Aerogels for Biomedical and Environmental Applications. Molecules 2019,24, 591–641. [CrossRef]

13.

Andlinger, D.J.; Bornkeßel, A.C.; Jung, I.; Schröter, B.; Smirnova, I.; Kulozik, U. Microstructures of Potato Protein Hydrogels and

Aerogels Produced by Thermal Crosslinking and Supercritical Drying. Food Hydrocoll. 2021,112, 106305. [CrossRef]

14.

Plazzotta, S.; Alongi, M.; De Berardinis, L.; Melchior, S.; Calligaris, S.; Manzocco, L. Steering Protein and Lipid Digestibility by

Oleogelation with Protein Aerogels. Food Funct. 2022,13, 10601–10609. [CrossRef] [PubMed]

15.

Nguyen, P.T.T.; Do, N.H.N.; Goh, X.Y.; Goh, C.J.; Ong, R.H.; Le, P.K.; Phan-Thien, N.; Duong, H.M. Recent Progresses in

Eco-Friendly Fabrication and Applications of Sustainable Aerogels from Various Waste Materials. Waste Biomass Valorization 2022,

13, 1825–1847. [CrossRef] [PubMed]

16.

Laroche, M.; Perreault, V.; Marciniak, A.; Mikhaylin, S.; Doyen, A. Eco-Efficiency of Mealworm (Tenebrio molitor) Protein Extracts.

ACS Food Sci. Technol. 2022,2, 1077–1085. [CrossRef]

17.

European Commission. Commission Implementing Regulation (EU) 2022/169 of 8 February 2022 Authorising the Placing on the

Market of Frozen, Dried and Powder Forms of Yellow Mealworm (Tenebrio molitor larva) as a Novel Food under Regulation (EU)

2015/2283 of the European Parliam. Off. J. Eur. Union 2022,2016, 48–119.

18.

Precup, G.; Ververis, E.; Azzollini, D.; Rivero-Pino, F.; Zakidou, P.; Germini, A. The Safety Assessment of Insects and Products

There of As Novel Foods in the European Union. In Novel Foods and Edible Insects in the European Union; Scaffardi, L., Formici, G.,

Eds.; Springer: Berlin/Heidelberg, Germany, 2022; p. C1. ISBN 9783031134944.

19.

Klost, M.; Ramirez-Huerta, M.I.; Drusch, S. Heat-Induced Gelation of Protein from Mealworm (Tenebrio molitor): Influence of PH

and Zinc Concentration. Food Hydrocoll. Health 2022,2, 100105. [CrossRef]

20.

Matheus, S.; Friess, W.; Mahler, H.C. FTIR and NDSC as Analytical Tools for High-Concentration Protein Formulations. Pharm.

Res. 2006,23, 1350–1363. [CrossRef]

21.

Jackson, M.; Mantsch, H.H. The Use and Misuse of FTIR Spectroscopy in the Determination of Protein Structure. Crit. Rev.

Biochem. Mol. Biol. 1995,30, 95–120. [CrossRef]

22.

Kocha´nczyk, T.; Drozd, A.; Kre

zel, A. Relationship between the Architecture of Zinc Coordination and Zinc Binding Affinity in

Proteins—Insights into Zinc Regulation. Metallomics 2015,7, 244–257. [CrossRef] [PubMed]

23.

Kr˛e˙

zel, A.; Maret, W. The Biological Inorganic Chemistry of Zinc Ions. Arch. Biochem. Biophys. 2016,611, 3–19. [CrossRef]

[PubMed]

24.

Nara, M.; Morii, H.; Tanokura, M. Coordination to Divalent Cations by Calcium-Binding Proteins Studied by FTIR Spectroscopy.

Biochim. Biophys. Acta-Biomembr. 2013,1828, 2319–2327. [CrossRef] [PubMed]

25.

Gerbino, E.; Mobili, P.; Tymczyszyn, E.; Fausto, R.; Gómez-Zavaglia, A. FTIR Spectroscopy Structural Analysis of the Interaction

between Lactobacillus Kefir S-Layers and Metal Ions. J. Mol. Struct. 2011,987, 186–192. [CrossRef]

Gels 2024,10, 275 9 of 9

26.

Deacon, G.B.; Phillips, R.J. Relationships Bteween the Carbon-Oxygen Stretching Frequencies of Carboxylato Complexes and the

Type of Carboxylate Coordination. Coord. Chem. Rev. 1980,33, 227–250. [CrossRef]

27.

Schroeter, B.; Jung, I.; Bauer, K.; Gurikov, P.; Smirnova, I. Hydrophobic Modification of Biopolymer Aerogels by Cold Plasma

Coating. Polymers 2021,13, 3000. [CrossRef] [PubMed]

28.

Raman, S.P.; Keil, C.; Dieringer, P.; Hübner, C.; Bueno, A.; Gurikov, P.; Nissen, J.; Holtkamp, M.; Karst, U.; Haase, H.; et al.

Alginate Aerogels Carrying Calcium, Zinc and Silver Cations for Wound Care: Fabrication and Metal Detection. J. Supercrit.

Fluids 2019,153, 104545. [CrossRef]

29.

Andreini, C.; Banci, L.; Bertini, I.; Rosato, A. Zinc through the Three Domains of Life. J. Proteome Res. 2006,5, 3173–3178.

[CrossRef]

30.

Kocyła, A.; Tran, J.B.; Kr˛e˙

zel, A. Galvanization of Protein–Protein Interactions in a Dynamic Zinc Interactome. Trends Biochem. Sci.

2021,46, 64–79. [CrossRef]

31.

Maret, W. Chemistry Meets Biology in the Coordination Dynamics of Metalloproteins. J. Inorg. Biochem. 2023,251, 112431.

[CrossRef]

32.

Arfat, Y.A.; Benjakul, S. Effect of Zinc Sulphate on Gelling Properties of Phosphorylated Protein Isolate from Yellow Stripe

Trevally. Food Chem. 2013,141, 2848–2857. [CrossRef] [PubMed]

33.

Basak, S.; Singhal, R.S. The Potential of Supercritical Drying as a “Green” Method for the Production of Food-Grade Bioaerogels:

A Comprehensive Critical Review. Food Hydrocoll. 2023,141, 108738. [CrossRef]

34.

Guastaferro, M.; Baldino, L.; Reverchon, E.; Cardea, S. Production of Porous Agarose-Based Structures: Freeze-Drying vs.

Supercritical CO2Drying. Gels 2021,7, 198. [CrossRef] [PubMed]

35.

Baudron, V.; Gurikov, P.; Smirnova, I.; Whitehouse, S. Porous Starch Materials via Supercritical-and Freeze-Drying. Gels 2019,

5, 12. [CrossRef] [PubMed]

36. El-Naggar, M.E.; Othman, S.I.; Allam, A.A.; Morsy, O.M. Synthesis, Drying Process and Medical Application of Polysaccharide-

Based Aerogels. Int. J. Biol. Macromol. 2020,145, 1115–1128. [CrossRef]

37.

Janssen, R.H.; Vincken, J.P.; Van Den Broek, L.A.M.; Fogliano, V.; Lakemond, C.M.M. Nitrogen-to-Protein Conversion Factors for

Three Edible Insects: Tenebrio Molitor, Alphitobius Diaperinus, and Hermetia Illucens. J. Agric. Food Chem. 2017,65, 2275–2278.

[CrossRef]

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